TURNOVER OF HEPARAN-SULFATE DEPENDS ON 2-O-SULFATION OF URONIC-ACIDS

To study how the pattern of sulfation along a heparan sulfate chain af fects its turnover, we examined heparan sulfate catabolism in wild-typ e Chinese hamster ovary cells and mutant pgsF-17, defective in 2-O-sul fation of uronic acid residues (Bai, X., and Esko, J. D. (1996) J. Bio l, Chem. 271, 17711-17717). Heparan sulfate from the mutant contains n ormal amounts of 6-O-sulfated glucosamine residues and iduronic acid a nd somewhat higher levels of N-sulfated glucosamine residues but lacks any 2-O-sulfated iduronic or glucuronic acid residues. Pulse-chase ex periments showed that both mutant and wild-type cells transport newly synthesized heparan sulfate proteoglycans to the plasma membrane, wher e they shed into the medium or move into the cell through endocytosis. Internalization of the cell-associated molecules leads to sequential endoglycosidase (heparanase) fragmentation of the chains and eventual lysosomal degradation. In wild-type cells, the chains begin to degrade within 1 h, leading to the accumulation of intermediate (1O-20-kDa) a nd small (4-7-kDa) oligosaccharides. Mutant cells did not generate the se intermediates, although internalization and intracellular trafficki ng of the heparan sulfate chains appeared normal, and the chains degra ded with normal kinetics. This difference was not due to defective hep aranase activities in the mutant, since cytoplasmic extracts from muta nt cells cleaved wild-type heparan sulfate chains in vitro. instead, t he heparan sulfate chains from the mutant ware relatively resistant to degradation by cellular heparanases. These findings suggest that 2-O- sulfated iduronic acid residues in heparan sulfate are important for c leavage by endogenous heparanases but not for the overall catabolism o f the chains.