Extended Polysialic Acid Chains ( n > 55 ) in Glycoproteins from Human Neuroblastoma Cells

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D['HIGlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of ["C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:polya-2,8-sialosyl sialyltransferase catalyzes the transfer of ["CINeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-a-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 'H-glycopeptides from the ['HI GlcN-labeled cells released short ['Hlsialyl oligomers (['HIDPl-6) which were degraded to ['HINeuNAc by exosialidase. Partial Endo-N digestion released a series of ['Hlsialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers (['H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of a-2,8linked ['HINeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with ["CINeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high M, and polydisperse (M, 100,000250,000) as demonstrated in sodium dodecyl sulfatepolyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with