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ABSTRACT: Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.
Journal of Biological Chemistry 09/2007; 282(31):22426-36. · 4.77 Impact Factor
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ABSTRACT: Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation is assessed for sequence determination of multiply sulfated oligosaccharide fragments of carrageenan obtained from partial depolymerization of the polysaccharides by either enzymatic digestion or mild acid hydrolysis. Carrageenan oligosaccharides with homogeneous disaccharide compositions were used to establish their fragmentation pattern, which was then applied to sequence determination of unusual oligosaccharides with either "hybrid" biose compositions or odd-numbered residues. As sulfate groups are labile, sulfate loss during collision-induced association was prevented by sodium adduction. The product ion spectra of [M - Na]- (where M represents the sodium salt of oligosaccharides) feature an extensive series of B- and C-type glycosidic cleavages, whereas the Y-type cleavage occurs mainly at the sulfated residues. The assignment of reducing or nonreducing terminal fragments was assisted by oligosaccharide reduction and the product ion spectra of the derived alditols. Due to the anionic nature of the sulfate present, high-sensitivity detection (1-5 pmol, using a hexasaccharide as an example) was obtained.
Analytical Chemistry 01/2007; 78(24):8499-505. · 5.86 Impact Factor
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ABSTRACT: Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).
Journal of the American Society for Mass Spectrometry 05/2006; 17(4):621-30. · 4.00 Impact Factor
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ABSTRACT: We previously reported sequence determination of neutral oligosaccharides by negative ion electrospray tandem mass spectrometry on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without the need of derivatization. In the present report, we extend our strategies to sialylated oligosaccharides for analysis of chain and blood group types together with branching patterns. A main feature in the negative ion mass spectrometry approach is the unique double glycosidic cleavage induced by 3-glycosidic substitution, producing characteristic D-type fragments which can be used to distinguish the type 1 and type 2 chains, the blood group related Lewis determinants, 3,6-disubstituted core branching patterns, and to assign the structural details of each of the branches. Twenty mono- and disialylated linear and branched oligosaccharides were used for the investigation, and the sensitivity achieved is in the femtomole range. To demonstrate the efficacy of the strategy, we have determined a novel complex disialylated and monofucosylated tridecasaccharide that is based on the lacto-N-decaose core. The structure and sequence assignment was corroborated by methylation analysis and 1H NMR spectroscopy.
Analytical Chemistry 04/2006; 78(5):1581-92. · 5.86 Impact Factor
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Angelina S Palma,
Ten Feizi,
Yibing Zhang,
Mark S Stoll, Alexander M Lawson,
Esther Díaz-Rodríguez,
María Asunción Campanero-Rhodes,
Júlia Costa,
Siamon Gordon,
Gordon D Brown,
Wengang Chai
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ABSTRACT: Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.
Journal of Biological Chemistry 04/2006; 281(9):5771-9. · 4.77 Impact Factor
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ABSTRACT: Twenty-two neutral O-linked oligosaccharides ranging from monosaccharides to octasaccharids were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometery, methylation analysis and 1H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(β1–6)[Gal(β1–3)]GalNAcol or GlcNAc(β1–6)[GlcNAc(β1–3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAc1–6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(β1–6){GalNAc(1–3)[Fuc(1–2)]Gal(β1–4)GlcNAc(β1–3)}GalNAcol (Fuc, l-fucose). Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal glycoproteins has been established by classical immunochemical studies.
European Journal of Biochemistry. 03/2005; 203(1‐2):257 - 268.
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ABSTRACT: Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-β-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 chromatography. Three linear and six branched oligosaccharides of poly(N-acetyllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: and (b) there was no evidence for the terminal branch-point sequence: Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi T. (1984) J. Biol. Chem. 259, 6586–6592], the endo-β-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point β-galactosidic linkages on erythrocyte membrane glycopeptides.
European Journal of Biochemistry. 03/2005; 168(3):585 - 593.
