Characterization of glycopeptides labelled from d-[2-3H]mannose and l-[6-3H]fucose in intestinal epithelial cell membranes during differentiation

Abstract

The labeled glycopeptides obtained by pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labeled fractions were eluted in the following order from Bio-Gel P-6. Fraction I, which was excluded from the gel, was labeled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained complex asparagine-linked oligosaccharides since it was labeled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment and resistant to endo-.beta.-N-acetylglucosaminidase H. Fraction III contained ''high-mannose'' asparagine-linked oligosaccharides, which were labeled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-.beta.-N-acetylglucosaminidase H and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl .alpha.-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were apparently precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labeled complex oligosaccharides to high-mannose oligosaccharides, 3 h after [3H]mannose injection, was greater in crypt than in villus-cell lateral-basal membranes. Luminal membranes of crypt and villus cells were greatly enriched in labeled complex oligosaccharides compared with the labeling in lateral-basal membranes. Intestinal epithelial cells are apparently polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labeled membrane glycopeptides, but no major qualitative change, were observed.