INTRODUCTION

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Heparin-like glycosaminoglycans (HLGAGs)¹ play an intricate role in the extracellular matrix, regulating a wide variety of biological functions (1, 2). HLGAGs are highly sulfated, complex, acidic polysaccharides consisting of alternating uronic acid (L-iduronic or D-glucuronic acid) and D-glucosamine residues connected through 1-4 linkages. Variations in the degree and distribution of sulfation result in a high degree of chemical heterogeneity in HLGAGs. Three enzymes that degrade HLGAGs (heparin and heparan sulfate), viz. heparinases I, II, and III from Flavobacterium heparinum, recognize unique sequences of sulfation and uronic acid epimerization in HLGAGs with a high degree of specificity (3-5). Heparinases have important clinical applications such as in the monitoring of heparin levels in blood (approved by the FDA) (6), neutralization of heparin in blood (in phase III clinical trials), and in production of low molecular weight heparins for use in humans. In addition, heparinases I and III are potent inhibitors of neovascularization (7). More importantly, heparinases have proven to be useful tools in understanding the important physiological roles of HLGAGs (8, 9).

Our group has cloned, sequenced, and expressed in Escherichia coli the genes for heparinases I, II, and III from F. heparinum (10-13). We subsequently carried out extensive biochemical studies to investigate structure-activity relationships of heparinase I and to understand the mechanism of heparin degradation (14-16). Earlier we showed that cysteine 135, in a highly positively charged environment, is catalytically active in heparinase I (14). We proposed that one possible role of the positively charged active site would be to lower the pK_a of the cysteine such that it is present as a thiolate anion in the enzyme active site. As an anion, cysteine 135 could initiate catalysis by acting as a base for proton abstraction from the substrate heparin. In another study, using a combination of chemical and proteolytic digests of heparinase I in direct binding and competition assays, we identified and mapped a primary heparin binding site in heparinase I spanning residues 196-221 of the heparinase I primary sequence (15). This region contains two positively charged clusters (residues 197-204 and 207-212) as well as a calcium binding consensus motif (residues 207-220) (15). Interestingly, these positively charged clusters conform to the Cardin-Weintraub heparin binding consensus sequence (17). In a parallel study we showed that histidine 203, contained in one of the positive clusters of the primary heparin binding site, is critical for catalytic activity in heparinase I (16). This provided compelling evidence for the positively charged heparin binding site being in close proximity to the active-site cysteine 135, and hence we proposed that cysteine 135 and histidine 203 together form part of the catalytic domain in heparinase I (16).

The above taken together raises the question as to the specific role(s) of the positively charged clusters in heparinase I. Potentially, the basic clusters in the heparin binding site could either provide the necessary charge complimentarity for specific heparin binding and/or bias the active-site reactivity (15). Alternatively, given the proximity of these residues to cysteine 135 and histidine 203, specific residue(s) within the putative heparin binding site could play a more direct role in catalysis. In this study we address the role of positive charge in the primary heparin binding site in heparinase I activity using extensive site-directed mutagenesis experiments.

	EXPERIMENTAL PROCEDURES

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Chemicals and Materials

Heparin (porcine intestinal mucosa, average molecular mass of 12 kDa and activity of 157 USP units/mg) was from Hepar (Franklin, OH). Urea, dithiothreitol, and acetonitrile were from Allied Chemicals (Deerfield, IL). Other chemicals were from Mallinckrodt Chemical Works (Chesterfield, MO). Molecular mass standards were obtained from Life Technologies. E. coli BL21(DE3) host was from Novagen, WI. Molecular biology reagents and their sources are listed in the appropriate sections below.

Heparinase I: Protein Analyses

Heparinase I from F. heparinum was purified as described previously (10, 18). The purified heparinase I was collected and lyophilized (VirTis freeze mobil model 12; VirTis Inc., NY). Heparinase I used for activity measurements was extensively desalted using a Centricon P-30 (Amicon, Beverly, MA). Protein concentration was determined using Micro BCA reagent (Pierce) relative to a bovine serum albumin standard.

Mutagenesis, Expression, and Purification of r-heparinase I

The recombinant and mutant heparinases I were expressed without the putative F. heparinum leader sequence, i.e. as a construct ([-L] r-heparinase I) that reads Met-Glu²²-Glu²³- (10). To facilitate purification, the heparinase I gene was expressed using the pET-15b system (Novagen). This construct has a polyhistidine tag and a thrombin cleavage site in a 21-amino acid N-terminal leader sequence (14).

