[6] Analysis of oligosaccharides by gel filtration

Yamashita, Katsuko; Mizuochi, Tsuguo; Kobata, Akira

doi:10.1016/0076-6879(82)83008-5

Cited by 350 publications

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“…The radioactive oligosaccharides were applied to a Mono Q column equilibrated with 5 mM acetate, Neu5AcA2→3[Galβ1→4GlcNAcβ1→6(Galβ1→3)]GalNAc OT , Neu5AcA2→6(Galβ1→3)GalNAc OT, Neu5AcA2→3Galβ1→4-pH 4.0, and eluted with a 5 mMϪ300 mM acetate, pH 4.0, gradient. Fractionation of the radioactive oligosaccharides by Bio-GlcNAcβ1→6(Neu5AcA2→3Galβ1→3)GalNAc OT , and Neu5A-cA2→6(Neu5AcA2→3Galβ1→3)GalNAc OT , were reported to be Gel P-4 column chromatography was performed as described previously [37]. Sugar composition analysis was performed ac-eluted at the same positions as peaks A1, A2, A3 and A4, respectively [43].…”

Section: A In (A) (B)mentioning

confidence: 99%

Structural study of the O‐linked sugar chains of human leukocyte tyrosine phosphatase CD45

Furukawa

Funakoshi

Autero

et al. 1998

European Journal of Biochemistry

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The O-linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium-labeled oligosaccharides by β-elimination in the presence of NaB 3 H4. Mono Q column chromatography revealed that they comprise neutral (64%) and acidic (36%) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio-Gel P-4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15% of which are modified with poly (N-acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB 3 H 4 treatment. Bio-Gel P-6 column chromatography of a mixture of the tritium-labeled glycopeptide/oligosaccharides obtained by pronase-digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.Keywords : human leukocyte; CD45 ; O-linked sugar chain.CD45 is a receptor-like leukocyte cell surface glycoprotein and may be involved in apoptosis of T-lineage cells [25]. Similarly, mannose residues of CD45 expressed on mouse immature with protein tyrosine phosphatase activity in its cytoplasmic domain [1Ϫ6]. Analysis of mature T-cell and B-cell lines deficient thymocytes are recognized by the serum mannan-binding protein, and may be important for the development and maturation in the expression of CD45 revealed that the activation signals mediated by T-cell antigen receptor and B-cell antigen receptor of thymocytes [26], presumably by interaction with DEC-205, a membrane-bound protein which is homologous to the macroare impaired [7Ϫ11]. Similarly, in CD45-gene disrupted mutant mice, the maturation of T cells was severely disturbed and the phage mannose receptor, expressed on thymic dendritic cells [27]. The sialylated N-linked sugar chains of CD45 contain only peripheral T cells failed to respond to T-cell-antigen-receptorϪ mediated signaling [12]. These results indicate that CD45 is in-A-2,6-linked sialic acid residues [28] and may be involved in cell adhesion mediated by the Ig superfamily member CD22 [29], a dispensable for exerting antigen-specific activation of T and B cells. CD45 exists as multiple isoforms generated from the sialic-acid-binding lectin [30]. Binding studies of oligosaccharides with different sialylation levels to a column immobilized single gene by alternate splicing of exons which encode a part of the extracellular domain [13Ϫ16]. The expression of distinct with CD22-IgG chimeric protein revealed that CD22 has higher affinity toward highly sialylated complex-type oligosaccharides CD45 isoforms varies among T-cell subgroups and through Tcell ontogeny, but all the isoforms contain the identical trans- [31]. Structural studies of the N-linked sugar chains of CD45 from human peripheral...

