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Carbohydrates in protein. 6. Studies on the carbohydrate-peptide bond in hen's-egg albumin

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... m.p. 170-172 °C (lit. 166-168 °C).267 ...
Thesis
Glycosylation is a prevalent form of post translational modification, believed to occur on over 50% of human proteins. Homogeneous forms of glycoproteins are essential for developing an understanding of how activity is mediated at a structural level. As biological origins of glycoproteins give rise to complex mixtures of glycoforms, homogeneous glycoprotein production has become an important goal. As chemical protein synthesis is often limited to sequences of 30-50 residues, access to large native glycoproteins is currently restricted to fragment based approaches. Protein semi-synthesis enables the preparation of larger proteins which can be difficult to obtain through chemical synthesis alone. Consequently, a general semi-synthetic strategy towards N-glycoproteins has been proposed and demonstrated on Interferon-β-1 (IFNβ), a 166 residue glycoprotein. A three fragment strategy was designed, relying on the chemical synthesis of a short glycopeptide segment and recombinant expression of the two flanking domains. Homogeneity was established through the chemical synthesis of a glycopeptide containing a natively linked N-acetylglucosamine (GlcNAc), also enabling the selective transfer of complex oligosaccharides. After cloning and expression, the recombinant fragments were functionalised to allow assembly of the protein using Native Chemical Ligation. These desired protein modifications were achieved through the application of highly chemoselective reactions. These reactions were also applied towards the generation of N-glycopeptides compatible with the ligation strategy. Further to this, existing methods enabling the direct synthesis of functionalised N-glycopeptides were also explored. After glycopeptide synthesis, endoglycosidase A enabled the transfer of oligosaccharides to the N-acetylglucosamine motif. This has allowed the preparation of the desired IFNβ glycopeptide as well as a glycosylated variant of glucagon like peptide-1. To expand the utility of endoglycosidase methodology, a novel sugar nucleotide was synthesised to facilitate the incorporation of a sialyl galactose mimic onto N-glycans. The resulting oligosaccharides may serve as novel substrates for endoglycosidases in the preparation of N-glycoprotein mimics.
... Also plotted in this figure is the presumed number of good hits, computed using the estimated FDR, for each ICScore threshold. It is clear that while the number of presumed good, matched spectra increases rapidly for small values (5,10,20) of the ICScore, as this threshold is further relaxed, there are few additional good matches made for larger ICScore values (100, 200, 500, …), even though the total number of matched spectra continues to rise. Furthermore, the additional spectra matched with high ICScores represent a significantly lower ratio of presumed good to random matches. ...
Article
Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something which detached N-glycan and de-glycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy which takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false-discovery-rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at false-discovery-rate 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at false-discovery-rate 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS.
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Digestion of bovine glomerular basement membranes with purified collagenase resulted in the solubilization of over 90% of the carbohydrate and peptide portions. After further digestion of the solubilized material with Pronase, the carbohydrate units, with only a few amino acid residues attached, were isolated by gel filtration and ion exchange chromatography. Characterization of the glycopeptides separated by these fractionation procedures indicated the presence in the basement membrane of two distinct types of carbohydrate units. One type is a disaccharide unit containing glucose and galactose, while the second type is a heteropolysaccharide consisting of galactose, mannose, hexosamines, sialic acids, and fucose, with an average molecular weight of 3500. The carbohydrate of the basement membrane appears to be equally distributed between these two types of units. There are approximately 10 disaccharide units for every heteropolysaccharide unit in the membrane. The carbohydrate-peptide linkage of the disaccharide unit was shown to be a glycosidic linkage involving the hydroxyl group of hydroxylysine, whereas the attachment of the heteropolysaccharide unit to the peptide most likely involves asparagine. Periodate oxidation studies indicated that approximately 70% of the hydroxylysine residues of the basement membrane are involved in the linkage of the disaccharide unit.
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When passed repeatedly down a Sephadex G-25 column, pronase digests of ovalbumin gave glycopeptide fractions with hexose: hexosamine ratios varying progressively from 5:4.8 to 5:2.5. The bulk of the material approximated to a 5:3 ratio, but showed continuous dispersion on recycling on Sephadex.The preparation of pronase contained hexose and yielded a glucose-containing glycopeptide on autodigestion. Errors from this source were avoided by purifying the proteolytic enzyme before use. Pronase was examined for some of the commoner glycosidase activities, with negative results.
