The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.
The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans. Previously, amino acids located in the active site of heparinase I have been identified and mapped. In an effort to further understand the mechanism by which heparinase I cleaves its polymer substrate, we sought to understand the role of calcium, as a necessary cofactor, in the enzymatic activity of heparinase I. Specifically, we undertook a series of biochemical and biophysical experiments to answer the question of whether heparinase I binds to calcium and, if so, which regions of the protein are involved in calcium binding. Using the fluorescent calcium analog terbium, we found that heparinase I tightly bound divalent and trivalent cations. Furthermore, we established that this interaction was specific for ions that closely approximate the ionic radius of calcium. Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3-sulfonate (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and calcium was essential for proper functioning of the enzyme. Preincubation with either calcium alone or calcium in the presence of heparin was able to protect the enzyme from inactivation by these modifying reagents. In addition, through mapping studies of Woodward's reagent Kmodified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu 207 -Ala 219 ) and CB-2 (Thr 373 -Arg 384 ), in heparinase I that not only are specifically modified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calciumcoordinating consensus motif.Heparin-like glycosaminoglycans, such as heparin and heparan sulfate, are acidic polysaccharides that play a role in many central biological processes, such as cell proliferation and signaling (1, 2). However, attempts to determine whether heparinlike glycosaminoglycans are involved in a particular biological process have been hampered by the lack of tools available to study these substrates.One such tool, under development in our laboratory, is the heparinases, bacterially derived lyases. Heparinase I from Flavobacterium heparinum is a 43-kDa enzyme that cleaves primarily heparin-like regions of heparin-like glycosaminoglycans (i.e. regions containing a high degree of sulfation with primarily iduronic acid as the uronic acid component) (3, 4). Heparinase I has been used in a variety of circumstances to highlight the importance of heparin-like glycosaminoglycans in such diverse biological processes as angiogenesis (5) and development (6).To extend the capabilities of the heparinases, we have undertaken a series of biochemical studies aimed at identifying important functional residues as well as elucidating the enzyme's mode of action. Through a combination of chemical modification, proteolytic mapping, and site-directed mutagenesis, we have identified Cys 135 (7), His 203 (8), and Lys 199 (9) as amino acid residues important for heparinase ...
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...
The three heparinases derived from Flavobacterium heparinum are powerful tools for studying heparin-like glycosaminoglycans in major biological processes, including angiogenesis and development. Heparinase II is unique among the three enzymes because it is able to catalytically cleave both heparin and heparan sulfatelike regions of heparin-like glycosaminoglycans. Toward understanding the catalytic mechanism of heparin-like glycosaminoglycan degradation by heparinase II, we set out to investigate the role of the histidines of heparinase II in catalysis. We observe concentration-dependent inactivation of heparinase II in the presence of the reversible histidine-modifying reagent diethylpyrocarbonate (DEPC). With heparin as the substrate, the rate constant of inactivation was found to be 0.16 min ؊1 mM ؊1 ; with heparan sulfate as the substrate, the rate constant was determined to be 0.24 min ؊1 mM ؊1 . Heparinase II activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase II by DEPC is specific for histidine residues and that three histidines are modified by DEPC. Substrate protection experiments show that heparinase II preincubation with heparin followed by the addition of DEPC resulted in a loss of enzymatic activity toward heparan sulfate but not heparin. However, heparinase II preincubation with heparan sulfate was unable to protect heparinase II from DEPC inactivation for either of the substrates. Proteolytic mapping studies with Lys-C were consistent with the chemical modification experiments and identified histidines 238, 451, and 579 as being important for heparinase II activity. Further mapping studies identified histidine 451 as being essential for heparin degradation. Site-directed mutagenesis experiments on the 13 histidines of heparinase II corroborated the chemical modification and the peptide mapping studies, establishing the importance of histidines 238, 451 and 579 in heparinase II activity.Heparin-like glycosaminoglycans (HLGAGs) 1 are one of the major components of the extracellular matrix. Increasingly, evidence points to the fact that HLGAGs serve a critical regulatory role in numerous biological functions (1, 2). The chemistry and structure of HLGAGs are beginning to be well understood (3). HLGAGs are linear, sulfated, acetylated, polysaccharides consisting of 1-4-linked derivatives of hexosamine and uronic acid (1, 4).At present, however, there is little understanding, at the molecular level, of how specific sequences of HLGAGs modulate a given biological process (1, 2). The considerable chemical heterogeneity of HLGAGs provides the major challenge in determining these sequence-function relationships (3, 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 Dglu...
This study represents one of the first efforts to examine substance use among American Indian (AI) youth in an Eastern city. As part of a school-based study in metropolitan Columbus, Ohio, 596 self-identified AI youth (grades 6-12) completed surveys describing their use of alcohol, cigarettes, smokeless tobacco, marijuana and inhalants. Net of gender, grade and family structure, AI youth were more likely than their white peers to regularly use most substances while overall prevalence resembled estimates from studies of urban AI youth in the Western United States. These findings highlight the complex interactions of geography, identity and risk behavior among ethnic minority adolescents.
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