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NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin
Abstract
Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]
... The frequent appearance of GalNAc in sulfate-deficient glycans suggests an alternative hypothesis to explain reduced sulfation; human sialyltransferases compete more effectively with sulfotransferase in the human pituitary, leading to preferential addition of Neu5Ac to GalNAc. As N-glycan branches terminated with Neu5Ac-GalNAc were first reported for hLH oligosaccharides, finding this type of glycan is not unprecedented (36). ...
... In fact, hLH possesses the greatest abundance of sialic acid of all characterized mammalian LH preparations (23,36,37). Moreover, structure 23 is part of a series of 15 GalNAccontaining, biantennary glycans observed in at least one of the six hFSH preparations (structures 10-25, Figures 3A,B). ...
... Structures 31 and 32 were the most abundant oligosaccharides derived from recombinant, urinary, and pituitary hFSH ( Figure 3C). As 85-100% core-fucosylated glycans are found on the other human pituitary hormone LHβ and TSHβ subunits, structure 31 most likely reflects FSHα subunit glycosylation, while structure 32 reflects FSHβ subunit glycosylation (36,38). The 4th high abundance glycan cluster, comprising structures 38-42, includes triantennary oligosaccharides possessing only two sialic acid residues. ...
Human follicle-stimulating hormone (FSH) exhibits both macro- and microheterogeneity in its carbohydrate moieties. Macroheterogeneity results in three physiologically relevant FSHβ subunit variants, two that possess a single N-linked glycan at either one of the two βL1 loop glycosylation sites or one with both glycans. Microheterogeneity is characterized by 80 to over 100 unique oligosaccharide structures attached to each of the 3 to 4 occupied N-glycosylation sites. With respect to its receptor, partially glycosylated (hypo-glycosylated) FSH variants exhibit higher association rates, greater apparent affinity, and greater occupancy than fully glycosylated FSH. Higher receptor binding-activity is reflected by greater in vitro bioactivity and, in some cases, greater in vivo bioactivity. Partially glycosylated pituitary FSH shows an age-related decline in abundance that may be associated with decreased fertility. In this review, we describe an integrated approach involving genetic models, in vitro signaling studies, FSH biochemistry, relevance of physiological changes in FSH glycoform abundance, and characterize the impact of FSH macroheterogeneity on fertility and reproductive aging. We will also address the controversy with regard to claims of a direct action of FSH in mediating bone loss especially at the peri- and postmenopausal stages.
... NMR provides quantitation and the least ambiguity in structure determination, provided sufficient material of acceptable purity is available for study. The same 14 bi-antennary oligosaccharides were observed at each hLH glycosylation site (Weisshaar et al. 1991b). The relative proportion of each glycan differed at each site, and α-subunit glycans lacked core-linked fucose, while LHβ glycans possessed it (Fig. 4). ...
... Human LH possessed significant sialic acid content, in contrast to other mammalian LH prepartions, in which sialic acid is rare . A novel charateristic of hLH glycans was sialic acid linked to Gal residues by both α(2-6) and α(2-3) linkages (Weisshaar et al. 1991b). In CG, all sialic acids were α(2-3) linked. ...
... The plasma disappearance of endogenous LH is slower in postmenopausal than in young women (Sharpless et al. 1999), which is likely due to the increased sialylation of the gonadotropin that occurs after the menopause (Wide 1985a, b;Wide et al. 2007). The mechanism for this shift is probaby more complicated than solely the addition of GalNAc instead of Gal to partially synthesized glycans because, as discussed above, sialylated GalNAc has been reported for hLH (Weisshaar et al. 1991b). Differences in halflives among the different glycosylation variants that comprise circulating FSH also explain why in vivo and in vitro administration of a more acidic/sialylated mix of FSH isoforms or sequential administration of more acidic/sialylated FSH followed by a less sialylated variant more effectively stimulates follicular maturation and 17β-estradiol production than a poorly sialylated preparation (Colacurci et al. 2014;Nayudu et al. 2002;West et al. 2002). ...
The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin hormone (CG) play an essential role in reproduction. LH and FSH are synthesized in the gonadotropes of the anterior pituitary gland, while CG is synthesized by the placental syncytiotrophoblasts. Gonadotropins, together with thyroid-stimulating hormone (TSH) synthesized by the thyrotropes of the adenohypophysis, belong to the glycoprotein hormone family. The glycoprotein hormones are complex heterodimers consisting of a common α-subunit non-covalently associated with a β-subunit, which is structurally unique in its peptide sequence to each member of the family and that confers binding specificity at the receptor level. Both subunits are decorated with oligosaccharide chains, whose number vary depending on the particular glycoprotein hormone, and that are involved in many functional aspects, including folding and secretion of the heterodimer, as well as plasma half-life and bioactivity of the hormone at the target cell. The synthesis and secretion of gonadotropins are regulated by the concerted action of several endocrine, paracrine, and autocrine factors of diverse chemical structure, the main player being the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). Gonadotropins interact with their cognate receptors (the FSH receptor and the LH/CG receptor) in the ovary and the testes. In the ovary, FSH regulates the growth and maturation of the ovarian follicles as well as estrogen production by the granulosa cells, whereas in the testes FSH stimulates the Sertoli cells lining the seminiferous tubules to influence spermatogenesis. The target cells of LH are the theca cells of the ovarian follicles and the corpus luteum, where it promotes the synthesis of sex steroid hormones and the ovulatory process. In the testes, LH stimulates Leydig cell steroidogenesis, mainly testosterone production, to promote sexual maturation and function, and spermatogenesis. Mutations in the β-subunit genes of LH and FSH leading to gonadotropin deficiency are very rare. When they occur in LHβ, they are clinically manifested by lack of pubertal maturation and infertility in men and infertility in women, whereas mutations in FSHβ may lead to azoospermia in men and absent or partial puberty and infertility in women. Several natural and recombinant preparations of gonadotropins are currently available for therapeutic purposes. Given that glycosylation is well known to vary in a cell- and tissue-specific manner, the main difference between natural and the currently available recombinant preparations massively produced in Chinese hamster ovary cells for commercial purposes lies in the abundance of some of the carbohydrates that comprise the complex glycans attached to the protein core. Because of the functional and pharmacological similarities between natural and recombinant compounds, both may be employed in the clinical arena to treat diseases characterized by gonadotropin deficiency as well as infertility.
... Evidence supporting the existence of urinary LH cf is based on the detection of immunoreactive material resembling human chorionic gonadotrophin subunit (hCG cf) in mid-menstrual and postmenopausal urine (Iles et al. 1992, Neven et al. 1993. Green & Baenziger (1988a,b) and subsequently found to be present on LHβ by Weisshaar et al. (1991). N-acetylglucosamine (M); mannose (•); N-acetylgalactosamine ("); galactose ( ); sialic acid (m); n=1 or 2. ...
... The asparagine-linked oligosaccharides on intact human LH have previously been elucidated by Green & Baenziger (1988a,b) using conventional methodologies including radiolabelling reducible groups with NaB[ 3 H] 4 , release of oligosaccharides by N-glycanase digestion, sequential digestions with endo-and exoglycosidase enzymes followed by HPLC and ionexchange separations. In a second study by Weisshaar et al. (1991), the dissociation of the dimeric hormone was followed by the release of oligosaccharides by hydrazinolysis from LH and from tryptic glycopeptides of LH . The subsequent characterisation of the N-linked sugars largely by one and two dimensional protein nuclear magnetic resonance spectroscopy ( 1 H-NMR) permitted the attribution of the oligosaccharides to individual attachment sites. ...
... These studies, carried out before the genetic variants were known, found no evidence of an extra glycosylation site. Our data, by contrast, while finding evidence of fragments of many of the sugars described by Green & Baenziger (1988a,b) and Weisshaar et al. (1991), could not have been analysed without the assumption that a second carbohydrate moiety was present on the molecule. We found that virtually all the glycosylation on peptides bearing a mutation at 15 arises from either two separate sugars or from a strong possibility of there being two sugars on that peptide. ...
Metabolism of the human chorionic gonadotrophin (hCG)- and LHbeta-subunits (hCGbeta, LHbeta) terminates with the urinary excretion of core fragment (hCGbetacf, LHbetacf) molecules that retain antigenic shape and constituent N-linked carbohydrate moieties. We have previously demonstrated the resolved mass spectra of hCGbetacf, from which the carbohydrate moieties present at two N-linked glycosylation sites were identified. LHbetacf was subjected to the same mass spectrometric analysis. As LHbeta shares 82% homology with hCGbeta but possesses only one glycosylation consensus site a simpler spectral fingerprint of LHbetacf glycoforms was expected. LHbetacf was reduced with dithiothreitol and analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Glycoforms were predicted by subtracting the peptide mass from the m/z values of the observed peaks and then sequentially subtracting the masses of the monosaccharide residues of hCGbeta N-linked carbohydrates reported in the literature. The mass spectra of LHbetacf revealed a broad single peak ranging from m/z 8700 to 10 700. Following reduction, this peak was replaced by a set of partially resolved peaks between m/z 4130 and 5205 corresponding to glycosylated forms of the peptide LHbeta6-40. A peak at m/z 4252.2 corresponded to the non-glycosylated peptide LHbeta55-93. Remaining peaks indicated that the pooled sample comprised a wide set of glycoforms, contained LHbetacf with two N-linked carbohydrate moieties and indicated evidence of further glycosylation due to amino acid substitution in polymorphic variants. This is evidence that a single nucleotide polymorphism alters the post-translational modification of a protein and hence its structural phenotype.
... The two hormones are synthesized in the pituitary, secreted and circulate in blood as individual spectra of large numbers of isoforms (12,14). The FSH and LH isoforms isolated from pools of human pituitaries differ in their N-glycan compositions (15)(16)(17)(18). In addition, FSH has been reported to exist in human pituitary and urinary preparations as two major glycoforms designated tetraglycosylated and di-glycosylated hFSH (19,20). ...
... The average numbers of SA and SU residues per gonadotrophin molecule in each serum sample were estimated as previously described (12). The method is based upon previous observations (15)(16)(17)(18) that negatively charged terminal SA and SU residues on the N-glycans determine the variation of the electric charge of human FSH and LH. ...
... We suggest that, in analogy with FSH, the LHdi consists of an a-subunit with two N-glycans and a non-glycosylated LH b-subunit. It has been shown that the N-glycan at the position Asn30 of the LH b-subunit has a very high abundance of SU residues (17). A lack of this N-glycan on the LH b-subunit is compatible with the finding of a negative correlation between the percentage of di-glycosylated LH forms and the average number of SU residues per LH molecule. ...
Background
Glycosylation and glycan composition are of fundamental importance for the biological properties of FSH and LH. The aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum FSH and LH throughout the normal menstrual cycle.
Methods
Serum samples were collected from 79 healthy women with regular menstrual cycles. The mean numbers of anionic monosaccharide (AMS), sialic acid (SA), and sulfonated N-acetylgalactosamine (SU) residues per FSH and LH molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins.