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ABSTRACT: Mucins from the pooled gastric juice of Lewis-positive secretors were investigated to establish their glycosylation patterns with particular reference to the type and abundance of the glycan-core structures. Following reductive β-eliminination, the neutral glycan alditols from these mucins were fractionated by ion exchange and size-exclusion chromatographies and subjected to structural analyses. It was possible to gain insights into the core sequences of the neutral O-linked glycan alditols by matching (a) composition data from liquid secondary-ion mass spectrometry of the native alditol fractions, (b) specific structural information on the core sequences by thin-layer-chromatography mass spectrometry of alditol-derived neoglycolipids and (c) data from electron-impact mass spectrometry of permethylated glycan alditols or their partially methylated alditol acetates. The predominant core structures detected among the neutral glycans representing about 77% (by mass) of the total carbohydrates released from gastric mucins were core 1, Galβ1-3GalNAc (Ac, acetyl) and core 2, Galβ1–3(GlcNAcβ1–6)GalNAc in the approximate ratio 1:2. Core 3, GlcNAcβ1–3GalNAc, and core 4, GlcNAcβ1–3(GlcNAcβ1–6)GalNAc, were much less abundant (< 10%), while core 5, GalNAcα1–3GalNAc, core 6, GlcNAcβ1–6GalNAc, and a recently described sequence GalNAcα1–6GalNAc (core 7) were not detected. This investigation also addressed the question of the presence of the sequence Galβ1–6GalNAc which has been reported previously to occur as a core-structure element in gastric mucins. This was greatly assisted by the availability of the authentic chemically synthetised disaccharide alditol which, when converted into a neoglycolipid after mild periodate oxidation, gives diagnostic ions in mass spectrometry and can be detected with high sensitivity. No evidence was found for the presence of this unusual sequence among the oligosaccharides in gastric mucins.
European Journal of Biochemistry. 03/2005; 217(2):645 - 655.
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ABSTRACT: We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.
Archives of Biochemistry and Biophysics 03/2005; 434(1):116-27. · 2.93 Impact Factor
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01/2005: pages 747 - 760; , ISBN: 9783527602438
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ABSTRACT: We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso-lacto-N-octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES-MS) and NMR spectroscopy. The branching pattern and blood group-related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high-sensitivity ES-MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H-NMR. Three new structures were identified. The nonfucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc and Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6[Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3]Galbeta1-4Glc, both containing an internal Lex epitope, are also novel structures.
European Journal of Biochemistry 04/2004; 271(6):1172-86. · 3.58 Impact Factor
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Methods in Enzymology 02/2003; 362:160-95. · 2.04 Impact Factor
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ABSTRACT: On-line thin-layer chromatographic separation and electrospray mass spectrometry (TLC/ESI-MS) has been accomplished by direct linking of a commercial overpressure TLC instrument, OPLC 50, and a Q-TOF mass spectrometer. Mass spectrometric detection sensitivity and chromatographic resolution achieved by this configuration were assessed using acidic glycolipids as examples. Under the optimized conditions, a sensitivity of 5 pmol of glycosphingolipid was readily demonstrated for TLC/ESI-MS and 20 pmol for TLC/ESI-MS/MS production scanning to derive the saccharide sequence and long chain base/fatty acid composition of the ceramide. Initial preconditioning of TLC plates is necessary to achieve high sensitivity detection by reducing chemical background noise. Plates can be used repeatedly (at least 10 times) for analysis, although this may result in a minor reduction in TLC resolution. Following solvent development, separated components on the TLC plates can be detected in the conventional way by nondestructive staining or UV absorption or fluorescence and can be stored for on-line TLC/ESI-MS analysis at a later stage without reduction in mass spectrometric detection sensitivity and chromatographic resolution. Aspects for further improvement of OPLC instrumentation include use of narrower TLC plate dimensions and refined design of the eluate exit system.
Analytical Chemistry 02/2003; 75(1):118-25. · 5.86 Impact Factor
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ABSTRACT: We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.
Nature Biotechnology 11/2002; 20(10):1011-7. · 23.27 Impact Factor
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ABSTRACT: We previously reported that sequence and partial linkage information, including chain and blood-group types, of reducing oligosaccharides can be obtained from negative-ion electrospray CID MS/MS on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without derivatization (Chai, W.; Piskarev, V.; Lawson, A. M. Anal. Chem. 2001, 73, 651-657). In contrast to oligonucleotides and peptides, oligosaccharides can form branched structures that result in a greater degree of structural complexity. In the present work we apply negative-ion electrospray CID MS/MS to core-branching pattern analysis using nine 3,6-branched and variously fucosylated oligosaccharides based on hexasaccharide backbones LNH/LNnH as examples. The important features of the method are the combined use of CID MS/MS of singly and doubly charged molecular ions of underivatized oligosaccharides to deduce the branching pattern and to assign the structural details of each of the 3- and 6-branches. These spectra give complimentary structural information. In the spectra of [M - H]-, fragment ions from the 6-linked branch are dominant and those from the 3-linked branch are absent, while fragment ions from both branches occur in the spectra of [M - 2H]2-. This allows the distinction of fragment ions derived from either the 3- or 6-branches. In addition, a unique D2beta-3 ion, arising from double D-type cleavage at the 3-linked glycosidic bond of the branched Gal core residue, provides direct evidence of the branching pattern with sequence and partial linkage information being derived from C- and A-type fragmentations, respectively.
