Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides

Stoll, Mark S.; Mizuochi, Tsuguo; Childs, R A; Feizi, Ten

doi:10.1042/bj2560661

Cited by 107 publications

(39 citation statements)

References 18 publications

(14 reference statements)

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“…Binding of monoclonal antibody 10E4 to NGLs chromatographed on aluminum-backed HPTLC plates (Merck) was assayed essentially as described previously (17) except that Plexigum was not used. In brief, after blocking nonspecific binding sites with 1% casein (Pierce) in Trisbuffered saline, 10 mM Tris-HCl buffer, 150 mM NaCl, pH 8.0 (TBS), the chromatogram was overlaid with 10E4 antibody (10 g/ml) in 1% casein; antibody binding was detected using protein-LA-peroxidase (1/500 of stock) in 1% casein followed by FAST TM 3,3Ј-diaminobenzidine peroxidase substrate.…”

Section: Methodsmentioning

confidence: 99%

10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

Leteux

Chai

Nagai³

et al. 2001

Journal of Biological Chemistry

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The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrP Sc , even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity.

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Section: Methodsmentioning

confidence: 99%

10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

Leteux

Chai

Nagai³

et al. 2001

Journal of Biological Chemistry

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“…Neoglycolipid Synthesis-Pure glycolipid-derived oligosaccharide fractions containing either Le X or pseudo-Le Y glycans (80 g each) as well as lacto-N-fucopentaose III (LNFPIII; 100 g; Dextra Laboratories, Reading, UK) were used for synthesis of neoglycolipids by coupling to 1,2-sn-dipalmitoylphosphatidylethanolamine via reductive amination (36). Resulting products were analyzed by MALDI-TOF-MS.…”

mentioning

confidence: 99%

DC-SIGN Mediates Binding of Dendritic Cells to Authentic Pseudo-LewisY Glycolipids of Schistosoma mansoni Cercariae, the First Parasite-specific Ligand of DC-SIGN

Meyer

Liempt

Imberty

et al. 2005

Journal of Biological Chemistry

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“…The mixture was heated at 50" for 2 h. Sodium cyanoborohydride (40 pg; 635 nmol) in ethanol (4 pl) was added and the mixture heated at 50°C for a further 16 h. The solvent was then evaporated under a stream of nitrogen. The reference oligosaccharides 0 3 and 0 7 were conjugated to the lipid as described previously [8].…”

Section: Conjugation Of Oligosaccharides To Dipulmito~lglycerophosphomentioning

confidence: 99%

Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

Stoll

Hounsell

Lawson

et al. 1990

European Journal of Biochemistry

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In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4 -C5 bond of core N-acetylgalactosaminitol. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin-layer chromatography and can be sensitively analysed by liquid-secondary-ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the Nacetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di-to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.The microscale structural profiling of N-linked chains of glycoproteins using hydrazinolysis, Bio-Gel P4 chromatography and specific enzyme digestion is well documented [l]. However, methods for microscale sequencing of 0-linked chains of mucin-type glycoproteins have not been developed. These chains are difficult to purify and analyse because of considerable heterogeneity in their backbone and peripheral regions and the occurrence of several types of branching at the core region N-acetylgalactosamine [2, 31. In the course of characterising neoglycolipid products [4] derived from mucin oligosaccharide alditols after mild periodate oxidation and conjugation by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have observed that oligosaccharides branched at the core N-acetylgalactosaminitol each give two derivatives. We describe here evidence that the mild periodate oxidation step specifically cleaves the C4 -C5 bond of N-acetylgalactosaminitol disubstituted at C3 and C6 or monosubstituted at C3. Reductive amination of the resulting aldehyde groups gives derivatives characteristic of each branch which can be analysed with high sensitivity by thin-layer chromatography/liquid-secondary-ion mass spectrometry. Studies of a series of oligosaccharide fractions derived from human meconium glycopeptides [3,5,6] show that this approach has considerable potential for deriving sequence

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Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides

Cited by 107 publications

References 18 publications

10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

DC-SIGN Mediates Binding of Dendritic Cells to Authentic Pseudo-LewisY Glycolipids of Schistosoma mansoni Cercariae, the First Parasite-specific Ligand of DC-SIGN

Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

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