Results
Di-glycosylated glycoforms (FSHdi, LHdi) were detected in serum in addition to tetra-glycosylated FSH (FSHtetra) and tri-glycosylated LH (LHtri). FSHdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. FSHtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. There were lower concentrations of LHdi than LHtri, except at midcycle when the opposite occurred. The mean numbers of SA and SU residues per molecule of FSH and LH in serum showed four different patterns during the cycle, all with highly significant (P < 0.0001) differences between levels at different phases of the cycle. The pattern of SA residues on FSH was ‘M’-shaped, and that of SU on LH ‘V’-shaped.
Conclusion
Serum FSH and LH governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. This new information on the FSH and LH molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women.
... One of the striking differences between FSH and LH, apart from their β subunits, is that the majority of carbohydrates on FSH are terminated with sialic acid whereas the vast majority carbohydrates on LH are terminated in sulfate. In this regard, hLH glycosylation is an exception, as 28% of the glycans are terminated with sialic acid (Fig. 5) [52]. In the original model for glycoprotein clearance, sialic acid prevented recognition of the terminal galactose residues by Ashwell's lectin, the asialo-glycoprotein receptor (ASGP-R) [53], thus prolonging hFSH half-life in the blood. ...
... The most abundant glycan found at each site is shown along with its relative abundance, if known. The hCG and hLH glycans were determined by nmr [52,108], therefore, monosaccharide sequence and linkage were unambiguous. The hFSH glycans were determined by glycopeptide mass spectrometry [62,67] leading to ambiguities in branch location and sialic acid linkages, as indicated. ...
... A. The α2-6-linked sialic acid does not block access to these. B. The α2-3-linked sialic acid blocks access to both hydroxyls by replacing the C-3 hydroxyl with a glycosidic bond and potentially steric hindrance of the C-4 hydroxyl [52], FSH largely lacks hybrid glycans, and possesses complex: biantennary, triaantennary, and tetraantennary glycans [56,62,69]. In hFSH, triantennary and tetraantennary glycans amplify heterogeneity as mono-, di-, and tri-sialyl variants of triantennary and tetraantennary glycans are found [62]. ...
The synthesis and secretion of the gonadotropic hormones involves coordination of signal transduction, gene expression, protein translation, post-translational folding and modification and finally secretion. The production of biologically active gonadotropin thus requires appropriately folded and glycosylated subunits that assemble to form the heterodimeric hormone. Here we overview recent literature on regulation of gonadotropin subunit gene expression and current understanding of the assembly and secretion of biologically active gonadotropic hormones. Finally, we discuss the therapeutic potential of understanding glycosylation function towards designing new forms of gonadotropins based on observations of physiologically relevant parameters such as age related glycosylation changes.
... The expression of specific glycoforms of hCG during pregnancy disorders and cancer could offer targets for clinical diagnosis. The N-glycans of hCG have been studied by a variety of methods including chromatography, NMR and mass spectrometry (MS) [21][22][23][24]. They have been reported to consist mainly of mono-and bi-antennary complex type structures with terminal sialic acid and with or without core fucosylation [1,17,24,25]. ...
... The N-glycans of hCG have been studied by a variety of methods including chromatography, NMR and mass spectrometry (MS) [21][22][23][24]. They have been reported to consist mainly of mono-and bi-antennary complex type structures with terminal sialic acid and with or without core fucosylation [1,17,24,25]. Some glycans have been specifically associated with malignancy [26][27][28]. ...
Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. “Hyperglycosylated” variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes “hyperglycosylation” is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks’ gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation–time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.
... For over three decades, NMR spectroscopy has served as a pivotal analytical tool for the atomic resolution characterization of glycoproteins [85][86][87] , where the problem is not only to identify the presence of a glycan, but also to pinpoint its chemical structure. Multinuclear multidimensional NMR techniques have been shown to be applicable for studying glycoproteins without need for isotope-labeling 88,89 , making them powerful alternatives to mass spectrometry for obtaining complementary chemical information on glycan modifications in proteins. ...
... Additionally, N-linked glycans have one sugar residue that is linked to Asn via the N-glycosidic bond. The "anomeric" carbon for this residue is bonded to an oxygen and a nitrogen (instead of two oxygens for the canonical anomeric carbon) and this -CH resonates at yet another unique region around 5.0/80-85 ppm, separated not only from other protein and glycan peaks, but also from other anomeric carbons ( Figure 6B) 88 The constituent sugar residues of N-and O-glycans are typically identified and their resonances assigned using a combination of homonuclear ( 1 H-1 H) and heteronuclear ( 1 H-13 C) correlation-, TOCSY-and NOESY-based NMR methods 86,88,93,94 . The sugar units themselves, as well as the linkage between these units, result in fingerprint patterns in 2D NMR datasets that have been extensively used for characterizing glycans. ...
The diversity of the cellular proteome substantially exceeds the number of genes coded by the DNA of an organism because one or more residues in a majority of eukaryotic proteins are post-translationally modified (PTM) by the covalent conjugation of specific chemical groups. We now know that PTMs alter protein conformation and function in ways that are not entirely understood at the molecular level. NMR spectroscopy has been particularly successful as an analytical tool in elucidating the themes underlying the structural role of PTMs. In this Perspective, we focus on the NMR-based characterization of three abundant PTMs: phosphorylation, acetylation, and glycosylation. We detail NMR methods that have found success in detecting these modifications at a site-specific level. We also highlight NMR studies that have mapped the conformational changes ensuing from these PTMs as well as evaluated their relation to function. The NMR toolbox is expanding rapidly with experiments available to probe not only the average structure of biomolecules but also how this structure changes with time on time scales ranging from picoseconds to seconds. The atomic resolution insights into the biomolecular structure, dynamics, and mechanism accessible from NMR spectroscopy ensure that NMR will continue to be at the forefront of research in the structural biology of PTMs.
... The 'H-NMR data of the NeuSAca2-6GalNAcfll-4GlcNAcPl-2Manal-3 branch fit those reported for the identical branch in the diantennary reference oligosaccharide alditols N 2 B and NUB-6' [24], whereas the 'H-NMR data of the GalNAcPl-4GlcNAcfll-2Manal-6 branch match those of compounds N1.3, N1.4A and N3.3. ...
... Recently, a tyvelosylated variant, namely Tyvl-3GalNAc1-4[Fuc1-3]GlcNAc has been detected in tetraantennary N-glycans of the excretory/secretory antigens of the parasite Trichirzellu spiralis [34]. With respect to u-PA, be- The (SO~)-4GalNAc~1-4GlcNAc~lstructural element has been found previously in pituitary glycohormones, like lutropin [24] or thyrotropin 13.51, but also in human urinary Tamm-Horsfall glycoprotein [ 171 and bovine pro-opiomelanocortin [32]. The element has been suggested as a modulator of the circulatory half-life of glycoproteins by recognition of a (SO;)-4Gal-NAc-specific hepatic cell receptor [36]. ...
The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl--glucosaminy)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri′-antennary structures. The glycans contain predominantly GalNAc1-4GlcNAc instead of Gal1-4GlcNAc elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (α1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4−)-4GalNAc1-4GlcNAc1-2 antennae.
... A novel human (h) FSH glycosylation variant, which possesses only αsubunit oligosaccharides has been recently reported [117,118]; this particular variant predominates in young women and it is significantly more bioactive in vitro than the tetraglycosylated form of the hormone. As in other multicellular eukaryote glycoproteins, oligosaccharide structures on glycoprotein hormones are highly variable [115,[119][120][121][122][123][124][125] and play an important role in determining several properties of the hormones [107][108][109][110]126]. For example, in human and equine FSH more than 35 glycans may be identified using mass spectometry of isolated glycopeptides [125,127];~90% of the total glycans in hFSH are sialylated or sulfated [122], and the heterogeneity of this glycoprotein is primarily determined by the variability in these negatively charged species [106,128]. ...
... For example, in human and equine FSH more than 35 glycans may be identified using mass spectometry of isolated glycopeptides [125,127];~90% of the total glycans in hFSH are sialylated or sulfated [122], and the heterogeneity of this glycoprotein is primarily determined by the variability in these negatively charged species [106,128]. Human FSH contains relatively high amounts of sialic acid-enriched oligosaccharides in their corresponding mono-, di-, tri-or tetra-antennary structures, thus confering an overall negative charge to the molecule [115,116,123,124,129], whereas hLH is sialylated to a lesser extent, is primarily sulfated, and thus is less negatively charged than hFSH [130][131][132]. Differ-ences in the extent of sialylation and sulfation among these gonadotropins are of paramount importance for determining the in vivo bioavailability and net bioactivity of the hormone; terminal sialic acid prolongs whilst sulfate residues shortens the half-life of the glycoprotein in blood. ...
Gonadotropins play a central role in the control of male and female reproduction. Selective agonists and antagonists of gonadotropin receptors would be of great interest for the treatment of infertility or as non steroidal contraceptive. However, to date, only native hormones are being used in assisted reproduction technologies as there is no pharmacological agent available to manipulate gonadotropin receptors. Over the last decade, there has been a growing perception of the complexity associated with gonadotropin receptors' cellular signaling. It is now clear that the Gs/cAMP/PKA pathway is not the sole mechanism that must be taken into account in order to understand these hormones' biological actions. In parallel, consistent with the emerging paradigm of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. Small molecule ligands, modulating antibodies interacting with the hormones and glycosylation variants of the native glycoproteins have all demonstrated their potential to trigger such selective signaling. Altogether, the available data and emerging concepts give rise to intriguing opportunities towards a more efficient control of reproductive function and associated disorders.
... Restriction of core fucosylation primarily to b-subunit oligosaccharides has been reported for other glycoprotein hormones (69)(70)(71). Asn 78 glycans appear less accessible in folded FSHa as indicated by limited PNGaseF sensitivity in folded a-subunit (7). One could argue these glycans are less accessible to Golgi FUT8 because only folded proteins enter this compartment. ...
Follicle-stimulating hormone (FSH), an α/β heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHβ subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHβ band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks βAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHβ glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.
... It has been shown that Gal(β1− 4)GlcNAc(β-)-specific α2,6-sialyltransferase can also act on the nonreducing terminal residue, GalNAc(β1−4)GlcNAc(β-), to form NeuAc(α2−6)GalNAc(β1−4)GlcNAc(β-), 54 found in the majority of mono-and disialylated N-glycans at Asn 78 of human luteinizing hormone. 55 The Sd a antigen determinant, NeuAc(α2−3)[GalNAc(β1−4)]Gal(β1−4)GlcNAc(β-), was also present based on the ions at m/z 670.27 and m/z 873.35 (Hex 1 HexNAc 2 NeuAc 1 ) ( Figure 4B). This determinant was previously found on the Asn 232 of UMOD. ...
The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.
... The N-glycosylation of TSH alpha and beta-subunit polypeptides occurring co-translationally in the rough endoplasmic reticulum (ER) and the branching of the glycans and their decorations with terminal SA and SU residues in the Golgi of specific human anterior pituitary gland cells are schematically illustrated in Figure 1; nomenclature, pathways and design from references (10)(11)(12)(13)(14). ...
Context
In severe primary hypothyroidism (sPH), the serum TSH levels are elevated with an increased degree of sialylation. The circulating TSH comprises two different TSH glycoforms: TSHdi with two and TSHtri with three N-glycans and methods have developed to determine their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylglactosamine (SU) residues.