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Benzyl 2-acetamino-2-deoxy-α-D-glucopyranosiduronic acid was obtained by direct oxidation of the corresponding glucoside, and converted to esters (methyl, ethyl and benzyl) and amides (methyl, ethyl, phenyl and benzyl). Condensation of the uronic acid with aminoacid benzyl-esters (gly, L-ala, L-asp, and L-glu) by dicyclohexylcarbodiimide gave the corresponding amides, some of which were hydrogenated to N-(2-acetamino-2-deoxy-D-glucuronyl)-aminoacids. Acid hydrolysis of the peptides were examined.
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Comparison of the lipase-catalyzed cleavage of polar esters derived from ethylene glycol proved 2-methoxyethyl (ME) esters most favorable protecting groups for the carboxylic function of peptides and glycopeptides. They combine high substrate acceptance and iligh yields of hydrolysis with favorable physicochemical properties and advantageous solubility. The application of this polar ester as protecting group was extended to N-glycosylated amino acids and N-glycopeptides. The selective removal of ME esters by lipases was achieved under mild conditions (pH 7.0 and 37°C), leaving all other linkages including peptide bonds and other ester protecting groups unaffected.
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A synthesis of N-glycosyl amides by the acylation of glycosyl isothiocyanates with carboxylic acids is proposed. The by-products are N,N'-bisglycosylureas and N,N'-bisglycosylthioureas.
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Three asparagine derivatives serving as intermediates in the synthesis of glycopeptides have been synthesized. 2,3,6-Tri-O-acetyl-N-[1-benzyl N-(benzyloxy-carbonyl)-L-aspart-4-oyl]-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranosylamine and the 2-acetamido-2-deoxy-β-D-glucopyranosylamine analog were respectively obtained from the fully acetylated lactose and lactosamine (2-amino-2-deoxy-4-O-β-D-galactopyranosyl-D-glucose) via the halides, azide, and amine, followed by condensation with 1-benzyl N-(benzyloxycarbonyl)-L-aspartate.2-Acetamido-2-deoxy-β-D-glucopyranosyl azide was tritylated at O-6, the product was fully acetylated, the azide was reduced to the amine, and this was condensed with 1-benzyl N-(benzyloxycarbonyl)-L-aspartate; the resulting asparagine derivative was detritylated, and the product was condensed with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, to give 2-acetamido-N-[1-benzyl N-(benzyloxy-carbonyl)-L-aspart-4-oyl]-2-deoxy-3,4-di-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranosylamine.
Article
Glycopeptides are partial structures of the connecting regions of glycoproteins and, like these, always contain glycosidic bonds between the carbohydrate and peptide parts. Glycoproteins are not only widely distributed but are also decisive factors in post-translational biological selectivity, especially in biological recognition. Targeted syntheses of glycopeptides require stereoselective formation of the glycosidic bonds between the carbohydrate and the peptide parts and protective group methods that enable selective deblocking of only one functional group in these polyfunctional molecules. These heavy demands have been met by the well-established use of benzylic protective groups, which can be removed by hydrogenolysis, combined with the use of base-labile 2-phosphonioethoxycarbonyl (Peoc) or 9-fluorenylmethoxycarbonyl (Fmoc) protective groups or of bromoethyl esters, which can be removed under neutral conditions. The acidolysis of tert-butyloxycarbonyl (Boc) groups and of tert-butyl esters has also been successfully used, although, under acidic conditions, anomerization or rupture of the glycosidic bonds may occur, especially when nucleophiles are present. The stable, two-stage 2-(pyridyl)ethoxycarbonyl (Pyoc) protective groups allow a more reliable synthesis of complex glycopeptides since they can be removed, after modifications, under mild conditions. Particularly suitable for the synthesis of sensitive glycopeptides are the stable allyl protective groups. They can be removed from the complex glycopeptides in a highly selective and effective manner by means of noble-metal catalysts under practically neutral conditions. These methods have been employed to synthesize glycopeptides corresponding to partial structures of interesting glycoproteins. Deprotected glyopeptides representing tumor-associated antigen structures can be coupled to bovine serum albumin, which serves as a biological carrier molecule, without the necessity of using an artificial coupling component (spacer).