Journal of the American Society for Mass Spectrometry 07/2002; 13(6):670-9. · 4.00 Impact Factor
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ABSTRACT: An important characteristic of malaria parasite Plasmodium falciparum-infected red blood cells (IRBCs) is their ability to adhere to host endothelial cells and accumulate in various organs. Sequestration of IRBCs in the placenta, associated with excess perinatal and maternal mortality, is mediated in part by adhesion of parasites to the glycosaminoglycan chondroitin sulfate A (CSA) present on syncytiotrophoblasts lining the placental blood spaces. To define key structural features for parasite interactions, we isolated from CSA oligosaccharide fractions and established by electrospray mass spectrometry and high performance liquid chromatography disaccharide composition analysis their differing chain length, sulfate content, and sulfation pattern. Testing these defined oligosaccharide fragments for their ability to inhibit IRBC adhesion to immobilized CSA revealed the importance of non-sulfated disaccharide units in combination with 4-O-sulfated disaccharides for interaction with IRBCs. Selective removal of 6-O-sulfates from oligo- and polysaccharides to increase the proportion of non-sulfated disaccharides enhanced activity, indicating that 6-O-sulfation interferes with the interaction of CSA with IRBCs. Dodecasaccharides with four or five 4-O-sulfated and two or one non-sulfated disaccharide units, respectively, comprise the minimum chain length for effective interaction with IRBCs. Comparison of the activities of CSA and CSB oligo- and polysaccharides with a similar sulfation pattern and content achieved from partial desulfation demonstrated that glucuronic acid rather than iduronic acid residues are important for IRBC binding.
Journal of Biological Chemistry 07/2002; 277(25):22438-46. · 4.77 Impact Factor
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ABSTRACT: A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.
European Journal of Biochemistry. 12/2001; 267(6):1795 - 1804.
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Nature 03/1996; 380:559. · 36.28 Impact Factor
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ABSTRACT: A synthetic peptide [TAP25, (T1aAPPAHGVT9S10APDT14RPAPGS20)T1bAPPA5b] corresponding to one repeat (T1a–S20) and five overlapping amino acids (T1b–A5b) of the MUC1 core protein served as an acceptor substrate for in vitro glycosylation. TAP25 was glycosylated using the detergent-solubilized UDP-GalNAc: polypeptide N -acetylgalactosaminyltransferases from the breast carcinoma cell line T47D, the colon carcinoma cell line HT29 and from human premature skim milk. The glycosylated peptides were isolated by ultrafiltration, purified by reverse-phase HPLC and further analysed by liquid secondary ion mass spectrometry (LSIMS). Three different glycosylation species, mono-, di- and triglycosylated peptides were identified. Automated Edman sequencing and LSIMS of proteolytic fragments independently revealed the sites of GalNAc incorporation and confirmed that the threonine residues Thr9 and Thr1b are the preferred sites of glycosylation independent of the enzyme source, while Thr14 remained non-glycosylated even with the enzyme preparation from milk. In addition, evidence was obtained that at least 20% of the glycosylated peptides exhibited GalNAc incorporation at Ser20. On the basis of kinetic studies a preferred sequence of GalNAc addition to the three acceptor sites has been concluded (Thr9→Thr1b→Ser20). Although Thr14 within the PDTRP motif of the tandem repeats remained non-glycosylated, the introduction of GalNAc into adjacent positions significantly decreased the immunoreactivity of antibodies SM-3, HMFG-1 and HMFG-2 defining overlapping epitopes of this motif. It is assumed that glycosylation at Thr9, Thr1b and Ser20 distorts the peptide conformation of the binding epitope.
European Journal of Biochemistry. 03/1995; 229(1):140 - 147.
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ABSTRACT: A diversity of high-affinity Oligosaccharide ligands are identified
for NKR-P1, a membrane protein on natural killer (NK) cells which contains an
extracellular Ca2+-dependent lectin domain. Interactions of such
oligosaccharides on the target cell surface with NKR-P1 on the killer cell
surface are crucial both for target cell recognition and for delivery of
stimulatory or inhibitory signals linked to the NK cytolytic machinery.
NK-resistant tumour cells are rendered susceptible by preincubation with
liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or
virally infected cells in vivo may be a therapeutic possibility.
11/1994; 372(6502):150-157.