Mutagenesis-- The mutations were introduced via 12-cycle polymerase chain reaction, as described previously by the method of Higuchi (19). All the mutant genes were cloned into pET-15b and were sequenced to verify the mutations, as described previously (14).

Expression and Purification-- The constructs were transformed into BL21(DE3) (Novagen), and the proteins were purified as described previously (14). SDS-polyacrylamide gel electrophoresis was carried out using precast 12% gels and a Mini Protean II apparatus and stained with the Silver Stain Plus kit (14).

Heparinase I Activity Assays

UV 232-nm Assay-- The UV 232-nm assay was performed essentially as described previously (10, 20). The enzyme activity was directly measured from the increase in absorbance at 232 nm as a function of time. All assays were performed at 30 °C, and the heparin concentration was fixed at 2 mg/ml (100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0). Activity is expressed as IU (µmol of product formed/min using  = 3,800 M¹).

HPLC of Heparin Oligosaccharides-- Heparin (2 mg/ml) was incubated with native heparinase I from F. heparinum, r-heparinase I, and mutant enzymes in 100 mM MOPS, 5 mM calcium acetate buffer, pH 7.0, for 18 h. The reaction was then subjected to anion-exchange HPLC to resolve the oligosaccharide products, as described (10).

Heparin-POROS Chromatography-- About 30-40 µg of -L r-heparinase I and the various mutant enzymes were injected into a heparin-POROS (4.6 × 100 mm) column (PerSeptive BioSystems, Framingham, MA) connected to a BioCAD system (PerSeptive BioSystems). Proteins were eluted using a linear gradient of 0-1-M NaCl in 10 min (10 mM Tris, 1 mM EDTA, pH 7.0) and monitored at 210 nm. EDTA was added to chelate any calcium ions that may be present in the buffers.

Strategy for Site-directed Mutagenesis Studies

The primary heparin binding site of heparinase I (15) contains two basic clusters, HB-1 (F¹⁹⁷KKNIAHD²⁰⁴) and HB-2 (E²⁰⁷KKDKD²¹²). The basic cluster HB-2 conforms to a XBBXBX heparin binding consensus sequence (21), whereas HB-1 partially conforms to the XBBBXXBX consensus sequence. Further, HB-2 also is part of a putative calcium binding site, suggesting that a ternary complex of heparin and calcium with heparinase I may primarily involve HB-2. In addition, the primary sequence of heparinase I contains another basic cluster HB-3 (K³³¹NKKPQK³³⁷) in the C terminus (10) that resembles a Cardin-Weintraub consensus sequence.

Fig. 1 describes the different mutations in heparinase I made in this study. To map the residues critical for heparinase I activity, the primary heparin binding site (residues 196-221) and tryptic peptide 4 (residues 132-141) that bound heparin in our earlier study (15) were chosen as targets for mutagenesis studies. The positive charge in the primary heparin binding site was altered in the following manner. Basic residues from the two consensus sequences, lysines 198 and 199 (HB-1) and lysines 208 and 209 (HB-2), were first jointly altered to alanines (neutral), aspartic acids (negative charge), and arginines (positive charge); lysine 211 (HB-2) was changed to an alanine, whereas catalytically active histidine 203 (HB-1) was altered to an alanine in an earlier study (16). If a double mutation had an effect on catalytic activity, the lysines were individually changed to alanines to examine the possibility of one of the residues having a dominant effect on enzyme activity. Asparagine 200 was changed to a lysine to investigate the effect of increasing the net positive charge in HB-1 and to an alanine to probe the functionality of asparagine 200. A possible role for the C-terminal basic cluster (HB-3) was addressed by jointly altering lysines 333 and 334 to alanines. Furthermore, lysines in the vicinity of HB-1 and HB-2 in the heparin binding site were altered to alanines to probe the functionality of these basic residues. Finally, the role of the two positively charged residues, lysine 132 and arginine-141 in tryptic peptide 4 (K¹³²GIC ... R¹⁴¹, which contains the active-site cysteine 135) (15), was examined by individually altering each residue to alanine.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the various heparinase I mutations undertaken for this study. The primary heparin binding site (residues 196-221) and tryptic peptide 4 (Td 4; residues 132-141) that were found to bind heparin in our earlier study (15) were chosen as targets for mutagenesis studies. The positive charge in the primary heparin binding site was altered in the following manner. Basic residues from the two consensus sequences, lysines 198 and 199 (HB-1) and lysines 208 and 209 (HB-2), were first jointly altered to alanines (neutral), aspartic acids (negative charge), and arginines (positive charge); lysine 211 (HB-2) was changed to an alanine, whereas the catalytically active histidine 203 (HB-1) was altered to an alanine in an earlier study (16). Since the double mutants in HB-1 affected enzyme activity, lysines 198 and 199 were also individually changed to alanines to examine the possibility of one of the residues having a dominant effect on catalytic activity. Asparagine 200 was changed to a lysine to increase the positive charge in HB-1 and to an alanine to probe the functionality of this residue. Other basic residues in the heparin binding site, viz. lysines 205 and 214, were also individually altered to alanines. Finally, to investigate the role of the two positively charged residues, lysine 132 and arginine 141, in tryptic peptide 4, they were individually altered to alanines.