Article
(I) is hydrolysed in 2 M HC1 at a rate which is approximately twice that a which aspartic acid is released during the earlier stages of the reaction. The rate of release of aspartic acid from hen's egg albumin glycopeptide occures at a rate which is about 60% of that at which the amino acid is released from Compound I. The rates of release of aspartic acid become much lower as reaction proceeds further, as also does the rate of formation of ammonia. These data, together with considerations of the kinetics for the release of glucosamine from Compound I, suggest that there are two major pathways for the hydrolysis of Compound I.
Article
Recent progress in synthetic methods and structure determination has resulted in several new developments in the chemistry of glycosyl azides. A review on these synthetically useful intermediates was published by Micheel and Klemer more than 30 years ago but newer treatments of related topics scarcely take notice of these compounds despite their definite importance as precursors to glycosyl amines and heterocyclic derivatives such as 1,2,3-triazoles.
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The products of hydrazinolysis of the 1-N-acetyl and 1-N-(l-β-aspartyl) derivatives of 2-acetamido-2-deoxy-β-d-glucopyranosylamine could not be converted quantitatively into 2-amino-2-deoxy-d-glucose under mild conditions. Proton and 13C-n.m.r. measurements indicated that the hydrazone of 2-amino-2-deoxy-d-glucose was a major product of the hydrazinolysis of 2-acetamido-1-N-acetyl-2-deoxy-β-d-glucopyranosylamine. Control experiments showed that acetohydrazide is slowly converted into 4-amino-3,5-dimethyl-1,2,4-triazole under-the conditions of hydrazinolysis, and that 2-amino-2-deoxy-d-glucose reacts slowly with acetohydrazide in dilute acetic acid. The implications of these results in relation to the hydrazinolysis of glycopeptides and glycoproteins are discussed.
Article
A group of novel analogs of 2-acetamido-1-N-(β-L-aspartyl)-2-deoxy-β-glucospyranosylamine (1), the glycopeptide junction in immunoglobulins, were synthesized as potential regulators of the biosynthesis, secretion, and function of immunoglobulins. The 2-amino and carboxyl groups in the aspartyl moiety were incorporated into hydantoin, thiohydantoin, and dioxopiperazine systems, and converted into an N2-toluenesulfonamide to mimic the neighboring peptide-linkages of this juncture. The amide linkage in 1 was replaced by glycosidic and by a sulfonamide linkage. The latter represents a new type of glycosylamine, the chemical stability of which was examined. The o.r.d. and c.d. spectra of these novel glycosyl derivatives are compared.
Article
Full-text available
The kinetics of the incorporation in vivo of 14C-glucosamine into the protein-bound hexosamine and sialic acid of nine rat liver subcellular fractions and of plasma indicates that hexosamine is incorporated into glycoprotein in the channels of both the rough and smooth surfaced endoplasmic reticulum, whereas sialic acid is incorporated primarily within the smooth surfaced endoplasmic reticulum. Puromycin in vitro releases between 13 and 52% of acid-insoluble 14C-glucosamine incorporated into rat liver ribosomes in vivo, indicating that some glucosamine is incorporated into nascent ribosome-bound polypeptide. Evidence is presented that membrane-bound ribosomes are the site of this glucosamine incorporation. The conclusion is drawn that hexosamine is first incorporated into newly synthesized polypeptide while it is still attached to the ribosome on which it is being assembled and that further hexosamine residues are incorporated after detachment from the ribosome as the polypeptide traverses the channels of the rough and smooth surfaced endoplasmic reticulum on its way out of the cell into the plasma. Sialic acid, which is usually found in terminal positions of glycoprotein prosthetic groups, is incorporated primarily during the final stages of this synthetic process while the glycoprotein is within the smooth surfaced endoplasmic reticulum. Morris hepatoma 5123 TC, which is deficient in membrane-bound ribosomes, shows a markedly altered pattern of 14C-glucosamine incorporation into glycoprotein.
