GLYPICAN (HEPARAN-SULFATE PROTEOGLYCAN) IS PALMITOYLATED, DEGLYCANATED AND REGLYCANATED DURING RECYCLING IN SKIN FIBROBLASTS

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositol-anchored heparan-sulfate p roteoglycan) that is resistant to inositol-specific phospholipase C an d incorporates sulfate and glucosamine into heparan sulfate chains (Fr ansson, L.-Angstrom. et al., Glycobiology, 5, 407-415, 1995). We have now investigated structural modifications of recycling glypican, such as fatty acylation from [H-3]palmitate, and degradation and assembly o f heparan sulfate side chains, Most of the H-3-radioactivity was recov ered as lipid-like material after de-esterification, To distinguish be tween formation of heparan sulfate at vacant sites, elongation of exis ting chains or degradation followed by re-elongation of chain remnants , cells were pulse-labeled with [H-3]glucosamine and then chase-labele d with [C-14]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [H-3]-labeled heparan-sulfa te degradation products (molecular mass, 20-80 kDa) showing that the s ide chains were degraded during recycling, The degradation products we re initially glucuronate-rich, but became more iduronate-rich with tim e, The glypican proteoglycan formed during the chase was degraded eith er with alkali to release intact side chains or with heparinase to gen erate distally located chain fragments that were separated from the co re protein, containing the proximally located, covalently attached cha in remnants, All of the [C-14]-radioactivity incorporated during the p ulse was found in peripheral chain fragments, and the chains formed we re not significantly longer than the original ones, We therefore concl ude that newly made heparan-sulfate chains were neither made on vacant sites, nor by extension of existing chains but rather by re-elongatio n of degraded chain remnants, The remodeled chains made during recycli ng appeared to be more extensively modified than the original ones.