All mutant r-heparinases were constructed in the pET-15b expression system (Novagen, WI) and expressed in the BL21(DE3) host as described previously (14). The level of protein expression for all the r-heparinases was identical in the BL21(DE3) host (data not shown). Kinetic parameters k_cat and K_m were determined for all the mutants as described previously (11). The product profiles of heparin degradation were characterized using anion-exchange HPLC (POROS) to resolve the oligosaccharide products as described (10).

	RESULTS AND DISCUSSION

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HB-1 Mutagenesis-- Table I lists the kinetic parameters obtained for wild-type r-heparinase I and all the mutant enzymes. When lysine 198 and lysine 199 from HB-1 of the heparin binding site were jointly altered to alanines (creating a double mutant K198A/K199A), the enzyme activity was drastically affected, with a 20-fold reduction in k_cat. A similar effect was observed for the K198D/K199D double mutant enzyme. Interestingly, when the lysines 198 and 199 were jointly changed to arginines (K198R/K199R), the enzyme activity remained unaltered, as observed by the k_cat value (Table I), suggesting that the positive charge of these residues is important for heparinase I activity. When the heparin degradation reactions of the double mutants K198A/K199A and K198D/K199D were allowed to go to completion and the products were resolved using anion-exchange HPLC, the product profiles were altered when compared with the control r-heparinase I product profile (Fig. 2, a-c); a significant fraction of higher order fragments were observed. The heparin degradation product profile of the K198R/K199R double mutant (Fig. 2d) was similar to the wild-type enzyme.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Anion-exchange HPLC separation of oligosaccharides. Heparin (2 mg/ml) was incubated with -L and mutant heparinases I in 100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0, for 18 h as described under "Experimental Procedures." The reaction was then subjected to anion-exchange HPLC using a POROS Q/M column with a salt gradient of 0-2 M NaCl in 10 min and monitored at 232 nm. A shows the product profile of heparin degradation by wild-type r-heparinase I, B shows the product profile of heparin degradation by K198A/K199A, C shows the product profile of heparin degradation by K198D/K199D, D shows the product profile of heparin degradation by K198R/ K199R.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Anion-exchange HPLC separation of oligosaccharides. Heparin (2 mg/ml) was incubated with -L and mutant heparinases I in 100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0, for 18 h as described under "Experimental Procedures." The reaction was then subjected to anion-exchange HPLC using a POROS Q/M column with a salt gradient of 0-2 M NaCl in 10 min and monitored at 232 nm. A shows the product profile of heparin degradation by wild-type r-heparinase I, B shows the product profile of heparin degradation by K198A, C shows the product profile of heparin degradation by K199A, D shows the product profile of heparin degradation by K211A.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Anion-exchange HPLC separation of oligosaccharides. Heparin (2 mg/ml) was incubated with -L and mutant heparinases I in 100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0, for 18 h as described under "Experimental Procedures." The reaction was then subjected to anion-exchange HPLC using a POROS Q/M column with a salt gradient of 0-2 M NaCl in 10 min and monitored at 232 nm. A shows the product profile of heparin degradation by wild-type r-heparinase I, B shows the product profile of heparin degradation by N200K, C shows the product profile of heparin degradation by N200A, D shows the product profile of heparin degradation by K333A/K334A.

