The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases

Heparanases are mammalian endoglucuronidases that degrade heparan sulfate ( HS) glycosaminoglycans to short 5-6 kDa pieces. In the Golgi, WS glycosamin oglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate r esidues (S-domains), separated by regions that are nearly devoid of sulfate . Structural analysis of the short HS chains produced by Chinese hamster ov ary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave t he chain at junctions between these regions. To look more closely at whethe r the spacing of S-domains on the glycosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chai ns synthesized by the proteoglycan synthesis mutant, pgsE-606, PgsE-606 cel ls are deficient in the modification enzyme N-deacetylase/N-sulfotransferas e I, and synthesize HS chains that have fever N- and O-sulfate groups and i duronate residues compared to wild-type (Bame et al,, (1991), J. Biol. Chem ., 266, 10287), HS glycosaminoglycans were isolated fl om wild-type and pgs E-606 cells and separated into populations based on sulfate content. Compar ed to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606-1, 606-2, and 606-3, with 1, 4, and 8 S-domains, respectiv ely. The spacing of the S-domains on the pgsE-606 HS chains is similar to t he spacing the modified sequences on wild-type HS, indicating that each mut ant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains, When incubated with partially purified CHO heparanase s, only the portion of the mutant HS chains that had S-domains were degrade d. Structural analysis of the heparanase-products confirmed that both the n umber and the arrangement of S-domains on the HS glycosaminoglycan are impo rtant for heparanase susceptibility. The structure of the different pgsE-60 6 HS chains also suggests mechanisms for the placement of S-domains when th e glycosaminoglycan is synthesized.