Article
1. Ovalbumin was shown, by starch gel electrophoresis, to exist in two genetically different forms, A and B. It was suggested that these are determined by two alleles at one locus, named Ov . 2. Ovalbumin of each genetic type is electrophoretically heterogeneous. Dephos-phorylation of each type with human prostatic phosphatase and calf intestinal phosphatase removed some of the heterogeneity, but some remained. 3. The genetic difference was shown not to reside in the fragment released from ovalbumin by the proteolytic enzyme subtilisin. 4. The genetic difference was evident in the ovalbumin present in the fluid contained in right oviduct cysts.
Article
Crystalline 2-acetamido-2-deoxy-β-D-glucosylamine was prepared deacetylation of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide followed by catalytic hydrogenation. An improved synthesis of 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-β-D-glucopyranosylamine is described. The configuration and conformation of its derivatives are discussed on the basis of the n.m.r. and o.r.d. spectra.ZusammenfassungDurch Deacetylieren und anschliessende katalytische Hydrierung von 2-Acetamido-3,4,6-tri-O-acetyl-2-desoxy-β-D-glukopyranosyl-azid wurde kristallines 2-Acetamido-2-desoxy-β-D-glukopyranosylamin synthetisiert. Eine verbesserte Methode zur Synthese von 2-Acetamido-1-N-(4-L-aspartyl)-2-desoxy-β-D-glukopyranosylamin wird beschrieben. Die Konfiguration und die Konformation der Derivate werden auf Grund von N.m.r.- und O.r.d.-Daten diskutiert.
Article
Three glycopeptides, obtained in quantity from ovalbumin by exhaustive digestion with Pronase and purified by ion-exchange chromatography and gel filtration, had mannose-2-acetamido-2-deoxyglucose-aspartic acid ratios of 5:4:1, 6:2:1, and 5:2:1. The structures of the glycopeptides have been investigated by sequential digestion with purified exo-glycosidases, Smith degradation, and selective acetolysis, and by methylation analysis of the glycopeptides and their degradation products. The resulting data indicated the structures to be α-d-Manp-(1→6)-[α-d- Manp-(1→3)]-α-d-Manp-(1→6)-[β-d-GlcNAcp-(1→4)]-[β-d-GlcNAcp-(1→2)-α-d- Manp-(1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, α-d- Manp-(1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→2)-α-d-Manp- (1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, and α-d-Manp- (1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→3)]-β-d-Manp-(1→4)- β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn. The glycopeptides had a common-core structure consisting of five mannose and two hexosamine residues, but the two larger glycopeptides were not homologous.
Article
This chapter discusses the synthesis of β-aspartyl-N-acetylglucosaminadase from epididymis. The structure of the carbohydrate–protein linkage in ovalbumin is established as 1-β-aspartyl-2-acetamido-l,2-dideoxy-D-glucosylamine by a comparison of the synthetic compound with that isolated from an ovalbumin glycopeptide. The β-aspartylglycosylamine linkage in this compound is cleaved by the N-glycosidase to N-acetylglucosamine and asparagine. The N-acetylglucosamine liberated by the enzyme is determined colorimetrically by a modification of the Morgan–Elson reaction. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter. All operations are conducted between 0° and 4° unless specified otherwise. Precipitates are removed by centrifugation at 2° at 25,000 g for 30 minutes. The sheep epididymis is stored at –20 ° prior to use. The preparation is stable to both lyophilization and freezing after the butanol extraction. The enzyme does not release asparagine from glycopeptides obtained from α1-glycoprotein, fetuin, or ovalbumin after pronase treatment, even after prolonged incubation.
Article
Properties of non-dialyzable β-glucuronidase inhibitor isolated from guinea-pig urine were examined by using the calf liver β-glucuronidase (ketodie). It was a heat stable inhibitor of β-glucuronidase. Kinetic studies indicated that the substance functions as a non-competitive inhibitor and it was a relatively specific inhibitor for β-glucuronidase. The inhibitory activity of purified inhibitor on β-glucuronidase was stronger than those of acid mucopolysaccharides. chondroitin sulfate A, B, and C, heparin, and hyaluronic acid. Electrophoretic pattern of the purified inhibitor was completely different from those of the latter substances.