Objective
Characterize N-glycosylation and glycan composition of circulating TSH molecules and determine the effects during Levothyroxine treatment in patients with sPH.
Design, Subjects, Main Outcome Measures
Serum samples were obtained from 25 patients with sPH, from 159 euthyroid individuals and from twelve women during treatment with Levothyroxine for sPH. Degrees of N-glycosylation and concentrations of TSHdi and TSHtri as well as their contents of AMS, SA and SU residues were determined.
Results
The circulating TSH molecules in sPH patients had lower degrees of N-glycosylation, higher of sialylation and lower of sulfonation than in euthyroid individuals. Levothyroxin restored sialylation and sulfonation of the glycans already at low FT4 levels, while degree of N-glycosylation was not restored until the FT4 levels were normal.
Conclusions
The majority of TSH molecules in severe primary hypothyroidism were less N- glycosylated, more sialylated and less sulfonated, compared with euthyroid individuals. This glycan pattern favors a prolonged half-life in the circulation combined with lower in-vitro biopotency at the target cells. During Levothyroxine treatment of sPH patients, the sialylation and sulfonation of glycans were restored already at low FT4 levels, while N-glycosylation of TSH was not restored until the FT4 levels were normal.
... Both α-Neu5Ac-(2 → 6)-β-D-GalpNAc-(1 → 4)-β-D-GlcpNAc-(1 → R and α-Neu5Ac-(2 → 3)-β-D-GalpNAc-(1 → 4)-β-D-GlcpNAc-(1 → R motifs are naturally occurring elements of animal glycoconjugate glycans. Typical examples of the (α2-6)-sialylation are found in human lutropin [31], Bowes melanoma tissue plasminogen activator [32], human urokinase [33], bovine mammary gland derived glycoproteins [34][35][36], and recombinant human protein C expressed in human kidney 293 cells [37]. Typical examples of the (α2-3)sialylation are shown for a thrombin-like serine protease, ancrod, from the viper Agkistrodon rhodostoma [38] and a thrombin-like enzyme, batroxobin, from the snake Bothrops atrox moojeris [39]. ...
... As LH and FSH glycan populations are quite different in their branching patterns, their paths may remain separate. Their exposure in the medial Golgi to oligosaccharide branch-initiating GlcNAc transferases I to VI (132) appears to differ, resulting in more extensive branching of FSH glycans than LH (133). Adding the seven-residue, C-terminal LHb peptide to the FSHb C terminus directed the mutant FSH to the regulated secretory pathway (134). ...
Follicle-stimulating hormone glycosylation varies in two functionally important aspects, microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity arising from partial FSHβ subunit glycosylation. While advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, that exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHβ glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. In order to resolve these alternatives, well-characterized FSH glycoform preparations are necessary.
... LH molecules with two or three sulfonated N-acetylgalactosamine (SO(3)-GalNAc) residues show shorter half-lives than less sulfonated LH, suggesting their rapid removal by hepatic Kupffer cells, whereas higher-sialylated gonadotropin isoforms have extended half-lives, likely due to the masking of the sulfonated oligosaccharides to S4GGnM binding and sequestration (136,137). However, rather than SO(3), sialic acid may be linked to LH GalNAc residues, likely resulting in extended half-life anyway (138). ...
Luteinizing hormone (LH) and chorionic gonadotropin (CG) are glycoproteins fundamental for sexual development and reproduction. Since they act on the same receptor (LHCGR), there is a general consensus that LH and hCG are equivalent. However, separate evolution of LHβ and hCGβ subunits occurred in primates, resulting in two molecules sharing ∼85% identity and regulating different physiological events. Pituitary, pulsatile LH production results in a ∼90 min half-life molecule targeting the gonads, to regulate gametogenesis and androgen synthesis. Trophoblast hCG, the “pregnancy hormone”, exists in several isoforms and glycosylation variants with long half-lives (hours), angiogenic potential, and acts on luteinized ovarian cells as a progestational. The different molecular features of LH and hCG lead to hormone-specific LHCGR binding and intracellular signaling cascades. In ovarian cells, LH action is preferentially exerted through kinases, pERK1/2 and pAKT, resulting in irreplaceable proliferative/anti-apoptotic signals and partial agonism on progesterone production in vitro. In contrast, hCG displays notable cAMP/PKA-mediated steroidogenic and pro-apoptotic potential, which is masked by estrogen action in vivo. In vitro data are confirmed by large dataset from assisted reproduction, since the steroidogenic potential of hCG positively impacts on the number of retrieved oocytes, while LH impacts pregnancy rate (per oocyte number). Interestingly, Leydig cell in vitro exposure to hCG results in qualitatively similar cAMP/PKA and pERK1/2 activation as compared to LH, as well as testosterone. The supposed equivalence of LH and hCG is debunked by such data highlighting their sex-specific functions, thus deeming it an oversight caused by incomplete understanding of clinical data.
... The N-glycosylation occurs in the rough endoplasmic reticulum (ER) [see schematic drawing in Figure 7 with nomenclature, pathways, and design from refs. (22)(23)(24)(25)]. Dolichol is a special lipid that works as a carrier of the oligosaccharide precursor. ...
Background: The natural ovarian stimulation is mediated by four gonadotrophin glycoforms: FSHtri with three, FSHtetra with four, LHdi with two, and LHtri with three N-glycans. The aim of the study was to determine the serum concentrations of the four glycoforms and their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU) residues throughout the menstrual cycle.
Methods: Serum samples were collected from 78 healthy women with regular menstrual cycles. The serum glycoform molecules were identified by their distributions at electrophoreses. Analyses were also performed after removal of terminal SA. The hormones were measured with time-resolved sandwich fluoroimmunoassays.
Results: The concentration profiles of the four glycoforms were markedly different. FSHtri, which had a 3-fold higher biopotency than FSHtetra, had peak levels on cycle day 5 and at midcycle and nadirs on cycle days 9 and 21–23. FSHtetra had a raised level on cycle days 5–12, followed by a decrease. LHdi and LHtri had similar patterns, but the peak/nadir ratio was much more pronounced for LHdi than for LHtri, 18 versus 4. The numbers of SA residues per molecule were at a maximum around midcycle when the corresponding numbers of SU were at a minimum. The SU/SA ratio was at a minimum on cycle day 12.
Conclusion: The results indicate that the LHdi and the FSHtri molecules play major roles in the natural ovarian stimulation. The SU/SA ratios per molecule favoured a prolonged circulatory half-life of all glycoforms at the midcycle phase. The observations may lead to more successful inductions of ovulation in anovulatory women.
... These three hormones are not three single entities. Instead, each hormone exhibits a considerable heterogeneity due to different degrees of glycosylation and differences in glycan composition (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The isoforms can be separated by electrophoresis due to variation in charge determined by their contents of two terminal anionic monosaccharides (AMS): sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). ...
Background: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones.
Methods: The hormones were separated with respect to AMS content per molecule by calibrated 0.1% agarose suspension electrophoreses. Glycoforms in separated fractions were then analyzed with respect to size by 180 calibrated Sephadex G-100 gel filtrations. The hormones were measured with time-resolved sandwich fluoroimmunoassays. All separations and assays were performed in veronal buffer at pH 8.7. Sera and fractions were also analyzed after removal of terminal SA.
Results: In addition to the fully glycosylated FSH, LH, and TSH, also tri-glycosylated FSH and di-glycosylated LH and TSH forms could be identified in serum samples. The low- and fully glycosylated hormones differed both with respect to size and to median number of AMS per molecule. Algorithms, based on the distributions by electrophoreses, were developed for each hormone to estimate percent low-glycosylated forms in serum. The median numbers of SA and SU per glycoform molecule were estimated using results obtained after desialylation.
Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.
... Takifugu rubripes (pufferfish) has also substituted this Thr for Met, whereas the other 83 species studied have the glycosylation site consensus sequence (Supplementary Table 3). No crystal structure of LH β is available but Weisshaar et al. (1991) describe the carbohydrate structures of hLH at Asn30 in the β chain, established by 1D and 2D 1 H-NMR spectroscopy, composition analysis, methylation analysis and fast-atom-bombardment mass spectrometry (FAB-MS). This is consistent with the analysis based on amino acid sequence alignment. ...
We have studied glycosylation patterns in glycoprotein hormones (GPHs) and glycoprotein hormone receptor (GPHR) extracellular domains (ECD) from different species to identify areas not glycosylated that could be involved in inter- or intra-molecular interactions. Comparative models of the structure of the TSHR ECD in complex with TSH and in complex with TSHR autoantibodies (M22, stimulating and K1-70, blocking) were obtained based on the crystal structures of the FSH-FSHR ECD, M22-TSHR leucine-rich repeat domain (LRD) and K1-70-TSHR LRD complexes. The glycosylation sites of the GPHRs and GPHs from all species studied were mapped on the model of the human TSH-TSHR ECD complex. The areas on the surfaces of GPHs that are known to interact with their receptors are not glycosylated and two areas free from glycosylation, not involved in currently known interactions, have been identified. The concave faces of GPHRs leucine rich repeats 3 to 7 are free from glycosylation, consistent with known interactions with the hormones. In addition, four other non-glycosylated areas have been identified, two located on the receptors' convex surfaces, one in the long loop of the hinge regions and one at the C terminus of the extracellular domains. Experimental evidence suggests that the non-glycosylated areas identified on the hormones and receptors are likely to be involved in forming intra- or inter-molecular interactions.
... Glycosylation macroheterogeneity, the presence or absence of one or more glycans at known glycosylation sites, and microheterogeneity, the structural heterogeneity of glycans attached to the same site, provide the basis for most glycoprotein structural heterogeneity [1]. For the glycoprotein hormones, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and chorionic gonadotropin (CG), microheterogeneity resulting from populations of 2-30 glycans decorating three or four N-glycosylation sites [2][3][4][5][6][7] has been the focus of attention for the last several decades [8][9][10][11][12][13]. ...
FSH glycosylation macroheterogeneity in pituitary and urinary hFSH samples was evaluated by Western blotting.
Microheterogeneity in two highly purified urinary and pituitary hFSH preparations was evaluated by nano-electrospray
mass spectrometry of peptide-N-glycanase-released oligosaccharides. An age-related loss of hypo-glycosylated hFSH in
individual female pituitaries was indicated by progressively reduced abundance of hFSH21 relative to hFSH24. Urinary
hFSH was evaluated as a potentially non-invasive indicator of glycoform abundance, as the only way for pituitary FSH to
reach the urine is through the blood. Both highly purified and crude postmenopausal urinary hFSH preparations possessed
the same amount of hFSH21 as postmenopausal pituitary gland FSH. Considerable microheterogeneity was encountered
in both pituitary and urinary hFSH glycan populations, as 84 pituitary hFSH glycan ions were observed as compared with
68 urinary hFSH glycans. The biggest quantitative differences between the two populations were reduced abundance of
bisecting GlcNAc-containing and fucosylated glycans, along with sulfated glycans in the urinary hFSH glycan population.
The relative abundance of sialic acid and glycan antenna did not rationalize the retarded electrophoretic mobilities of the
urinary hFSHb21- and a-subunit bands relative to the corresponding pituitary hFSH bands, as the most abundant glycans
in the former possessed only 2 more branches and the same sialic content as in the latter. Site-specific glycosylation
information will probably be necessary.
