Abstract
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, CHP-134. 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-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc 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-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the $Department of Biological Chemistry, University of California School of Medicine, Davis,California95616, the BDeDartment of Pediatrics
The presence of poly-a-2&sialosyl residues was demonstrated in human neuroblastoma cells, CHP-134
The chain length of polysialic acid was a minimum of 55 sialyl residues in these human cells
Summary
72% of the radioactivity, accounting for about half of the total 3H label in the original 3H-glycopeptide fraction, eluted from the HPLC column as [3H]sialyl oligomers with the profile expected for the limit digestion products produced from a-2,8-linked polysialic acid [41]. Sialyl polymer synthesis was detected in the E. coli Kl (EVll) sialyltransferase assay by the addition of a membrane fraction from CHP-134 cells This sialyltransferase is highly specific for transferring [‘4C]sialyl residues to exogenous acceptors containing at least three sialyl units in cw-2,8-linkage. Radioactivity eluting from the HPLC column after partial Endo-N digestion in the intermediate sialyl oligomer (Fig. 2, DP9-20; Fractions 20-50) and long [3H]sialyl polymer region (Fig. 2, DP20-55; Fractions 50-130) was pooled, desalted by dialysis, retreated for 18 h with 5 units of Endo-N, and analyzed by HPLC as described in the legend to Fig. 2. An additional 13.6% of the original 3H-glycopeptide fraction was sensitive to Endo-N digestion and was recovered as DPl-
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