Chapter
Each different type of connective tissues in the human organism shows a specific distribution of proteoglycans. Further differences exist in the proteoglycan molecule itself. Besides variations in the polysaccharide chain, there are also differently composed protein cores containing the same type of polysaccharide chain. As found by several investigators proteochondroitin sulfates and proteokeratan sulfates of different organs vary in amino acid distribution of their protein or peptide matrix covalently bound (Anderson et al., 1967; Greiling and Stuhlsatz, 1966; Greiling et al., 1967). On the other hand, no proteoglycan could be separated with only one type of polysaccharide chain, even after numerous purification procedures. The heterogeneity of the polysaccharide portion in the same proteoglycan was pointed out by several authors in different types of connective tissues: cornea (Greiling and Stuhlsatz, 1966; Anseth and Laurent, 1961; Berman, 1970), trachea (Tsiganos and Muir, 1967), nasal septum (Gerber et al., 1960; Sajdera and Hascall, 1969; Franek and Dunstone, 1967), rib cartilage (Kleine and Hilz, 1968), knee joint cartilage (Greiling and Stuhlsatz, 1969), aorta (Kröz and Buddecke, 1967) and skin (Fransson, 1970).
Article
Glycoproteins play an important role in biological processes, such as cell recognition, cell adhesion, immunogenic recognition, and so on. Additionally, the carbohydrate moieties of the glycoprotein contribute to the solubility and thermal stability of proteins and to protection against proteolysis (1). In order to study these mechanisms, preparation of glycoproteins is required. In this review, preparation of glycopeptide using conventional chemical and recent manners (chemo-enzymatic method, using of natural source) is described.
Article
This chapter discusses biosynthesis of animal glycoproteins. The term “glycoprotein” covers a multitude of different biopolymers with only a single common characteristic, the presence of both carbohydrate and protein joined in covalent linkage. Glycoproteins are defined as conjugated proteins containing as prosthetic group one or more heterosaccharides with a relatively low number of sugar residues, lacking a serially repeating unit and bound covalently to the polypeptide chain. The monosaccharide components of mammalian glycoproteins and proteoglycans are all synthesized by way of nucleotide sugars. In vivo, these compounds normally originate from glucose, but certain other monosaccharides may also serve as precursors of their respective nucleotide derivatives. All interconversions of glucose to the sugars found in glycoproteins occur at or prior to the nucleotide sugar stage. The l-iduronic acid residues of heparin are formed at the polymer level by epimerization of D-glucuronic acid residues. Because 5-epimerization of UDP-D-glucuronic acid to UDP-L-iduronic acid has been demonstrated, it is possible that different pathways exist for the formation of polymeric iduronic acid units.
Article
Since the discovery that sialic acid was the receptor site for influenza virus on the red blood cell (Gottschalk, 1960), recognition of the biological importance of glycoproteins and glycolipids has been increasing. Oligosaccharide moieties of these complex carbohydrates function as the primary antigenic determinants of blood-group substances, and may be involved in such diverse biological phenomena as contact inhibition and cell-cell adhesion of cultured cells, gamete recognition, transplant rejection, and recognition of specific receptor sites for hormones, viruses, and agglutinins. In addition, a large and increasing number of pathological conditions have been shown to be related to complex carbohydrates, including the glycosphingolipid storage diseases (see Chapter 11), cholera (Holmgren et al., 1973), herpes (Nahmias and Roizman, 1973), neoplasia (Burger and Martin, 1972), diabetes (Spiro and Spiro, 1971) hepatic cirrhosis (Marshall et al., 1974), and hemostasis (Barber and Jamieson, 1971). A better understanding of the chemistry and the mechanisms of biosynthesis of these complex molecules may provide information leading to the control of these diseases.
Article
This chapter discusses the methods by which several problems associated with carbohydrate portion of a glycoprotein can be approached. The problems include (1) the number, size, and composition of the carbohydrate units present in the glycoprotein; (2) the structure of these carbohydrate units in regard to their monosaccharide sequence, linkages, and branching; and (3) the chemical nature of the glycopeptide bond, the amino acid and sugar involved in this linkage, and the location along the peptide chain at which these attachments occur. The glycoproteins are a large and varied group of compounds and that only relatively, are the subject of detailed structural investigation. Sufficient variation in structural plan is found from these studies to make it evident that although a general approach to the study of the carbohydrate units of glycoproteins may be given, the details of the experiments are modified from protein to protein to meet the specific problems at hand. The carbohydrate portion of a glycoprotein with a minimum number of amino acids attached is obtained by extensive digestion with a protease of low specificity can be performed.