... The observed diversity of terminal glycosylation sequences suggests that the peripheral sugar residues are not essential for the biological action of these hormones. 22 This observation, together with the fact that structures lacking N-glycosylation exhibit almost complete loss of hormone function, 23 indicates that the underlying core structures may play a more relevant role in the biological activity of both hormones. 24 However, the entire β-hCG carboxy-terminus, which contains four O-glycosylation sites with mucin-type O-glycans ( Figure 1D), has been found to be a common epitope for hCG-based monoclonal antibodies, which can inhibit tumor cell growth and metastasis. ...
The gonadotropic hormones human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α subunit and a unique β subunit, that form non-covalent heterodimers. Structurally, β-hCG shares a high degree of sequence similarity with β-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogenous β-hLH and β-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of β-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (β-hCG), as well as its closely related homologue (β-hLH), as discrete glycoforms.
... Several studies on N-glycosylation of gonadotropins have shown that 1) although the a-subunit is common, maturation of oligosaccharides is dependent on the formation of the different heterodimer-complexes [20][21][22], and 2) glycosylation is site-specific even in the same subunit [23][24][25]. These facts indicate that N-glycosylation is influenced by the surrounding polypeptide chain(s) at a given site. ...
hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common α-subunit, but differ in their hormone-specific β-subunits. hCGβ is unique among β-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of α-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Cα) and another analog with CTP at the C-terminus (αC) of the α-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Cα and αC were expressed alone or with FSHβ-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [ 35S]methionine/cysteine, immunoprecipitation with anti-α or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Cα than that in αC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric αC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Cα was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).
... Since DSiaT is only expressed during embryonic development of the central nervous system [11], we can hypothesize that ST6GAL2 compensates the low expression of ST6GAL1 in brain for a precise unknown function, which could be conserved from invertebrate to human. However, pituitary hormones, glycodelin and urokinase, are the only glycoproteins known to exhibit terminal SLDN motifs141516. Moreover, α2,8-sialylation is the most, if not the only, type of sialylation depicted for neuronal functions such as cell surface interactions, neurogenesis or neuronal plasticity [17,18] and terminal α2,6-sialylation has not been yet reported in brain-related glycoconjugates. ...
... bovine LF, is an immunogenic structure and has been indicated in allergies [28,29] , possibly induced by tick bites [28]. A second epitope that is uncommon in human glycosylation, absent in human LF, but present in bovine LF, is the Gal- NAc(β1-4)GlcNAc (LacdiNAc) sequence, next to the Gal(β1-4)GlcNAc (LacNAc) epitope (known human glycoprotein examples are urokinase [30], lutropin [31], glycodelin [32], human protein C [33] , Tamm- Horsfall glycoprotein [34], and kallidinogenase [35]). Immunogenicity of this epitope has been shown in parasite infections [36], but not in relation to food allergies. ...
Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.
For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.
This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.
Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.
Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.
Context
A preponderance of basic luteinizing hormone (LH) molecules having elevated bioactivity was detected in the circulation of women with polycystic ovary syndrome (PCOS). Subsequent studies have shown that both LH and follicle-stimulating hormone (FSH) circulate as glycoforms differing in number of glycans: low-N-glycosylated glycoforms, LHdi and FSHtri, with high in-vitro bioactivity, and fully-glycosylated glycoforms, LHtri and FSHtetra, with high in-vivo bioactivity.
Objective
Characterize the glycosylation patterns on circulating gonadotropin glycoforms in women with PCOS.
Design, Subjects, Main Outcome Measures
Serum samples, collected from 8 women with PCOS were included. Concentration, sulfonation and sialylation of each glycoform were determined and compared with values of serum samples from healthy women: 22 women at follicular phase, 16 at mid- cycle and 15 after menopause.
Results
All the women with PCOS had higher LHdi serum levels compared with those of the follicular phase group. Median LHdi and median LHtri levels were significantly elevated in PCOS women. The percentage of LHdi was increased from 37 to 49 and that of FSHtri was decreased from 41 to 33. The LHdi, LHtri and FSHtetra glycoforms were more sialylated and both LH glycoforms less sulfonated in women with PCOS.
Conclusions
All women with PCOS had increased serum levels of LHdi, compared with those of follicular phase. The percentage of LHdi was increased and that of FSHtri decreased in women with PCOS. The increased LHdi leads to maintenance of the abnormal early follicular development of the polycystic ovary and the decreased FSHtri contributes to the arrested follicle growth.
The gonads (ovary, testis) produce gametes (oocytes and spermatozoa). The pituitary glycoprotein hormones luteinizing hormone (LH, lutropin) and follicle-stimulating hormone (FSH, follitropin) are gonadotropins because their targets are the gonads. Another hormone produced by the syncytiotrophoblast of the human placenta is human chorionic gonadotropin (hCG, choriogonadotropin), and its activity is like LH. This review describes the protein structure, gene structure, and regulation of biosynthesis of both the gonadotropins and their membrane bound receptors. The functions of both hormones and receptors are covered extensively, as well as naturally occurring mutations that have been identified in disease states and therapies derived from their study.
The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin hormone (CG) play an essential role in reproduction. LH and FSH are synthesized in the gonadotropes of the anterior pituitary gland, while CG is synthesized by the placental syncytiotrophoblasts. Gonadotropins, together with thyroid-stimulating hormone (TSH) synthesized by the thyrotropes of the adenohypophysis, belong to the glycoprotein hormone family. The glycoprotein hormones are complex heterodimers consisting of a common α-subunit non-covalently associated with a β-subunit, which is structurally unique in its peptide sequence to each member of the family and that confers binding specificity at the receptor level. Both subunits are decorated with oligosaccharide chains, whose number vary depending on the particular glycoprotein hormone, and that are involved in many functional aspects, including folding and secretion of the heterodimer, as well as plasma half-life and bioactivity of the hormone at the target cell. The synthesis and secretion of gonadotropins are regulated by the concerted action of several endocrine, paracrine, and autocrine factors of diverse chemical structure, the main player being the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). Gonadotropins interact with their cognate receptors (the FSH receptor and the LH/CG receptor) in the ovary and the testes. In the ovary, FSH regulates the growth and maturation of the ovarian follicles as well as estrogen production by the granulosa cells, whereas in the testes FSH stimulates the Sertoli cells lining the seminiferous tubules to influence spermatogenesis. The target cells of LH are the theca cells of the ovarian follicles and the corpus luteum, where it promotes the synthesis of sex steroid hormones and the ovulatory process. In the testes, LH stimulates Leydig cell steroidogenesis, mainly testosterone production, to promote sexual maturation and function, and spermatogenesis. Mutations in the β-subunit genes of LH and FSH leading to gonadotropin deficiency are very rare. When they occur in LHβ, they are clinically manifested by lack of pubertal maturation and infertility in men and infertility in women, whereas mutations in FSHβ may lead to azoospermia in men and absent or partial puberty and infertility in women. Several natural and recombinant preparations of gonadotropins are currently available for therapeutic purposes. Given that glycosylation is well known to vary in a cell- and tissue-specific manner, the main difference between natural and the currently available recombinant preparations massively produced in Chinese hamster ovary cells for commercial purposes lies in the abundance of some of the carbohydrates that comprise the complex glycans attached to the protein core. Because of the functional and pharmacological similarities between natural and recombinant compounds, both may be employed in the clinical arena to treat diseases characterized by gonadotropin deficiency as well as infertility.
Asn-linked oligosaccharides terminating with the sequence SO4-4GalNAcbeta1,4GlcNAcbeta1,2Manalpha (S4GGnM) are present on the glycoprotein hormones lutropin and thyrotropin, pro-opiomelanocortin, and tissue factor pathway inhibitor. The peptide motif ProXaaArg/Lys (PXR/K), which is recognized by a PXR/K-specific GalNAc-transferase, is present in each of these glycoproteins 6-9 residues NH2-terminal to an Asn glycosylation site. Both the PXR/K-specific GalNAc-transferase and a GalNAcbeta1,4GlcNAcbeta1,2Manalpha (GGnM)-4-sulfotransferase are required for synthesis of the S4GGnM sequence. Glycoproteins which do not contain the PXR/K motif but bear Asn-linked oligosaccharides terminating with GGnM or sialic acidalpha2,3/6GGnM have also been described, suggesting a distinct GalNAc-transferase may be responsible for their synthesis. We have examined a number of tissues and cultured cell lines for the transfer of sulfate to the trisaccharide acceptor GGnM and transfer of GalNAc to oligosaccharide acceptors on protein which do, human chorionic gonadotropin (hCG), and do not, transferrin (Trf), contain the PXR/K motif. The PXR/K-specific GalNAc-transferase and the GGnM-4-sulfotransferase are expressed in salivary gland, pituitary, lacrimal gland, kidney, and brain, and in the cell lines AtT-20, 293, SHSY5Y, and alphaT3. In contrast Bowes, EL-4, and B16L6 cell extracts transferred GalNAc to oligosaccharides acceptors on Trf but not on hCG. A number of tissues and cell lines displayed transfer of GalNAc to both hCG and to Trf suggesting that two distinct GalNAc-transferases were present. The GGnM-4-sulfotransferase was expressed in tissues and cell lines which expressed the PXR/K-specific GalNAc-transferase but not in cell lines expressing exclusively the Trf-specific GalNAc-transferase. Thus, the PXR/K-specific GalNAc-transferase and the GGnM-4-sulfotransferase are coordinately expressed in a number of tissues other than pituitary. The Trf-specific GalNAc-transferase may account for the presence of beta1,4-linked GalNAc on glycoproteins which do not contain the PXR/K motif.
The glycoprotein hormone family includes the pituitary hormones, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), of direct interest to reproductive physiology, and thyroid-stimulating hormone (TSH), as well as the chorionic gonadotropins. The biosynthesis of the glycoprotein hormones involves all the complexities of the protein biosynthetic mechanisms provided in nature, from genomic transcription, mRNA translation, cotranslational processing, to the final post-translational processing. The numerous intermediates in the post-translational processing in the Golgi apparatus, several of which may become final forms of the complex carbohydrate moieties, produce a significant heterogeneity in the secreted hormone. This heterogeneity is reflected in several isoforms of that particular glycoprotein hormone. These isoforms, to a degree, also correlate with the physiological state of the individual organism. Thus, the protein biosynthesis of glycoprotein hormones is a very complex process subject to many physiological controls, few of which are fully understood.
We have characterized two BHK cell lines (BHK-21B and BHK-21A) with different glycosylation properties. N-glycans synthesized by BHK-21B cells contain the typical N-acetyllactosamine motif (Gal(βl-4)GlcNAc-R) whereas BHK-21 A cells bear to a high amount nonsialylated terminal GalNAc(βl-4)GlcNAc-R moieties [1]. Due to the incapability of the endogenous oc2, 3-sialyltransferase to transfer NeuAc to the GalNAc(βl-4) GlcNAc-R structure recombinant glycoproteins produced by BHK-21 A cells are under-sialylated.
