Poly(N-acetyllactosaminyl) oligosaccharides in glycoproteins of PC12 pheochromocytoma cells and sympathetic neurons

Abstract

Endo-.beta.-galactosidase treatment of glycopeptides derived from the trypsinate and membranes of PC12 pheochromocytoma cells and cultured sympathetic neurons demonstrated the presence of poly-(N-acetyllactosaminyl) units of tri- and tetraantennary oligosaccharides, some of which have a core fucose residue and a 2,6-substituted .alpha.-linked mannose residue. Nerve growth factor induced differentiation of the PC12 cells led to a small but significant decrease in the proportion of these oligosaccharides. Poly-(N-acetyllactosaminyl) oligosaccharides were also identified in a major 230,000-Da cell-surface glycoprotein (the nerve growth factor inducible large external, or NILE, glycoprotein) of PC12 cells and appear to account for much or all of the difference in size between this glycoprotein as compared to the immunochemically cross-reactive 205,000-Da species present in postnatal brain. Glycoproteins containing poly(N-acetyllactosaminyl) oligosaccharides were selectively labeled by treatment of PC12 cells with endo-.beta.-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the presence of a number of distinct PC12 cell glycoproteins that contain these oligosaccharides and have apparent molecular weights in the range of 25,000-250,000. Treatment of PC12 cells with nerve growth factor (NGF) altered the relative labeling of several of the glycoprotein bands, with a time course similar to the effects on NGF on neurite outgrowth. However, most of the NGF effects on glycoprotein labelling are also seen in PC12 cells cultured in suspension, conditions under which neurite extension and certain other NGF effects do not occur, and they can be partially induced by long-term treatment with forskolin (which increases cellular levels of cyclic AMP) but not by epidermal growth factor.