Identification of oligo-N-glycolylneuraminic acid residues in mammal-derived glycoproteins by a newly developed immunochemical reagent and biochemical methods.
The occurrence of the alpha2-->8-linked oligomeric form of N-glycolylneuraminic acid (oligo-Neu5Gc) residues in mammalian glycoproteins was unequivocally demonstrated using a newly developed anti-oligo/poly-Neu5Gc monoclonal antibody as well as by chemical and biochemical methods. First, the antibody, designated mAb.2-4B, which specifically recognized oligo/poly-Neu5Gc with a degree of polymerization of >2, was developed by establishing a hybridoma cell line from P3U1 myeloma cells fused with splenocytes from an MRL autoimmune mouse immunized with dipalmitoylphosphatidylethanolamine-conjugated oligo/poly-Neu5Gc. Second, oligo-Neu5Gc was shown to occur in glycoproteins derived from pig spleen by Western blot analysis using mAb.2-4B, which was also confirmed by fluorometric high performance liquid chromatographic analysis of the product of periodate oxidation/reduction/acid hydrolysis of the purified glycopeptide fractions and by TLC and 600-MHz 1H NMR spectroscopic analysis of their mild acid hydrolysates. Finally, the ubiquitous occurrence of oligo-Neu5Gc chains as glycoproteinaceous components in Wistar rat tissue was immunochemically indicated. This is the first example demonstrating the diversity in oligo/poly-Sia structure in mammalian glycoproteins, where only poly-N-acetylneuraminic acid is known to occur. Such diversity in oligo/poly-Sia structure also implicates a diverged array of biological functions of this glycan unit in glycoproteins.
Sato
C
C
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan.
Kitajima
K
K
Inoue
S
S
Inoue
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D007118:Immunoassay; D008969:Molecular Sequence Data; D009438:Neuraminic Acids / Q000032:analysis* / Q000276:immunology; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000032:analysis* / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D012794:Sialic Acids / Q000032:analysis; D013154:Spleen / Q000737:chemistry; D013552:Swine
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D006023:Glycoproteins; D009438:Neuraminic Acids; D009844:Oligosaccharides; D012794:Sialic Acids; C021319:polysialic acid; C032592:N-glycolylneuraminic acid