Article
The presence of oligosaccharide chains covalently attached to the peptide backbone is the feature that distinguishes glycoproteins from other proteins and accounts for some of their characteristic physical and chemical properties. Glycoproteins occur in fungi, green plants, viruses, bacteria, and in higher animal cells where they serve a variety of functions. Connective tissue glycoproteins, such as the collagens and proteoglycans of various animal species, are structural elements as are the cell wall glycoproteins of yeasts and green plants. The submaxillary mucins and the glycoproteins in the mucous secretions of the gastrointestinal tract, which consist of numerous oligosaccharide chains attached at closely spaced intervals to a peptide backbone, serve as lubricants and protective agents. The body fluids of vertebrates are rich in glycoproteins secreted from various glands and organs. Constituents of blood plasma which are glycoproteins include the transport proteins transferrin, ceruloplasmin, and transcobalamin I as well as the immunoglobulins, all the known clotting factors, and many of the components of complement. Follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone (secreted by the pituitary) and chorionic gonadotropin are all glycoproteins as are the enzymes ribonuclease and deoxyribonuclease (secreted by the pancreas) and α-amylase (secreted by the salivary glands). Fungi secrete a number of glycoprotein enzymes, for example, Taka-amylase and invertase. Another group of glycoproteins are those which occur as integral components of cell membranes in a variety of species. Enveloped viruses contain surface glycoproteins that are involved in the attachment of the virus to its host, and in eukaryotic cells the histocompatibility antigens are membrane glycoproteins. There is a growing body of evidence to suggest that cell surface glycoproteins are involved in a number of physiologically important functions such as cell-cell interaction, adhesion of cells to substratum, and migration of cells to particular organs, for example, the “homing” of lymphocytes to the spleen and the metastasis of tumor cells to preferred sites.
Article
2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N2-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (21) and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N2-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (22), 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(glycine ethyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(phenylalanine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine were synthesized by condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with the appropriate protected amino acids and tri- and tetra-peptides. The amino acid sequences of 21 and 22 correspond to the protected amino acid sequences 34–37 and 34–38 of ribonuclease B that are adjacent to the carbohydrate-protein linkage.
Chapter
This chapter discusses the structural aspects of the glycoproteins and illustrates the way these are reflected in important biological properties of the molecules. The known or presumed functions of glycoproteins are diverse, spanning a wide range of vital biological activities. Almost all the proteins of plasma, with the notable exception of albumin, contain carbohydrate and fulfill varied roles such as transport, clotting, and antibody activity. Gonadotropins from both pituitary and placental origin are glycoproteins, as are thyroid-stimulating hormone and thyroglobulin—the thyroid hormone storage protein. A rapidly increasing number of proteins with enzyme activity, including various hydrolases, oxidases, and transferases, are being reported to contain covalently bound carbohydrate. These originate from a large variety of tissues and from organisms throughout the phylogenetic scale. The protective and lubricating roles of the glycoproteins from epithelial secretions are well known. The physiological function of many glycoproteins seems to be well established; the role which the carbohydrate plays in helping these proteins carry out their activities is in many cases much less clear and is discussed in this chapter.
Chapter
The chapter summarizes the progress that has been made in the area of “sugar nucleotide” metabolism. The chapter discusses the enzymic synthesis of glycosyl esters of nucleoside 5'-pyrophosphates. Some sugar nucleotides are produced by “cross reactions” of biosynthetic enzymes that have broad substrate- specificity. Synthesis of oligosaccharides, homopolysaccharides, heteropolysaccharides, and other compounds such as glycolipids, glycoproteins, blood group substances, teichoic acids etc are also discussed. The biosynthesis of most sugar nucleotides is under efficient feed-back control so that, in some cases, little accumulation of these compounds takes place, even when their utilization is blocked. Several new types of enzyme that degrade “sugar nucleotides” are reported.
