Differentially expressed patterns of glycosaminoglycan structure in heparan sulfate proteoglycans and free chains.

The metabolic relationships between heparan sulfate proteoglycans, free chains, and oligosaccharides in different cell locations were evaluated by comparing their glycosaminoglycan structure. Metabolically labeled heparan sulfate proteoglycans of BALB/c 3T3 cell layers and in conditioned medium were compared with the heparan sulfate free chains (modal mass = 10 kDa) and oligosaccharides (modal mass = 3 kDa) of the cells. Nonlytic, in situ digestion with heparitinase I indicated that 90% of proteoglycans, 70% of the free chains, and 20% of the oligosaccharides were enzyme accessible, but there was no evidence using competitive ligands for binding of the products to the cell surface via the glycosaminoglycan moieties. Structurally, the membrane proteoglycans were the most O-/N-sulfated and yielded more tri- and tetra-sulfated di- and tetra-saccharides by nitrous acid degradation. In contrast, the side chains of medium proteoglycans were less sulfated and more polydisperse in mass, suggesting that most medium proteoglycans are not processed from membrane precursors. The heparan sulfate free chains were of lower mass, less sulfated, and more heterogeneous in distribution of the anionic groups than were proteoglycan side chains. Corroborating analytical heparitinase I digestion indicated that generation of di- and tetra-saccharides proportionately increased from membrane proteoglycan, to cell free chain, to medium proteoglycan categories. Because the structural patterns of the heparan sulfate free chains did not reveal a clear relationship with the side chains of the major proteoglycans, their origin was further probed by [3H]BH4-labeling of the reducing terminus under varying stringencies. The end-labeled residues obtained by nitrous or strong acid hydrolysis of the free chains showed insignificant amounts of galactose and xylose, but rather glucosamine N-sulfate and a residue likely generated from glucuronate. The effective labeling that was achieved with weak alkali indicated that covalent oligopeptide is not present. In summary, the heparan sulfate free chains, which in part are components of the cell surface, are of relatively low mass, are unassociated with covalent peptide, and most probably have a disaccharide motif of glucosamine N-sulfate and a uronate residue at the reducing end. Taken together, these observations suggest that the free chains originate by processing of precursor heparan sulfate proteoglycans on the cell surface via an endoglycosidase acting on an N-sulfated portion of the original polymer.