HB-2 and Other Positive Charge Mutagenesis-- When lysines 208 and 209 were jointly changed to alanines, the enzyme activity (k_cat) was reduced by less than 1.5-fold (Table I), and the product profile was similar to wild-type heparinase I (Fig. 5). However, neither the K208R/K209R nor the K208D/K209D double mutations altered the activity and product profiles of the mutant enzymes relative to wild-type heparinase I (Fig. 5). Further, the K211A mutation (HB-2) did not affect the enzyme activity and product profile (Fig. 3d). Thus, compared with HB-1, the positive charge in HB-2 of the heparin binding site, within the limits of this study, does not seem to play a significant role in heparinase I activity. However, it must be pointed out that other residues of HB-2 are part of the putative calcium binding site in heparinase I (15). We are currently investigating the effect of this region in chelating calcium and exploring the role of different residues from the calcium binding consensus sequence in modulating heparinase I activity.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Anion-exchange HPLC separation of oligosaccharides. Heparin (2 mg/ml) was incubated with -L and mutant heparinases I in 100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0, for 18 h as described under "Experimental Procedures." The reaction was then subjected to anion-exchange HPLC using a POROS Q/M column with a salt gradient of 0-2 M NaCl in 10 min and monitored at 232 nm. A shows the product profile of heparin degradation by wild-type r-heparinase I, B shows the product profile of heparin degradation by K208A/K209A, C shows the product profile of heparin degradation by K208D/K209D, D shows the product profile of heparin degradation by K208R/K209R.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Anion-exchange HPLC separation of oligosaccharides. Heparin (2 mg/ml) was incubated with -L and mutant heparinases I in 100 mM MOPS buffer, 5 mM calcium acetate, pH 7.0, for 18 h as described under "Experimental Procedures." The reaction was then subjected to anion-exchange HPLC using a POROS Q/M column with a salt gradient of 0-2 M NaCl in 10 min and monitored at 232 nm. A shows the product profile of heparin degradation by wild-type r-heparinase I, B shows the product profile of heparin degradation by K132A, C shows the product profile of heparin degradation by R141A, D shows the product profile of heparin degradation by K205A.

Heparin Affinity Chromatography-- We have shown previously that, in the absence of calcium, native heparinase I from F. heparinum binds a heparin-POROS column and can be eluted at a salt concentration of about 500 mM (15). It should be pointed out that compared with other heparin-binding proteins, such as lipoprotein lipase or fibroblast growth factor, which elute at much higher salt concentrations of about 0.9-1.5 M NaCl (22, 23), heparinase has a lower affinity for heparin (15). We used heparin-POROS chromatography to investigate whether the mutations affected heparin binding and hence altered the elution profile. As shown in Table I, wild-type r-heparinase I elutes at a salt concentration of about 480 mM. The K198A/K199A and the K198D/K199D double mutants eluted at lower salt concentrations of about 448 and 445 mM, respectively. The individual mutants K198A and K199A also eluted at lower salt concentrations of about 448 and 452 mM, respectively. Furthermore, the K132A mutation eluted at a moderately lower salt concentration of about 460 mM. The K198/199R double mutant, however, eluted at about 475 mM, similar to wild-type enzyme. The HB-2 mutant enzymes (K208/209A, K208/209D, and K208/209R) also eluted at salt concentrations comparable to wild-type r-heparinase I (Table I). Increasing the positive charge in HB-1 (N200K) did not result in tighter heparin binding, with the mutant eluting at about 469 mM salt concentration.

The Heparin Binding Domain of Heparinase I-- The data presented in this study leads us to postulate that lysine 199 is important for catalysis and raises interesting possibilities for the role of lysine 199 in catalysis. It has been proposed as a general mechanism for polysaccharide lyases (24) that an important step in the catalytic mechanism is the neutralization of the negative charge on the C-6 carboxylate anion of the uronate in heparin. This function is most likely performed by a lysine residue by the formation of a salt bridge. Based on the above and the data presented here, it is possible that lysine 199 acts as an acid to stabilize the negative charge developing on the carboxylate of the uronate in heparin.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Schematic model of the catalytic domain of heparinase I. Cysteine 135 and histidine 203 were identified as catalytically critical residues in previous studies (14, 16). We propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic residues. Lysine 199, as a third residue in the catalytic mechanism, could play an additional role in stabilizing the negative charge of the uronate of substrate heparin or in stabilizing the thiol group of cysteine 135. Basic residues lysines 198 and 132 possibly aid in heparin binding through charge complimentarity.