Turnover of Heparan Sulfate Depends on 2-O-Sulfation of Uronic Acids

Abstract

To study how the pattern of sulfation along a heparan sulfate chain affects its turnover, we examined heparan sulfate catabolism in wild-type Chinese hamster ovary cells and mutant pgsF-17, defective in 2-O-sulfation of uronic acid residues (Bai, X., and Esko, J. D. (1996) J. Biol. Chem. 271, 17711-17717). Heparan sulfate from the mutant contains normal amounts of 6-O-sulfated glucosamine residues and iduronic acid and somewhat higher levels of N-sulfated glucosamine residues but lacks any 2-O-sulfated iduronic or glucuronic acid residues. Pulse-chase experiments showed that both mutant and wild-type cells transport newly synthesized heparan sulfate proteoglycans to the plasma membrane, where 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 (10 -20-kDa) and small (4 -7-kDa) oligosaccharides. Mutant cells did not generate these intermediates, although internalization and intracellular trafficking of the heparan sulfate chains appeared normal, and the chains degraded with normal kinetics. This difference was not due to defective heparanase activities in the mutant, since cytoplasmic extracts from mutant cells cleaved wild-type heparan sulfate chains in vitro. Instead, the heparan sulfate chains from the mutant were relatively resistant to degradation by cellular heparanases. These findings suggest that 2-O-sulfated iduronic acid residues in heparan sulfate are important for cleavage by endogenous heparanases but not for the overall catabolism of the chains.

Substrate Specificity of Heparanases from Human Hepatoma and Platelets

“…Moreover, heparin with low affinity for antithrombin was degraded by heparanase to about the same extent as high affinity heparin, despite an ϳ10-fold lower content of GlcN 3-O-sulfate groups (39). Finally, Bai et al (16) recently found aberrant turnover of HS in CHO cells defective in a sulfotransferase required for 2-O-sulfation of hexuronic acid residues and concluded that 2-O-sulfated IdceA units are important for cleavage of HS by intracellular heparanase. 3 The results of the present study suggest that the single 2-O-sulfated hexuronic acid unit required for substrate recognition by the heparanase may be either GlcUA or IdceA.…”

“…Endoglycosidases capable of partially depolymerizing HS chains have been demonstrated in a variety of cells and tissues, such as placenta (7), skin fibroblasts (8), platelets (9), melanoma (10), lymphoma (11), hepatocytes (12), CHO cells (13), and endothelial cells (14). Such enzymes, commonly referred to as "heparanases," contribute to the intracellular degradation of HS proteoglycans (15,16) but are also released into the extracellular matrix, where they appear to modulate several processes of pathophysiological importance. Heparanases thus contribute to the remodeling of extracellular matrix and basement membranes prerequisite to angiogenesis (17) and to the egress of metastatic tumor cells (18) and other types of blood-borne cells (19) from the vasculature.…”

“…Several attempts have been made to define the substrate recognition properties of heparanases from various sources (6,9,16,25,(27)(28)(29)(30)(31). The approaches taken have involved structural comparison between polysaccharides susceptible or resistant to an enzyme, sequence analysis of fragments generated by enzymatic cleavage, and studies of inhibitory effects of chemically modified heparin derivatives.…”

Heparin and Heparan Sulfate Biosynthesis

“…A CHO cell mutant, pgsF-17, defective in HS2ST, produces HS chains lacking not only IdoA(2-O-sulfate)-N -sulfoglucosamine and IdoA(2-O-sulfate)-N -sulfoglucosamine(6-O-sulfate) but also GlcA(2-O-sulfate)-N -sulfoglucosamine (83). When the HS2ST cDNA is transfected into the mutant cells, the synthesis of these structures is restored (84). A gene trap mutation for HS2ST causes renal agenesis and neonatal lethality in mutant mice (85).…”

Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I

“…35 S]GAGs recovered from the cultures 1-2 days after temperature shift presumably reflects partial turnover of membrane proteoglycans through endocytosis and lysosomal degradation (28,29).…”