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Section: A In (A) (B)mentioning

confidence: 99%

Structural study of the O‐linked sugar chains of human leukocyte tyrosine phosphatase CD45

Furukawa

Funakoshi

Autero

et al. 1998

European Journal of Biochemistry

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“…Third, the amount of presumed Gal␤1-4GlcNAcol, obtained after galactosylation, appeared to be higher for proteins from control red cells compared with those from red cells with 10% parasitemia. Fourth, the reported effective size of Gal␤1-4GlcNAcol on Bio-Gel P-4 was 2 GU or less (23), a figure not in agreement with the reported effective sizes of galactitol, N-acetylgalactosaminitol, and Hex␤1-4GlcNAcol of 1.5, 2.6, and 3.2, respectively (52), raising doubts about the authenticity of the characterized structure. Finally, whereas we and others (18,28) found that uninfected erythrocytes do not metabolically incorporate sugars into their proteins to significant levels, Dieckmann-Shuppert et al (23) reported that the level of 14 GlcN incorporation into proteins of control red cells was about half the level of incorporation into proteins of red cells with 10% parasitemia (27).…”

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Glycosylphosphatidylinositol Anchors Represent the Major Carbohydrate Modification in Proteins of Intraerythrocytic Stage Plasmodium falciparum

Gowda

Gupta

Davidson

1997

Journal of Biological Chemistry

134

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The nature and extent of carbohydrate modification in intraerythrocytic stage Plasmodium falciparum proteins have been controversial. This study describes the characterization of the carbohydrates in intraerythrocytic P. falciparum proteins and provides an overall picture of the nature of carbohydrate modification in the parasite proteins. P. falciparum strains were metabolically labeled with radioactive sugar precursors and ethanolamine at different developmental stages. The individual parasite proteins separated on SDS-polyacrylamide gels and whole parasite cell lysates were analyzed for the carbohydrate moieties. The results established the following: 1) glycosylphosphatidylinositol (GPI) anchors represent the major carbohydrate modification in the intraerythrocytic stage P. falciparum proteins; 2) in contrast to previous reports, O-linked carbohydrates are either absent or present only at very low levels in the parasite; and 3) P. falciparum contains low levels of N-glycosylation capability. The amount of N-linked carbohydrates in whole parasite proteins is ϳ6% compared with the GPI anchors attached to proteins based on radioactive GlcN incorporated into the proteins.The glycan cores of multiple parasite protein GPI anchors are all similar, consisting of protein-ethanolamine-phosphate-(Man␣1-2)6Man␣1-2Man␣1-6Man␣1-4GlcN. The fourth Man residues distal to GlcN of the GPI anchor glycan cores contain unidentified substituents that are susceptible to conditions of nitrous acid deamination. This unusual structural feature may contribute to the reported pathogenic properties of the P. falciparum GPI anchors.Malaria, a life-threatening disease caused by parasitic protozoa of the genus Plasmodium, is a major health problem throughout the tropical and subtropical regions of the world. Among the four species that infect humans, Plasmodium falciparum is the most virulent. New approaches such as vaccine development and novel therapeutic agents are urgently needed due to the emergence of parasite strains resistant to chloroquine and other commonly used drugs (1).Vaccines based on antigens of the blood stage parasite are under intensive study. A major focus has been on synthetic peptides or recombinant proteins of cell surface antigens (2-6). However, this approach has not been highly effective, although immunization with native cell surface proteins purified from the erythrocytic stage parasite is known to confer significant protective immunity (7-10). It is plausible that post-translational modifications play an important role in antigenicity. Accordingly, a basic understanding of such modifications may assist in the development of effective vaccines.Glycosylation is an often extensive post-translational modification of eukaryotic proteins. Carbohydrate moieties of glycoproteins perform a variety of functions including modulation of immunological properties, receptor-ligand interactions, sorting and localization of proteins, cell adhesion, and cell-cell communication (11). In addition, they contribute to protein conform...

