Heparinase II from Flavobacterium heparinum

Abstract

Heparinase II (no EC number) is one of three lyases isolated from Flavobacterium heparinum that degrade heparin-like complex polysaccharides. Heparinase II is unique among the heparinases in that it has broad substrate requirements and possesses the ability to degrade both heparin and heparan sulfate-like regions of glycosaminoglycans. This study set out to investigate the role of cysteines in heparinase II activity. Through a series of chemical modification experiments, it was found that one of the three cysteines in heparinase II is surfaceaccessible and possesses unusual chemical reactivity toward cysteine-specific chemical modifying reagents. Substrate protection experiments suggest that this surface-accessible cysteine is proximate to the active site, since addition of substrate shields the cysteine from modifying reagents. The cysteine, present in an ionic environment, was mapped by radiolabeling with N-[ 3 H]ethylmaleimide and identified as cysteine 348. Site-directed mutagenesis of cysteine 348 to an alanine resulted in loss of activity toward heparin but not heparan sulfate, indicating that cysteine 348 is required for heparinase II activity toward heparin but is not essential for the breakdown of heparan sulfate. Furthermore, we show in this study that cysteine 164 and cysteine 189 are functionally unimportant for heparinase II.Heparin-like glycosaminoglycans (HLGAGs) 1 are key components of the extracellular matrix that serve to regulate an array of biological functions (1, 2). HLGAGs are linear, sulfated, acetylated, polysaccharides consisting of 1-4-linked derivatives of hexosamine and uronic acid (3). One of the major challenges in elucidating a specific role for HLGAGs in certain biological systems is that the considerable chemical heterogeneity of HLGAGs has thwarted attempts to determine sequence-function relationships (4, 5). Heparin, one subset of HLGAGs, possesses predominantly L-iduronic acid with a high degree of sulfation (3, 4). Heparan sulfate, another subset of HLGAGs, is chemically similar to heparins, but contains less 2-O-sulfate and N-sulfate groups than heparin and also possesses a higher percentage of D-glucuronic acid within the polymer (3, 6).To elucidate the mechanism by which sequence or sequences within HLGAGs bind to and regulate components of the ECM, it is critical to develop biochemical methods of structure-function analysis (4). HLGAG-degrading enzymes, or heparinases, a family of polysaccharide lyases that catalyze the eliminative cleavage of HLGAGs, have shown promise as potential tools to determine specific HLGAG sequences involved in HLGAG-protein interactions (7). Toward development of the heparinases, we have cloned and recombinantly expressed heparinases I, II, and III from Flavobacterium heparinum (8 -11). In addition, we have carried out extensive biochemical characterization of heparinase I to determine the molecular basis of its substrate specificity (11-13). Such an understanding will facilitate both a general appreciation of the chemistry of polysaccharide l...

Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II

“…The strong preference of heparinase II for the unsulfated glucuronate-containing linkage of O2 is consistent with the observation that heparinase II might contain two active sites, one of which is heparinase I-like, cleaving primarily heparinlike linkages, whereas the other site is heparinase III-like and cleaves primarily heparan sulfate-like linkages (17). In accordance with this hypothesis, we have identified a particular cysteine residue, cysteine 348, which is critical for the breakdown of heparin but not heparan sulfate by heparinase II (17).…”

“…In addition, recombinant heparinase II and the heparinase mutant protein, C348A, where cysteine-348 is changed to alanine were expressed in Escherichia coli (17). Protein expression, isolation, and purification was carried out as described (17).…”

Crystal Structure of Heparinase II from Pedobacter heparinus and Its Complex with a Disaccharide Product

“…The results from these mapping studies indicated that of the three cysteines, present in HepII, Cys-348 was uniquely susceptible to chemical modification by sulfhydryl-reactive reagents. Mutation of this cysteine (C348A) resulted in a selective loss of activity toward heparin but not toward heparan-sulfate (24). Indeed, the HepII structure indicates that Cys-348 is the only cysteine with a surface-accessible sulfhydryl group.…”

“…Mutagenesis and Chemical Modification Studies-Previous structure-function studies of P. heparinus HepII have been carried out through the complementary use of chemical modification and site-directed mutagenesis (21,24,25). In particular, these studies focused on the potential catalytic role of cysteine and histidine residues.…”

“…Heparinase II shows greater catalytic efficiency for longer rather than shorter oligosaccharide substrates (21). Previous chemical modification and site-directed mutagenesis studies suggested that cysteine and histidine residues are involved in catalysis (24,25) and that the cleavage of the two substrates, heparin and heparan sulfate, occurs at separate but proximal active sites within a single HepII polypeptide chain (21).…”