Therefore we have transfected BHK-21 A cells harbouring a plasmid encoding human EPO with the human Golgi enzyme CMP-NeuAc:Gal(βl-4)GlcNAc-R a2,6-sialyltransferase (ST6N). Detailed structural analysis of Oligosaccharides from the affinity purified recombinant EPO (HPAEC-PAD-mapping and MALDI/TOF-MS) revealed a significant increased NeuAc content when compared to the parent BHK-21 A cells without ST6N activity. Methylation analysis corroborated these results. The newly introduced α2,6-sialyltransferase recognizes the terminal GlcNAc-R motif as a substrate. The cell line obtained thus exhibits a ‘human kidney-type’ glycosylation characteristic [2].
Follicle-stimulating hormone (FSH) is a member of the glycoprotein hormone family, which is a subfamily of the cystine knot growth factor superfamily [1,2]. The glycoprotein hormones are composed of heterodimeric glycoprotein subunits, a common α-subunit, and a hormone-specific β-subunit. While the α-subunit primary structure is identical for all glycoprotein hormones within the same species, the oligosaccharide populations differ in a hormone-specific manner [3–6]. Characterizing the oligosaccharides released from an α-subunit preparation can identify the hormone from which the subunit was derived [7]. There are 3 to 4 β-subunits in vertebrates, which combine with α-subunit to create either FSH, luteinizing hormone (LH), thyroid-stimulating hormone (TSH), or in primates and equids, chorionic gonadotropin (CG) [8]. As both glycoprotein hormone subunits are cystine knot proteins [9–11] the protein backbone is folded into a series of three loops, two relatively rigid hairpin loops on one side of the knot, designated L1 and L3, and a single, flexible loop on the other side [12], designated L2. Oligosaccharides are attached to all 3 loops in a subunit-specific pattern (Figure 1). FSH subunits possess two potential N-glycosylation sites on each subunit and all four are of the Asn-Xaa-Thr type, which exhibit very efficient carbohydrate attachment [13]. Indeed, the α-subunit is always glycosylated at both sites in all known glycoprotein hormones. Because FSH α and β subunits co-migrate during electrophoresis, it is difficult to detect missing N-glycans in this hormone. FSHβ-specific Western blots have revealed partial glycosylation in equine FSHβ, human FSHβ (hFSH β), rhesus FSH β, and Japanese macaque FSHβ [14–16]. During the past few years, we have studied partially glycosylated hFSH isolated from pituitary extracts, postmenopausal urine, and conditioned tissue culture medium containing recombinant hFSH. Each glycosylation site in hFSH is decorated with a population of N-glycans. When total glycans are removed from reduced, carboxy-methylated FSH subunits, 39–130 glycans are found in mass spectra. We have data from only one glycosylation site, αAsn52, which possessed 29 neutral core ions, and when decorated with various patterns of sialic acid grew to 109 unique glycan structures. Micro heterogeneity can affect electrophoretic mobility, for example, placental hCGα with hybrid and biantennary glycans migrated faster than pituitary hFSHα, with triantennary, biantennary and tetraantennary glycans, which complicated sorting out the hFSH variants that resulted from loss of one or more N-glycans [17].
The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N- and O-linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the α3-linked sialic acid residues followed by α6-sialylation, α3-galactosylation or α3-fucosylation of uncovered Galβ1→4GlcNAc (LacNAc) termini, or α3-sialylation of Galβ1→3GalNAc sequences. Also a form that carried GalNAcβ1→4-GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by β4-N-acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as-hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon α6-sialylation of the Galβ1→4GlcNAcβ1→2Manα1→3Man branch of the N-linked glycans. This was without a major effect on receptor binding. Further α6-sialylation, occurring at the Galβ1→4GlcNAcβ1→2Manα1→6Man branch, resulted in a bioactivity below a level found with as-hCG, concomitant with a decreased receptor binding affinity. Similarly α3-galactosylation of the Galβ1→4GlcNAcβ1→2-Manα1→6Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. α3-Fucosylation of native as well as as-hCG also led to a decreased activity. Re-α3-sialylation of the O-linked chains on as-hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAcβ→4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is concluded that the Galβ1→4GlcNAcβ1→2Manα1→3Man- rather than the Galβ1→ 4GlcNAcβ1→2-Manα1→6Man branch of the N-linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O-linked chains is of minor importance.
Gonadotropins are a family of three glycoprotein hormones (FSH, LH and hCG) essential for steroid production and reproductive functions. Over the past two decades, these glycoproteins either from extractive origin or produced by recombinant technology have been marketed for assisted reproductive techniques. Recombinant gonadotropins are produced by rodent cell lines which display glycosylation machinery different from human cells and often add undesired carbohydrate determinants which may alter protein folding, induce immunogenicity and overall reduce circulatory half-life of the drug. Notably, they fail to transfer sialic acid as N-acetylneuraminic acid (Neu5Ac) in a α 2,6-linkage as in the natural endocrine cells and this affects their activity and duration in blood. We have designed ST6Gal minigenes to optimize sialic acid transfer in the most common drug-approved cell line i.e the Chinese Hamster Ovary cells. We present herein various strategies that may be used to produce a2,6-sialylated gonadotropins. A level of 60-90% of sialylation may be routinely achieved depending on the enzymeminigene used to equip the producer clone.
Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1 → 6)-α-l-fucosylated structures in Fractions A and B. Sequences containing α-d-Galp-(1 → 3)-β-d-Gal on the α-d-Man-(1 → 6) antenna, and β-d-GalpNAc-(1 → 4)-β-d-GlcNAc and α-NeuAc-(2 → 6)-β-d-GalpNAc-(1 → 4)-β-d-GlcNAc on the α-d-Man-(1 → 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4 − 9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-β-d-glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.
In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the α-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element , which is part of the carbohydrate chains of human lutropin, has been prepared by chemical and chemo-enzymatic synthesis in the form of its propyl glycoside. Condensation of trichloroacetimidate with allyl gave after deacetylation allyl . Ethyl was converted into the galacto-derivative ethyl via an oxidation-reduction route, as well as via SN2-type substitution with acetate. The use of this galacto thioglycoside, after its conversion into the corresponding bromide, as GalN donor for condensation with the mentioned disaccharide derivative yielded after deacetylation allyl . Methylsulfenyl bromide-silver triflate promoted sialylation of this trisaccharide derivative with dithiocarbonate and subsequent deprotection resulted into the aimed tetrasaccharide structural element. Alternatively, this compound was prepared via a block synthesis, which, however, was not superior to the linear strategy. Finally, a stereoselective sialylation of synthetically prepared with CMP-Neu5Ac and rat liver α-2,6-sialyltransferase was accomplished affording the same tetrasaccharide structural element.
Recombinant human Protein C (rHPC), expressed in human kidney 293 cells, has a higher anticoagulant activity than plasma HPC, while its in vivo circulatory half-life is essentially unaltered compared to that of the natural protein. In seeking to elucidate the molecular basis for the improved efficacy of the recombinant antithrombotic drug, we focused on the carbohydrate moiety of rHPC. Protein C is a heavily post-translationally modified serine protease with four N -glycosylation sites. Glycosyl composition analysis of rHPC revealed a 5-fold higher fucose content and a 2-fold lower sialic acid content compared to plasma HPC. In addition, we found that rHPC contains N -acetylgaiac-tosamine (2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides, while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharides of rHPC were released by N -glycanase and separated into 25 fractions by high-pH anion-exchange chromatography. The most abundant oligosaccharides were structurally characterized by glycosyl composition and linkage analysis, in conjunction with 1H-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPC are: and Thus, many of the Asn-linked oligosaccharides in rHPC were found to terminate in GaINAcβ(1→4)GlcNAcβ(1→•), in NeuAca(2→6)GalNAcβ(1→4)GlcNAcβ(1→•), and/or in GaINAcβ(1→4) [Fuca(1→3)]GlcNAcB(1→•). Since the latter trisaccharide was first [Yan, S.B., Chao, B.Y. and Van Halbeek, H. (1992) J. Cell. Biochem ., 16D, 151] observed in the Asn-linked oligosaccharides of rHPC derived from human kidney 293 cells, we propose to label the GaINAcβ(1→4)[Fuca(1→3)]GlcNAcβ(1→•) terminal trisaccharide the PC-293 determinant. The PC-293-containing oligosac-charides may contribute to the higher anticoagulant activity of rHPC as compared to plasma HPC.
This chapter discusses mass spectrometry (MS) of carbohydrates and glycoconjugates. All mass spectrometers contain three basic components: the ion source, the mass analyzer, and the detector. Ions are produced in the ion source by one of the many ionization methods, such as electron ionization (EI), field desorption (FD), and chemical ionization (CI). The mass analyzer separates the ions according to their mass-to-charge ratio (m/z), and then the ions strike the detector and are converted to electrons, thereby producing an ion current proportional to the number of incident ions. A plot of the relative abundances of each ion versus its m/z value is a mass spectrum. There are several methods of introducing a sample into a mass spectrometer, depending on the type of sample to be analyzed and the ionization method used. The complete analysis of a glycoprotein involves the identification of the entire array of oligosaccharide structures attached to the protein and the quantitative assignment of each structure to its site of attachment on the polypeptide chain.
This chapter discusses the structure and function of the carbohydrate chains of the glycoprotein hormone family. The family of glycoprotein hormones consists of lutropin (LH), follitropin (FSH), and thyrotropin (TSH) synthesized in the anterior pituitary and chorionic gonadotropin which is synthesized only in the placenta of human, primate, and equine. The hormones are heterodimers composed of two non-covalently joined α and β subunits. Both subunits are glycosylated and contain N- and, in the case of the CGβ subunit, O-linked oligosaccharides which account for 15-40% of their mass. Assembly of the common α subunit, with any of the four β subunits, results in unique properties of each hormone that are reflected in binding to hormone-specific receptors and subsequent signal transduction. Gonadotropins regulate reproductive functions; LH stimulates steroidogenesis in ovarian granulosa and theca cells, and testicular Leydig cells. One function of hCG is to maintain high progesterone production in the corpus luteum to maintain pregnancy and hCG and LH bind to the same receptor.
Assay performance criteria of two immunoradiometric kits (LH-COATRIA bioMérieux, LH IRMA Immunotech) and of one radioimmunoassay (LH K-PR Cis bio international) were studied and the results obtained with the 3 methods in 155 serum samples were compared. They confirmed the improvements introduced by immunometric assays as regards precision, detection limits and specificity. Concerning accuracy, significant differences were observed about international standard (1er IRP 68/40) recoveries with these three kits. This preliminary study allowed to dissociate technicol discrepancies (matrix effects, calibration) from those related to hLH microheterogeneity. Despite level differences in results from the 155 serum samples, concentrations measured by Cis and bioMérieux kits did not lead to differing clinical conclusions. About 20% outlying hLH bioMérieux / hLH Immunotech and hLH Cis / hLH Immunotech ratios were found in the 155 serum samples assayed and could not be related to particular clinical features. hLH concentrations were found to be undetectable in two serum samples with the Immunotech technique, while they were found to be over 20 UI/I with the other kits; furthermore, hLH present in these serums was bioactive on cultured rat Leydig cells. Chromatofocusing, used to separate hLH isoforms from one of these two serums, did not allowed characterization of any special hLH forms which could explain the differences in immunoreactivity. Although 80% of the discrepancies in level observed with these three kits could be explained by calibration and matrix effects problems, the exact cause of the significant disagreements between results from some particular serums remains to be determined.