Article
Glycopeptides and glycoproteins have received much attention due to their biological interests in cell recognition and cell adhesion. Recently we have developed a new chemo-enzymatic synthetic strategy for glycopeptide and glycoprotein. In this article we describe our new methodology including 1) a new synthetic method of glycosylasparagine derivatives by the reaction of glycosyl azide and aspartic acid in the presence of tertially phosphine, 2) solid-phase synthesis of [Asn (GlcNAc)]-peptide without protecting the hydroxyl functions using dimethylphosphinothioic mixed anhydride method, 3) peptide elongation by peptide thioester method, and 4) transglycosylation reaction using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). By these methods, we synthesized eel calcitonin (32 amino acids) analog having natural complex-type sugar chain (11 sugar residues).
Article
A method for the quantitative determination of amide residues in nanomolar amounts of proteins is described, based on dilute acid hydrolysis at 100°, followed by isothermal gas-liquid chromatography of the ammonia released by on-column neutralisation of the hydrolysate and quantitation by means of a conductometric detector. Amide contents are given for twenty well characterised proteins, as well as for asparagine and glutamine.
Article
the synthesis of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosylamine (2), the key intermediate in glycopeptide synthesis, has been improved. The dimerization of 2 has been studied as a model for its activity in biological systems. The formation of β,⨿ and α,β dimers from 2 and their interconversions could be readily followed by 13C-n.m.r. spectroscopy, and a probable mechanism of their formation involving an acyclic immonium ion intermediate has been proposed.
Article
The synthesis is described of the glycotripeptide derivatives 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L--seryl-L-nitroarginyl-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine, 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-seryl-L-nitroarginyl-L-aspart-1-oyl-(1-p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and of the glycopentapeptide and glycohexapeptide derivatives 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucyl-L-threonyl-threonyl-Nε-tosyl-L-lysine-(p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glycopyranosylamine and 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucyl-L-threonyl-Nε-tosyl-L-lysyl-L-aspartic 1,4-di-p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine.
Article
When an unbuffered solution of ovine submaxillary gland mucoprotein, adjusted to pH 10.4, was heated for several hours at 80°, concomitant with the release of prosthetic groups () carboxyl groups at the residual OSM were unmasked. Since this observation suggested an ester type of linkage between the prosthetic group and the polypeptide chain, ovine submaxillary gland mucoprotein was submitted to treatment with LiBH4 in tetrahydrofuran, a reagent known to effect a reductive cleavage of ester linkages. To increase the solubility of ovine submaxillary gland mucoprotein in tetrahydrofuran, the size of the ovine submaxillary gland mucoprotein molecule was decreased by trypsin action and its dipolar ion character reduced by converting the free NH2-groups into their phenylthiocarbamyl derivatives. After treatment with LiBH4 of the ovine submaxillary gland mucoprotein frafments thus prepared, only 12% of the total dicarboxylic acids were recovered, indicating that about 80% of the prosthetic groups are involved in a glycosidic-ester linkage to the free carboxyl groups of aspartyl and glutamyl residues.The prosthetic group released on reductive cleavage of the ester bond was isolated. It was further shown that about 18% of the total N-acetylneuraminic acid remained bound within ovine submaxillary gland mucoprotein even after 4 h heating in 0.01 N NaOH, suggesting an alkali-stable O-glycosidic linkage of about 18% of the prosthetic groups to serine and/or threonine residues.
Article
material. The studywas initiated partlyinordertoinvestigate thenature oftheprotein-carbohydrate linkage, butthisgoal couldnot be achievedby themethodsthen available. Someprogress hasbeenmade inthis problemby more recentstudies and Johansen, Marshall & Neuberger (1960) havegiventhemost probable values forthemannose,glucosamine and acetyl contents ofthewholeprotein andofaglycopeptide isolated fromit.Theprobability thatthese aretheonlysugarspresentwasindicated. Itis thepurposeofthispapertodescribe thepreparationand some oftheproperties ofthisglycopeptide, andtoconsider thenatureofthechemical bondlinking thecarbohydrate to theprotein. A briefdescription ofthisworkwas reported earlier (Johansen, Marshall & Neuberger, 1958). Cunningham, Nuenke& Nuenke (1957)and Jevons(1958) havealsogivenshortaccountsof their findings onthesamesubject.