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“…The organic phases were combined and concentrated. The residue was purified on a silica gel column 3 SiOTf (31 mL, 0.17 mmol) under N 2 atmosphere at − 15°C. The mixture was stirred under these conditions for 1 h, at the end of which time TLC (5:1 petroleum ether-EtOAc) indicated that starting material 1 was completely consumed.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…2 Different types of milk since then have been analyzed for their oligosaccharides that have immunological properties. 3 Recently, Deepak and coworkers 4 isolated a novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk with the following structure:A six-fold increase in haemagglutinating antibody (HA) titre and a two-fold increase in plaque-forming cell (PFC) count were reported in treated animals as compared with untreated controls. To investigate structure -activity relationships, we planned to prepare a monomer and a trimer of the above-mentioned pentasaccharide.…”

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Concise synthesis of a buffalo milk pentasaccharide derivative

Pan

2002

Carbohydrate Research

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An efficient synthesis of buffalo milk pentasaccharide derivative via a 3 + 2 strategy is described. The use of a trisaccharide isopropyl thioglycoside as a latent glycosyl donor and the application of two well-defined regioselective glycosylations significantly simplified the target preparation. © 2002 Elsevier Science Ltd. All rights reserved.Keywords: Glycosylation; Antigens; Thioglycosides; Oligosaccharides; Regioselectivity Milk, which contains numerous complex carbohydrate components, is known as a good source of oligosaccharide antigens and bifidus factors for breastfed newborns. 1 Monoclonal antibodies of several tumour cell lines or carbohydrate antigens have provided evidence that membrane glycoproteins or glycolipids, which may function as differentiation antigens or tumour-associate antigens, also occur as free oligosaccharides in human milk. 2 Different types of milk since then have been analyzed for their oligosaccharides that have immunological properties. 3 Recently, Deepak and coworkers 4 isolated a novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk with the following structure:A six-fold increase in haemagglutinating antibody (HA) titre and a two-fold increase in plaque-forming cell (PFC) count were reported in treated animals as compared with untreated controls. To investigate structure -activity relationships, we planned to prepare a monomer and a trimer of the above-mentioned pentasaccharide. We present here the concise chemical synthesis of this pentasaccharide derivative.The efficient chemical synthesis of complex oligosaccharides requires highly convergent strategies in which well-designed glycosyl donors and acceptors are assembled involving a minimum of steps of reactions. Accordingly, the target molecule was retrosynthetically disconnected into a disaccharide acceptor 5 and a trisaccharide donor 14 as shown in Scheme 1. Thus, compound 1 5 was condensed with 3 6 in the presence of N-iodosuccinimide (NIS) and catalytic amount of trimethylsilyl triflate (TMSOTf) to afford disaccharide 4 in 89% yield. The stereochemical outcome is induced by the neighbouring-group participation of donor 1 and confirmed by the 1 H NMR spectroscopy of 4 (H-1: l 4.49 ppm, J 1,2 8.0 Hz). Hydrolysis of 4 in aqueous 90% trifluoroacetic acid (TFA) furnished disaccharide acceptor 5 in good yield (84.5%). Convergently, 1 was reacted with p-methoxyphenol (MPOH) with the promotion of NIS -TMSOTf in dry CH 2 Cl 2 to give 2, which was treated with 90% TFA to generate diol 6 (63.7% for two steps). Regioselective coupling of 6 with glucosamine imidate 7 in anhydrous CH 2 Cl 2 in the presence of TMSOTf produced b-(1 3)-linked disaccharide 8 in 72% yield. Acetylation of 8 with acetic anhydride in pyridine gave 9 showing H-4 at l 5.63 ppm (J 4,3 3.4 Hz), which confirmed the correct regioselectivity. Ceric ammonium nitrate (CAN) promoted cleavage of anomeric MP of 9 was carried out smoothly in toluene -MeCN -H 2 O co-solvent system ( 10), followed by Schmidt activation using trichloro...

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[6] Analysis of oligosaccharides by gel filtration

Cited by 350 publications

References 4 publications

Structural study of the O‐linked sugar chains of human leukocyte tyrosine phosphatase CD45

Structural study of the O‐linked sugar chains of human leukocyte tyrosine phosphatase CD45

Glycosylphosphatidylinositol Anchors Represent the Major Carbohydrate Modification in Proteins of Intraerythrocytic Stage Plasmodium falciparum

Concise synthesis of a buffalo milk pentasaccharide derivative

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