One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. Accurate comparison of glycoforms and quantification of glycosites is an essential step in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group (gPRG) study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked and it has been observed that prostate cancer tissues and cell lines, contain more antennae than the benign form. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact use of PSA as a biomarker. We studied standard peptide based proteomics/glycomics methodologies including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences in glycoforms between two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods, respectively.
Bovine whey protein products are used as a base ingredient in infant formulae, to optimise the amino-acid pattern to a more human-like composition. Although the protein composition of bovine milk has been studied in detail, glycosylation details of commercial whey protein products are missing. To this end both the N- and O-glycans of such a protein concentrate were sequentially released, the N-glycans enzymatically and the O-glycans chemically (reducing and non-reducing conditions). For the structural analysis of the N- and O-glycans a combination of MALDI-TOF-MS, one-dimensional (1)H NMR spectroscopy, Wisteria floribunda agglutinin affinity chromatography, HPAEC-PAD profiling, and HPLC-FD profiling (2-aminobenzamide derivatives), together with exoglycosidase treatments, were used. A mixture of over 60 N-glycans and 10 O-glycans was characterized, giving a detailed insight into the glycosylation of a bovine whey protein product, Deminal® 90, which is applied as an ingredient for infant formulae.
CD36 is a glycoprotein included in the bovine milk fat globule membrane derived from mammary secretory epithelial cells during lactation. Asparagine-linked sugar chains were quantitatively released from CD36 as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with (NaBH4)-H-3 and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with serial chromatography on immobilized lectin columns including a Wistaria floribunda agglutinin (WFA)-agarose column. WFA is known to bind oligosaccharides terminating with either an alpha- or beta-N-acetylgalactosamine residue. Structural studies of oligosaccharides in each fraction by sequential exoglycosidase digestion as well as methylation analysis revealed that CD36 contains high mannose-type, hybrid-type, and bi-, tri-, and tetraantennary complex-type sugar chains. A portion of the hybrid-type and the complex-type sugar chains which bound to a WFA-agarose column (28% of all oligosaccharides) contained the GalNAcbeta1-->4GlcNAc group(s) instead of the Galbeta1-->4GlcNAc group(s) in their outer chain moieties. Like oligosaccharides found in human luteinizing hormone [Weisshaar, G., Hiyama, J., Renwick, A. G., & Nimtz, M. (1991) Eur. J. Biochem. 195, 257-268], some of the GalNAcbeta1-->4GlcNAc groups found in the CD36 oligosaccharides were sialylated as the Neu5Acalpha2-->6GalNAc group. Furthermore, most of the hybrid-type sugar chains of CD36 with the Gal/GalNAcbeta1-->4GlcNAcbeta1-->2 outer chain on their Manalpha1-->3 arm contained an unusual Manalpha1-->2Manalpha1-->3 group on their Manalpha1-->6 arm.
A high pH anion exchange chromatographic (HPAEC) system for the separation of isomeric sialo-oligosaccharide products was developed. Employing this system, using Galβ1→4GlcNAcβ1→2Manα1→6Manβ1→4GlcNAc as a substrate, a Galβ1→4GlcNAc-R α2→3-sialyltransferase activity was detected for the first time in human liver. This activity is expressed together with the prevalent α2→6-sialyltransferase. Furthermore, in addition to the major α2→3-sialyltransferase, a low but distinct activity of α2→6-sialyltransferase was detected in human placenta. This activity could not be found by methods based on methylation analysis or high resolution NMR spectroscopy. It is concluded that HPAEC, in combination with the use of the pentasaccharide as an acceptor substrate, is suited for the specific detection of minor, Galβ1→4GlcNAc-specific sialyltransferase activities.
Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz1H-NMR spectroscopy.
The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation.
The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells) with glycosyltransferases, nucleotide-sugar synthases and transporters appears to be sufficient to guarantee complex-type glycosylation of recombinant proteins with a high degree of terminal α2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydrate motifs are not synthesized by these cells since they lack the proper sugar-transferring enzymes (e.g. α1-3/4 fucosyltransferases, α2-6 sialyltransferases). Glycosylation engineering of these hosts by stable transfection with genes encoding terminal human glycosyltransferases allows to obtain products with tailored (human tissue-specific) glycosylation in high yields.
Using site-directed mutagenesis, unglycosylated polypeptides can be successfully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to different loop regions of acceptor proteins.
The genetic engineering of glycoproteins and of host cell lines are considered to provide a versatile tool to obtain therapeutic glyco-products with novel/improved in-vivo properties, e.g. by introduction of specific tissue-targeting signals by a rational design of terminal glycosylation motifs.
The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In the accompanying paper (Green, E.D., and Baenziger, J.U. (1987) J. Biol. Chem. 262, 25-35), we elucidated the structures of the anionic asparagine-linked oligosaccharides found on the bovine, ovine, and human pituitary glycoprotein hormones. In this study, we determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear alpha-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by greater than 10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (alpha 2,3 versus alpha 2,6). In addition to differences in the proportion of sulfated and sialylated structures on LH and FSH, there were site-specific variations in the amount of mono- and disulfated oligosaccharides at different glycosylation sites on LH alpha-beta dimers. The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.
For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N
4-(N-acetyl--glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz1H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAc2-3Gal1-4GlcNAc1-2Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man1-3]Man 1-4GlcNAc1-4[Fuc1-6]0-1GlcNAc and Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man 1-3]Man1-4GlcNAc1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAc2-3Gal1-3[NeuAc2-6]GalNAc, NeuAc2-3Gal 1-3GalNAc and NeuAc2-3Gal1-3[NeuAc2-3Gal1-4GlcNAc1-6]GalNAc.
The role of the carbohydrate part of human chorionic gonadotropin (hCG) was investigated by measuring the ability of hCG derivatives lacking various sugar residues to bind to rat Leydig cells and stimulate them to synthesize testosterone and cyclic adenosine 3':5'-monophosphate (cyclic AMP). Whereas sequential removal of the sialic acid, galactose, N-acetylglucosamine, and mannose residues led to a progressive increase in the effective dose of the hormone required to stimulate steroidogenesis, it resulted in a marked loss in the ability of the hormone to stimulate cyclic AMP accumulation. Low doses of the glycosidase-treated hormone derivatives were additive with hCG when their ability to stimulate testosterone synthesis was analyzed. Nevertheless, the glycosidase-treated derivatives were potent inhibitors of hCG-induced cyclic AMP accumulation, suggesting that removal of the sugars did not influence binding of the hormone to the cell as much as it reduced the ability of the bound hormone to activate adenyl cyclase. This hypothesis was further supported by our finding that the hCG derivatives were highly effective inhibitors of 125I-hGC binding to the intact cells. Removal of sialic acid and galactose enhanced the inhibition, whereas removal of all the sugar residues only decreased the inhibition slightly. The degree of these effects was comparatively small. The possibility that steroidogenesis and cyclic AMP accumulation are altered independently by hCG stimulation is discussed.
The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.
Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCG alpha was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the alpha genes (wild-type and mutant) and the hCG beta gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and less than 20% is secreted. However, the presence of hCG beta stabilizes this mutant and allows approximately 45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the alpha subunit but does affect dimer secretion; under conditions where this mutant or hCG beta was in excess, less than 30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCG alpha asparagine-linked oligosaccharides with respect to the stability and assembly of hCG.
The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.
The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.
The structure of the polypeptide chains and oligosaccharide moieties of the alpha and beta subunits of pituitary and placental glycoprotein hormones are known. The dimeric polypeptide structure (but not the carbohydrate) is important for binding of the hormone to specific receptors. The N-linked but not O-linked carbohydrates, on the other hand, are required in some manner to activate the effector system. Hormones with depleted carbohydrate content (deglycosylated hormones) interact with receptor but are unable to activate intracellular events. Because of such discordant properties, these forms act as competitive inhibitors of hormone action. Through a combination of chemical deglycosylation procedures and site-directed mutagenesis, the first site of N-glycosylation from the NH2 terminus of the common alpha subunit has been identified to be more critical for glycoprotein hormone signal transduction. Control of glycosylation by the endocrine milieu could contribute to regulation of hormone function by secreting variable forms of agonist/antagonist.
The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form. In bovine pituitary an extra O-linked oligosaccharide is added to free alpha subunit, and this modification has recently been detected at an analogous position (threonine 39) on human alpha subunit secreted by choriocarcinoma cells. To assess the contribution of N-linked and O-linked oligosaccharides to the heterogeneity of human free alpha subunit, we have compared free alpha with human chorionic gonadotropin alpha secreted by explants and cultured cytotrophoblasts of human first trimester placenta. We have also examined the free and combined forms of human alpha subunit expressed in transfected C-127 mouse mammary tumor cells. Processing of the alpha subunit in placental and C-127 cells was similar. Tryptic mapping of placental-derived and transfected alpha subunits indicated that O-glycosylation at threonine 39 was not a major modification. In the presence of the oligosaccharide processing inhibitor swainsonine the difference in size between the free and combined forms of alpha was eliminated in both placental and C-127 cells, indicating that the two forms of alpha differed in their N-linked oligosaccharides. Furthermore, the oligosaccharides of free alpha subunits from placental and transfected cells were resistant to endoglycosidase H, but the combined forms of alpha were partially sensitive to the enzyme. Thus, in human first trimester placenta and mouse C-127 cells, combination of alpha with human chorionic gonadotropin beta alters the processing of N-linked oligosaccharides on alpha subunit.
The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In the accompanying paper (Green, E.D., and Baenziger, J.U. (1987) J. Biol. Chem. 262, 25-35), we elucidated the structures of the anionic asparagine-linked oligosaccharides found on the bovine, ovine, and human pituitary glycoprotein hormones. In this study, we determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear α-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by > 10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (α2,3 versus α2,6). In addition to differences in the proportion of sulfated and sialylated structures on LH and FSH, there were site-specific variations in the amount of mono- and disulfated oligosaccharides at different glycosylation sites on LH α-β dimers. The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.
The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.
The influence of quaternary structure on glycosylation was evaluated in a macrophage-like cell line, P388D1. This cell line simultaneously synthesizes two structurally related glycoproteins, Mac-1 and LFA-1. Mac-1 and LFA-1 each contain two subunits in noncovalent association in an alpha 1 beta 1 structure. The beta-chain polypeptides of these two glycoproteins have identical primary structures while their alpha-chain polypeptides are distinct. For both Mac-1 and LFA-1, the association of the alpha- and beta-chains occurs prior to any Golgi-mediated processing of the oligosaccharide moieties on either one of the subunits. To evaluate the effects of differential subunit association on the site-specific glycosylation of the beta-chain, [3H]glucosamine-labeled oligosaccharides were isolated from the beta-chain of Mac-1 and LFA-1 and were compared by a variety of enzymatic and chromatographic techniques. Reverse-phase high performance liquid chromatography analyses of tryptic-chymotryptic glycopeptides suggest that each beta-chain has at least five glycosylation sites. Structural analysis of oligosaccharides from each corresponding glycopeptide fraction of the beta-chains of Mac-1 or LFA-1 (comparing their glycosidase sensitivities, behavior on serial lectin affinity chromatography, size heterogeneity, extent of sialylation, and branching) indicates that the LFA-1 beta-chain is glycosylated substantially differently on at least four of its sites, compared to the corresponding sites of the Mac-1 beta-chain, even though they are simultaneously synthesized in the same cells. Thus, these data demonstrate that quaternary structure can influence the site-specific glycosylation of a protein, even when the polypeptide structure and the cellular glycosylation machinery remain constant.
Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.
Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.
By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Man alpha 1----3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N-acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1----3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme.
Lutropin (LH), follitropin (FSH), and thyrotropin (TSH) from pituitary and human chorionic gonadotropin (hCG) from placenta are a family of glycoprotein hormones, each with an alpha and beta subunit. The alpha subunits of all four hormones have the same amino acid sequence, whereas biological specificity is determined by their unique beta subunits. The carbohydrate compositions of these hormones indicate the structures of their Asn-linked oligosaccharides are not identical. Sulfate is present on most, but not all, of these hormones, and for bovine LH is attached to GalNAc (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We used a reconstituted cell-free system to study sulfation of bovine (b) and human (h) glycoprotein hormones and its relationship to glycosylation. Exogenously added bLH, bTSH, bFSH, hLH, and hTSH are sulfated exclusively on the oligosaccharides of both alpha and beta subunits. The distribution of sulfated oligosaccharide structures varies among the hormones and appears to result from differences in the extent and/or pathway of oligosaccharide processing. Significant amounts of disulfated, dibranched complex oligosaccharides are present on all the sulfated hormones. Human FSH is not susceptible to sulfation unless first treated with neuraminidase. The sulfated oligosaccharides obtained from bovine FSH and desialylated human FSH are unlike those of the other hormones. Therefore, there is differential processing of the oligosaccharides on pituitary hormones. For FSH and LH, which are believed to be synthesized in the same cell, we would suggest that the unique beta subunits may regulate processing of all oligosaccharides present on the alpha-beta dimers.
Hen ovomucoid is characterized by a high degree of microheterogeneity of its carbohydrate moieties, as was recently demonstrated by hydrazinolysis in combination with high performance liquid chromatography (Paz Parente, J., Strecker, G., Leroy, Y., Montreuil, J., and Fournet, B. (1982) J. Chromatogr., in press). This approach resulted in 17 oligosaccharide-alditol fractions; the major one could be purified to homogeneity. Primary structural analysis of this fraction was carried out by methylation analysis, partial acid hydrolysis, and 500-MHz 1H NMR spectroscopy. Combination of these techniques enabled the unambiguous determination of a novel type of asparagine-bound carbohydrate chain: (formula, see text).
This chapter deals with the use of nuclear magnetic resonance (NMR) spectroscopy in the study of mono– and oligosaccharides. The importance of NMR spectroscopy in the study of carbohydrates has increased tremendously. This has occurred primarily because the introduction of pulsed Fourier transform (FT) NMR spectrometers has made the measurement of I3C NMR spectral parameters easy, which is particularly important for the study of carbohydrates in aqueous solutions. In addition, pulsed NMR instruments have increased the sensitivity of 'H NMR spectra by several orders of magnitude and facilitated the measurement of relaxation times and nuclear Overhauser enhancement (NOE) factors. The chapter discusses the way to assign NMR parameters and the way to use these values in the study of carbohydrates. Moreover, the chapter illustrates that the assignment of the NMR signals is a prerequisite for the application of NMR spectroscopy in structural investigations of carbohydrates. Because assignment techniques have been described in many reviews and monographs, special emphasis is given to the problems associated with the assignment of signals in the NMR spectra of carbohydrates and their derivatives.
Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid-stimulating hormone, and human follicle-stimulating hormone are closely related family of proteins which share a common α-subunit. However, their sugar moieties are quite different.
hCG contains five acidic asparagine-linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N-1 and N-2) and one monoantennary (N-3) complex-type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N-1, N-2, and N-3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the α- and β-subunits of hCG revealed that α contains 1 mol each of N-2 and N-3, while β contains 1 mol each of N-l and N-2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones.
hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocareinoma patients contain five additional neutral oligosaccharidcs. In contrast, hCGs from invasive-mole patients contain three of the live oligosaccharidcs, specifically found in choriocarcinoma hCGs.
The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fucαl→6GlcNAc group and by ectopic expression and subsequent modification of N-acetylglucosaminyltransferase IV. The appearance of tumor-specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.
Human chorionic gonadotropin (hCG) purified from placenta, like urinary hCG, is shown to have the sialylated forms of three neutral oligosaccharides: Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAc (N-1), Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-2) and Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-3). Gel permeation chromatographic analysis of oligosaccharides released from α- and β-subunits of placental hCG has revealed that the α-subunit has one each of sialylated N-2 and N-3, while the β-subunit has one each of sialylated N-1 and N-2.
We have developed methods for rapid fractionation of anionic oligosaccharides containing sulfate and/or sialic acid moieties by high-performance liquid chromatography (HPLC). Ion-exchange HPLC on amine-bearing columns (Micropak AX-10 and AX-5) at pH 4.0 is utilized to separate anionic oligosaccharides bearing zero, one, two, three, or four charges, independent of the identity of the anionic moieties (sulfate and/or sialic acid). Ion-exchange HPLC at pH 1.7 allows separation of neutral, mono-, di-, and tetrasialylated, monosulfated, and disulfated oligosaccharides. Oligosaccharides containing three sialic acid residues and those bearing one each of sulfate and sialic acid, however, coelute at pH 1.7. Since the latter two oligosaccharide species separate at pH 4.0, analysis at pH 4.0 followed by analysis at pH 1.7 can be utilized to completely fractionate complex mixtures of sulfated and sialylated oligosaccharides. Ion-suppression amine adsorption HPLC has previously been shown to separate anionic oligosaccharides on the basis of net carbohydrate content (size). In this study we demonstrate the utility of ion-suppression amine adsorption HPLC for resolving sialylated oligosaccharide isomers which differ only in the linkages of sialic acid residues (α2,3 vs α2,6) and/or location of α2,3- and α2,6-linked sialic acid moieties on the peripheral branches of oligosaccharides. These two methods can be used in tandem to separate oligosaccharides, both analytically and preparatively, based on their number, types, and linkages of anionic moieties.
The three-dimensional structures are presented for representative N-linked oligosaccharides drawn from four major structural classes. The nuclear magnetic resonance evidence leading to these structures is summarized and the conclusions are compared with those obtained from potential energy calculations and from the crystal structures of fragments. The major finding is that the Man alpha 1-3 arm structure is invariant throughout the series, whereas the Man alpha 1-6 arm has two possible orientations in some structures and a single orientation in others. Finally, the three-dimensional structures permit a rationalization of the substrate specificity of several key enzymes in the biosynthetic pathway DA - 19831220IS - 0714-7511LA - engPT - Journal ArticleRN - 0 (Oligosaccharides)SB - IM
This chapter discusses the application of high-resolution, 1H-nuclear magnetic resonance (NMR) spectroscopy to the structural analysis of carbohydrates related to glycoproteins. Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the O-glycosylic chains are linked to the hydroxyl group of amino acid residues such as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). The high-resolution, 1H-NMR spectroscopy technique, in conjunction with methylation analysis, is extremely suitable for the structural studies of N-, as well as on O-, glycosylic glycans. For the interpretation of the 1H-NMR spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structural reporter groups” was developed. This means that the chemical shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit the assigning of the primary structure. This chapter presents literature data on the high-resolution, 1H-NMR spectroscopy of carbohydrates derived from glycoconjugates. It also discusses the results for carbohydrates related to the glycoproteins of N-glycosylic type.
The Asn‐linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one‐ and two‐dimensional 400‐MHz ¹ H‐NMR spectroscopy.
Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its α and β subunits (oLHα, glycosylated at Asn56 and Asn82; oLHβ glycosylated at Asn13). Oligosaccharides from intact oLHβ and from glycopeptides obtained after tryptic digestion of oLHα were released by hydrazinolysis and subsequently fractionated according to charge and size by anion‐exchange and ion‐suppression amine‐adsorption HPLC, respectively. ¹ H‐NMR analysis revealed, that monosulphated, mostly hybrid‐type, oligosaccharides predominate at both glycosylation sites of oLHα, whereas a disulphated, diantennary N ‐acetyllactosamine‐type structure accounts for more than 60% of total oligosaccharides in the β subunit. Furthermore, the saccharides attached to the β subunit are almost completely fucosylated (Fucα1–6) at the reducing terminal GlcNAc, whereas the sugar chains in oLHα are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56).
The results clearly indicate a distinct subunit‐ and site‐specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.
Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-β-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Manα1→6(Mam α1→3)Manα1→6(Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAcβ1→, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Manα1a1[6(Manα1→3)Manα→6 and Manα1→3Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcN Acβ1→, were found at Asn12, as previously published [Schluter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. and Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAcα2→3 or Galα1→3Galβ1→4GlcNAcβ1→2Manα1→6(NeuAcα2→6 or NeuAcα2→3Gal-β1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNacβ1→4GlcNAcβ1→ with some bifurcation at →6Manα1→6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Galβ1→3GalNAcα1→, monosialylated or disialylated at position 3 of Gal or/and position 6 of GalNAc.
A new preparative procedure is described for the efficient purification of LH and TSH from frozen human pituitary glands. LH and TSH were isolated simultaneously from crude preparations by hydrophobic-interaction chromatography followed by separation and purification by reverse-phase high-performance liquid chromatography in a single step. Highly purified hormones were prepared in good yields (45 mg LH and 20 mg TSH/1000 glands) and with high biological activities; the potencies of purified LH and TSH were 5·8 × NIH-LH-S1 equivalent in an ovarian ascorbic acid depletion assay and 7·1 U/mg against human TSH MRC Research Standard (T1 70/9) in the McKenzie assay respectively. Cross-contamination by other glycoprotein hormones was low.
Journal of Endocrinology (1990) 125, 493–500
Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
The gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) of pituitary origin, and chorionic gonadotropin (hCG, eCG) of placental origin, along with thyroid-stimulating hormone (TSH) constitute a family of glycoprotein hormones. The α-subunit is common to all of the hormones and shows considerable homology from one species to another. The β-subunits are unique for LH, FSH, and TSH. The ß-subunits of hLH and hCG are highly homologous (85%) through the first 114 residues, but hCG β differs in that it has a C-terminus extension rich in serine and proline. This offers a chemical basis for their common biological activities. Baboon chorionic gonadotropin, and eLH and chorionic gonadotropin ß-subunits also have C-terminal extensions. The glycoprotein hormone ß-subunits also show regions of homology between themselves and among different species. A wide range of chemical and enzymatic modifications have been employed in efforts to define residues and sequences in the ß-subunits that may be essential for receptor binding. In general, chemical modifications of reactive amino acids appear to be more tolerated in the ß- than in the α-subunits.