Article
THE usually accepted value for the amide nitrogen content of ovomucoid is 1.00 per cent1 or 21 residues per molecular weight of 28,800 gm. (ref. 2). This value was obtained by acid hydrolysis of the protein followed by distillation of the ammonia from mildly alkaline solution. It is known, however, that ovomucoid contains hexosamine and that the latter is deaminated to a greater or lesser extent by the methods used. It is thus likely that the reported values for the amide nitrogen content of ovomucoid, and those of other glycoproteins which have been determined by similar procedures, are too high.
Article
After prolonged digestion with Pronase, over 50% of the carbohydrate of the α1-glycoprotein was isolated as glycopeptide fractions with molecular weight near 1800. One of the fractions yielded asparagine (aspartic acid plus NH3) as the sole amino acid residue. Some of the aspargine had been cleaved from the carbohydrate unit (octasaccharide) by Pronase leaving N-acetylglucosamine in the reducing position. It was proposed that aspargine is joined through its amide group to form a 8-aspartyl N-acetylglucosaminylamine linkage. Some of the asparagine residues of the other fraction appear to be in peptide linkage with threonine which is N-terminal. This report does not concur with the proposal (Winzler and Inoue, 1961) that glutamic acid is joined to the amino group of glucosamine in the α1-glycoprotein. Recently glycopeptides in which aspartic acid predominated were isolated from the α1-glycoprotein (Kamiyama and Schmid, 1962).
Article
Ovomucoid was decomposed with barium hydroxide by the method of Stacey, the carbohydrate fragment thereby obtained was treated with acetic anhydride and strongly basic resin (carbonate form) to form an N-acetylated derivative, and purified by fractional precipitation with ethanol and chromatography through carbon-Celite mixture. These treatments separated the carbohydrate residue of ovomucoid into several fractions with different hexosamine/hexose ratio. These saccharide fragments possess amino acids, such as aspartic acid, alanine, and glycine. Removal of free amino acids by treatment with ion exchange resin and separation by paper electrophoresis afforded saccharide fractions which still retained amino acids, probably bonded with the saccharide.
Article
Treatment of bovine submaxillary mucoprotein with LiBH4 in tetrahydrofuran resulted in the reduction of approx. 83% of the total dicarboxylic acid residues and in the release of unreduced prosthetic groups previously identified as α-d-sialyl(2→6) N-acetylgalactosamine. When the acid hydrolysate of LiBH4-treated bovine submaxillary mucoprotein was fractionated according to Stein and Moore, fractions containing homoserine and α,δ-aminohydroxy-n-valeric acid were abtained. α,δ-aminohydroxy-n-valeric acid was identified by conversion to proline in acid medium; homoserine was identified by comparing its paper-chromatographic behaviour with that of authentic homoserine and by its resistance to periodate oxidation. From these data it was concluded that approx. 81% of the prosthetic groups in bovine submaxillary mucoprotein are joined through a glycosidic-ester linkage to the β-carboxyl group of aspartyl and the γ-carboxyl group of glutamyl residues respectively. The near completion of the reductive cleavage of these ester linkages was also indicated by the release of 84% of the prosthetic groups on alkali treatment of bovine submaxillary mucoprotein. The residual prosthetic groups are assumed to be linked by an O-glycosidic bond to serine and/or threonine. Homoserine and α,δ-aminohydroxy-n-valeric acid were synthesized by LiBH4 treatment of β-methyl aspartate and γ-methyl glutamate respectively.
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Bowman, 1946) has been separated from soya beans and purified by dialysis, freeze-drying and fractionation on a diethylaminoethylcellulose column (Birk, 1961a, b) Smith (1961) reported the isolation of a similar inhibitor from soya-bean-whey proteins
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An acetone-insoluble trypsin inhibitor (Bowman, 1946) has been separated from soya beans and purified by dialysis, freeze-drying and fractionation on a diethylaminoethylcellulose column (Birk, 1961a, b). Rackis, Sasame, Mann, Anderson & Smith (1961) reported the isolation of a similar inhibitor from soya-bean-whey proteins. Applebaum, Jankovic & Birk (1961) have shown that the
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