IDENTIFICATION OF OLIGO-N-GLYCOLYLNEURAMINIC ACID RESIDUES IN MAMMAL-DERIVED GLYCOPROTEINS BY A NEWLY DEVELOPED IMMUNOCHEMICAL REAGENT AND BIOCHEMICAL METHODS

The occurrence of the alpha 2-->8-linked oligomeric form of N-glycolyl neuraminic acid (oligo-Neu5Gc) residues in mammalian glycoproteins was unequivocally demonstrated using a nea ly developed anti-oligo/poly-N eu5Gc monoclonal antibody as well as by chemical and biochemical metho ds, First, the antibody, designated mAb.2-4B, which specifically recog nized oligo/poly-Neu5Gc with a degree of polymerization of >2, was dev eloped by establishing a hybridoma cell line from P3U1 myeloma cells f used with splenocytes from an MRL autoimmune mouse immunized with dipa lmitoylphosphatidylethanolamine-conjugated oligo/poly-Neu5Gc. Second, oligo-Neu5Gc was shown to occur in glycoproteins derived from pig sple en by Western blot analysis using mAb.2-4B, which was also confirmed b y fluorometric high performance liquid chromatographic analysis of the product of periodate oxidation/reduction/acid hydrolysis of the purif ied glycopeptide fractions and by TLC and 600-MHz H-1 NMR spectroscopi c analysis of their mild acid hydrolysates. Finally, the ubiquitous oc currence of oligo-Neu5Gc chains as glycoproteinaceous components in Wi star rat tissue was immunochemically indicated. This is the first exam ple demonstrating the diversity in oligo/poly-Sia structure in mammali an glycoproteins, where only poly-N-acetylneuraminic acid is known to occur, Such diversity in oligo/poly-Sia structure also implicates a di verged array of biological functions of this glycan unit in glycoprote ins.