The glycoprotein hormones lutropin (LH) and follitropin (FSH), which have common alpha-subunits but hormone-specific beta-subunits,
are both synthesized in the gonadotroph. However, they bear Asn-linked oligosaccharides that differ in structure. Those on
LH terminate with the sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, whereas those on FSH terminate with the
sequence sialic acid alpha-Gal beta 1----4GlcNAc beta 1----2Man alpha. A GalNAc-transferase was identified in bovine pituitary
membranes that recognizes features of the alpha-subunit peptide and adds GalNAc to its oligosaccharides with an apparent Michaelis
constant of 25 micromolar. The different patterns of glycosylation for LH and FSH indicate that access to the protein recognition
marker on the alpha-subunit is modulated by the associated beta-subunit. The tightly regulated synthesis of sulfated and sialylated
oligosaccharides on the pituitary glycoprotein hormones suggests these oligosaccharides have an important biological role.
The biological properties of recombinants of glycoprotein hormones in which the alpha and beta subunits were differentially deglycosylated have been investigated. Specific deglycosylation of the alpha subunit generated a recombinant that had more receptor-binding activity but did not produce hormone response in the target cells. The deglycosylated alpha + beta recombinant was also an antagonist of the action of the native hormone. Thus, the carbohydrates in the alpha subunit play a dominant role in the transduction of the hormone signal into the cell.
It has become evident that a reevaluation of the biological role of oligosaccharides is necessary. Oligosaccharides exist mainly in covalent association with proteins or lipids, consequently both intramolecular and intermolecular functions are simultaneously influenced by the carbohydrate. Changes in protein stability, rate of proteolysis, thermal stability, solubility, etc that occur when glycosylation variants are generated experimentally support the idea that intramolecular interactions involving both carbohydrate and polypeptide define the physical properties of a glycoprotein. Paradoxically, dramatic changes in the structures of the oligosaccharide moieties of glycoconjugates often are associated with oncogenic and ontogenic events without affecting cell viability. The structural and functional roles of oligosaccharide moieties of proteins are interdependent and controlled variation in the structures is necessary. At any developmental stage cells may have solved the biosynthetic problem of controlled variation by making not just one glycoprotein (i.e. protein with a single oligosaccharide at each site) but by coding for large repertoires of a protein, each variant with different covalently attached oligosaccharides. This pool of protein glycoforms would contain distinct members, each of which may have a unique spectrum of biological activities while maintaining the intramolecular characteristics necessary for structural integrity. Hence, a glycoprotein has composite activity, which a cell can control by varying the relative incidence of each glycoform. Displaying the entire spectrum of oligosaccharides simultaneously (i.e. microheterogeneity) may be necessary to avoid immunogenicity at later developmental stages. A more flexible mechanism that allows for the interdependence of the structural and functional roles of oligosaccharides can be envisaged if the polypeptide directs its own glycosylation. The biosynthesis of oligosaccharides while covalently attached to folded peptide domains appears to be controlled in part by polypeptide-oligosaccharide interactions involving stabilization of oligosaccharide conformers by peptide. Recognition of, or failure to interact with, specific oligosaccharide conformers (in addition to steric restraints) by processing enzymes, continues or diverges the biosynthetic pathway. Control of the expression of glycosidases and transferases by the cell gives rise to cell-type-specific glycosylation patterns. Clearly, the separation and isolation of individual glycoforms should provide researchers with neutral variants to probe the functional properties of the oligosaccharide moieties of glycoproteins. In this review we hope to place into perspective those experimental findings that are directing the current theories concerning the role of N-linked oligosaccharides in mediating protein-specific biological activity.
Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.
Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Björndal, Lindberg and Svennson, 1967; Björndal, Hellerqvist, Lindberg and Svensson, 1970).This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.
A rapid and simple gas-liquid chromatographic method for the determination of subnanomolar amounts of carbohydrates derived from glycoproteins is described. The procedure involves methanolysis in the presence of methyl acetate followed by removal of hydrogen chloride by coevaporation with t-butyl alcohol and trimethylsilylation. The method is also applicable to samples containing uronic acids and lipids.
The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz 1H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue.
The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues.
The three-dimensional structures are presented for representative N-linked oligosaccharides drawn from four major structural classes. The nuclear magnetic resonance evidence leading to these structures is summarized and the conclusions are compared with those obtained from potential energy calculations and from the crystal structures of fragments. The major finding is that the Man alpha 1-3 arm structure is invariant throughout the series, whereas the Man alpha 1-6 arm has two possible orientations in some structures and a single orientation in others. Finally, the three-dimensional structures permit a rationalization of the substrate specificity of several key enzymes in the biosynthetic pathway.
A version of the methylation analysis of complex carbohydrates by gas chromatography-mass spectrometry of the methylalditol acetates (H. Björndal, C. G. Hellerquist, B. Lindberg, and S. Svensson (1970) Angew. Chem. 82, 643-674) is described. With this version 100- to 500-pmol samples of N-glycosidically linked glycoprotein oligosaccharides may be analyzed. The method is based on the use of capillary columns which allow the separation of all partially methylated alditol acetates potentially obtained from this group of oligosaccharides and on their selective and sensitive detection by mass fragmentography after chemical ionization with ammonia.
A rapid method for the fractionation of anionic oligosaccharide and glycopeptide species on the basis of net carbohydrate content utilizing high-performance liquid chromatography has been developed. Amine-bearing bonded-phase columns are eluted with a mobile phase consisting of a water:acetonitrile gradient containing 3% acetic acid titrated to pH 5.5 with triethylamine. Phosphorylated and sialylated oligosaccharides within various charge classes differing in their hexose or hexosamine contents but bearing the same number of anionic species can be resolved without prior removal of the anionic moieties. Glycopeptides containing at least as many as six amino acids are also well fractionated on the basis of carbohydrate content. A variety of detection methods may be used and sensitivity in the subnanomole range is possible with fluorescent or radiolabeling techniques. This method offers a significant improvement in the rapidity and resolution attainable for the size fractionation of anionic complex carbohydrates.
The LH potencies of 12 preparations of highly purified human pituitary LH, from 6 laboratories, were estimated by 2 in vivo bioassays and an in vitro bioassay in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (coded 69/104); and by immunoassay in terms of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay (IRP; coded 68/40). The LH potencies varied between preparations, including the IRP (68/40), from 864 to 5740 IU/mg by seminal vesicle weight gain (SVW) assay; from 1510 to 11500 IU/mg by ovarian ascorbate depletion (OAAD) assay; from 4490 to 14500 IU/mg by in vitro (testicular interstitial-cell testosterone production) bioassay; and from 2030 to 9180 IU/mg by immunoassay. Estimates of protein content were based on the assumption that the absorbance of LH at 280 nm (A 1%1 cm) was 6.0.
The LH potency of most preparations was highest by in vitro bioassay and lowest by SVW assay. The correlation between activities determined by SVW and OAAD assays was more marked than that between estimates by OAAD assay and in vitro bioassay; there was no correlation between estimates by SVW assay and in vitro bioassay. The slopes of the log dose-response curves of preparations in the OAAD assay were positively correlated with their potencies by OAAD assay and negatively correlated with the slopes of their log dose-response curves in the SVW assay. The qualitative differences between preparations are considered to be a reflection of the heterogeneity of LH and of its modification by different purification procedures. The present data, together with the different patterns of heterogeneity found in some of these preparations by isoelectric focusing in a separate study, suggest that the more basic molecular forms of LH, which are preferentially purified during the isolation of LH free from FSH and TSH, have shorter plasma survival times than the more acidic forms.
The LH immunoreactivities of all preparations were significantly correlated with their potencies estimated by each of the in vivo bioassays but not with those estimated by in vitro bioassay. The ratios of in vitro bioactivity (in terms of IRP (68/40)): immunoreactivity varied between preparations from 0.53–1.5.
The FSH content of each preparation was less than 2% (w/w) by bioassay and immunoassay. Most preparations were more potent by in vitro bioassay than by in vivo bioassay, which contrasted with, and complemented, findings for purified FSH preparations. This indicated that, as in the case of LH, the more basic molecular species of FSH are associated with lower ratios of in vivo: in vitro bioactivity than are the more acidic species.
This study provides the most comprehensive comparison available of the activities of purified preparations of LH isolated from frozen and acetone-dried human pituitary glands in different experienced laboratories. These data are needed for selecting material for an international reference preparation of LH for immunoassay on the basis of high LH potency by in vivo bioassay, recommended by the WHO as a criterion for the identity of the hormone and for its freedom from contaminants. The consequences of the heterogeneity of LH are considered for the purification of the reference material and for the suitability of the latter for the various types of specimens which require LH assays.
The N-glycosidic carbohydrate chains of hen beta-ovomucoid were released from the protein by hydrazinolysis, and separated by HPLC. Primary structural analysis of 3 major fractions was conducted by applying 500-MHz 1H-NMR spectroscopy in combination with methylation analysis. One of the fractions investigated appeared to consist of an intersected penta-antennary structure extended with one Gal residue. The location of the latter in a certain branch could be established unambiguously by NMR. This structure is a novel member of the family of N-glycosidic carbohydrates of glycoproteins.
Several enzymic and chemical methods liberate asparagine-linked sugar chains as oligosaccharides. Among them, endo-β-N-acetylglucosaminidases have been used to elucidate the whole structures of highmannose type of sugar chains and all of the sugar chains of hen egg albumin, and to confirm the presence of hybrid-type asparagine-linked sugar chains. However, the substrate specificities of these enzymes have hampered their use for the study of complex-type asparaginelinked sugar chains, a most important series of this group. The method described in this chapter is the chemical procedure applicable to the study of asparagine-linked sugar chains without any limitations. The hydrazinolysis reaction is used for the isolation of whole asparagine-linked sugar chains as oligosaccharides by N-acetylation, instead of cleaving all glucosamine linkages by deamination. For determining an optimal condition to release oligosaccharides, the hydrazinolysis products of bovine IgG and of gltcoprotein-IV obtained from hen egg albumin are carefully examined.
We have developed a method for the rapid separation of anionic oligosaccharide species by high-performance liquid chromatography utilizing a MicroPak AX-10 ion-exchange column (Varian Associates) with the mobile phase consisting of 25–500 mm KH2PO4, pH4.0. Separation of oligosaccharides bearing zero, one, two, three or four sialic acid residues requires less than 45 min. Oligosaccharides containing mannose-6-PO4 moieties in monoester or diester linkage can also be analyzed in this system. Preparative separations of as much as 20 mg of oligosaccharide can be accomplished in a single chromatographic analysis with quantitative yields of oligosaccharide. This method should prove useful for the rapid isolation and characterization of anionic oligosaccharide species.
A detailed analysis of the proton magnetic resonance spectral parameters for the anomeric and C2 hydrogen resonances of 63 different glycopeptides and oligosaccharides of known structure reveals a general method for the determination of the primary structure of glycopeptides for most currently known classes of structures. In particular, a two-dimensional display formed by plotting mannosyl C1-H vs. C2-H chemical shifts demonstrates that these pairs of values are sensitive to long-range perturbation by remote substitution by hexoses as well as to direct substitution effects. A total of 41 Cl-H/C2-H chemical shift clusters have been defined which characterize unique structural microenvironments. On the basis, the sequence and branching pattern for most structures can be derived. Corroborative evidence is obtained from an examination of the chemical shifts of the galactosyl and N-acetylglucosaminyl anomeric hydrogens as well as other features of the spectrum.
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