(Received for publication, September 12, 1995; and in revised form, November 15, 1995)
From the
In this study we have identified the primary heparin binding
site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of
heparinase I were used in direct binding and competition assays, to map
the regions of heparinase I that interact specifically with heparin. We
find the heparin binding site contains two Cardin-Weintraub heparin
binding consensus sequences and a calcium co-ordination consensus
motif. We show that heparin binding to heparinase I is independent of
calcium (K of 60 nM) and that
calcium is able to activate heparinase I catalytically. We find that
sulfhydryl selective labeling of cysteine 135 of heparinase I protects
the lysines of the heparin binding sequence from proteolytic cleavage,
suggesting the close proximity of the heparin binding site to the
active site. Site-directed mutagenesis of H203A (contained in the
heparin binding site) inactivated heparinase I; however, a H203D mutant
retained marginal activity, indicating a role for this residue in
catalysis. The above results taken together suggest that histidine 203
(hence the heparin binding site) is immediately adjacent to the
scissile bond. We propose that the heparin binding site and active site
are in close proximity to each other and that the calcium coordination
motif, contained in the heparin binding site, may bridge heparin to
heparinase I through calcium in a ternary complex during catalysis.
Glycosaminoglycans, such as heparin, heparan sulfate and chondroitin sulfate, play a key role in the extracellular matrix (Jackson et al., 1991; Lindahl et al., 1994). Heparin is an acidic polysaccharide, characterized by a disaccharide repeating unit of hexosamine and uronic acid (L-iduronic or D-glucuronic acid) connected through 1-4 linkages. Heparin is heterogeneous due to the varying degree of modification of the functional groups in the disaccharide unit (Comper, 1981; Linhardt and Loganathan, 1989). Heparinases are heparin-degrading enzymes that cleave certain sequences of heparin/heparan sulfate specifically (Lohse and Linhardt, 1992; Ernst et al., 1995). Heparinases have aided in the understanding of important physiological roles of heparin, which include anticoagulation, angiogenesis, etc. (Linhardt et al., 1993; Sasisekharan et al., 1994; Ernst et al., 1995). Additionally, heparinases themselves have therapeutic and diagnostic applications such as heparin detection and removal (Langer et al., 1982; Bernstein et al., 1988; Baugh et al., 1992; Tejidor et al., 1993; Linhardt et al., 1993; Sasisekharan et al., 1994; Ernst et al., 1995).
Flavobacterium heparinum produces three heparinases, which are specific for different sequences of heparin (Linhardt et al., 1990; Lohse and Linhardt, 1992; Desai et al., 1993). While there are several reports on the substrate specificity of heparinases (Rice and Linhardt, 1989; Linhardt et al., 1990; Nader et al., 1990; Desai et al., 1993; Ernst et al., 1995), there is limited information on the molecular features of the enzymes that confer this specificity (Linhardt et al., 1986; Lohse and Linhardt, 1992; Sasisekharan et al., 1995; Ernst et al., 1995).
There have been
numerous recent reports on the high degree of specificity in the
binding interactions of heparin and heparan sulfate with cytokines,
growth factors and other molecules of the extracellular matrix (Jackson et al., 1991; Varki, 1993; Ernst et al., 1995). In
several heparin-binding proteins, the binding specificity is dependent
on certain sequences of the polysaccharide as well as characteristic
amino acid sequences of the protein (Jackson et al., 1991;
Lindahl et al., 1994). On the protein side, Cardin and
Weintraub identified two consensus sequences (XBBBXXBX or XBBXBX (B = basic residues; X = hydrophobic or
other residues)) found in many heparin-binding proteins (Cardin and
Weintraub, 1989). Subsequently, site-directed mutagenesis and binding
studies with synthetic or isolated peptides from several of these
proteins have confirmed that this consensus region often is involved in
binding specifically to heparin (Bae et al., 1994; Baird et al., 1988; Bober Barkalow and Schwarzbauer, 1991; Jackson et al., 1991; Smith and Knauer, 1987; Thompson et
al., 1994). On the polysaccharide side, oligosaccharides with
certain sequences of modifications in the disaccharide repeat have been
isolated and shown to confer binding specificity for antithrombin III,
bFGF, ()and others (Lindahl et al., 1984; Maccarana et al., 1993; Maimone and Tollefsen, 1990; Parthasarathy et al., 1994). This is parallel to the substrate specificities
of heparinases and suggests that the mechanism of substrate binding to
heparinases may be involved in generating specificity.
Earlier studies suggested the importance of basic residues in heparinase I activity (Comfort et al., 1989; Leckband and Langer, 1991). Recently, we found that cysteine 135 of heparinase I is catalytically active (Sasisekharan et al., 1995). In addition, the observation of a decrease in the rate of heparinase I inactivation by chemical modification of cysteine 135 in the presence of heparin, along with other experiments, led to the hypothesis that a heparin binding domain is in close proximity to cysteine 135 (Sasisekharan et al., 1995). In this study, we map and characterize the heparin binding domain of heparinase I and test the hypothesis of the binding domain being in proximity to the active site.
Following gel electrophoresis CNBr digests of heparinase I were blotted onto nitrocellulose sheets (Schleicher & Schüll) according to the method of Matsudaira(1993). Transfer was carried out in the following amperage sequence: 30 min at 50 mA, 30 min at 100 mA, and 60 min at 275 mA. The nitrocellulose membrane was stained for 1 min with 0.1% Ponceau S solution (Fluka, Buchs, Switzerland) in 0.1% acetic acid and then washed with 1% acetic acid for 1 min (Tempst et al., 1990).
For heparin hybridization, the membrane was further
processed by washing it with 10 mM TrisHCl, 10 mM NaCl and 0.5 mM EDTA, pH 7.0 (TNE-10) twice, and once
with the probing buffer. The heparin binding domain was probed using
I-labeled heparin at a concentration 10
cpm/ml in the probing buffer. The membrane was hybridized at 37
°C for 18 h followed by washing of the membrane to remove excess
radiolabel. The membrane was cut into bands corresponding to the
stained peptide fragments, and each band was counted for
I incorporation on a scintillation counter (model LS
3801, Beckman, Fullerton, CA). The bands that were positive for
I incorporation were washed with phosphate-buffered
saline, pH 7.0, and sequenced as described below. Alternatively, the
stained membrane was cut into bands before hybridization and the bands
were then washed in a microcentrifuge tube, prehybridized, and probed
individually. CNBr peptides were extracted from the nitrocellulose
membrane using essentially the procedure described by Matsudaira(1993)
for sequencing (see below).
A total of 2.5 nmol of heparinase I was digested and then
divided into five batches (500 pmol each; 20 µg) inclusive of
one control digest. For the competition experiments, heparin was added
in varying concentrations (5, 50, 100, and 200 µg) to the digests,
which were subsequently separated using RPHPLC, with a 20-min isocratic
run, followed by 0-80% acetonitrile (in 0.1% trifluoroacetic
acid) for 120 min, and monitored at 210 and 277 nm. Chondroitin sulfate
was used as control for nonspecific competition. In dot blot assays,
2.5 nmol of heparinase I digested with trypsin were separated by RPHPLC
as described above. The peptide fragments were collected in
microcentrifuge tubes and blotted onto nitrocellulose paper. The blots
were washed with TNE-10 twice and then probed with
I-labeled heparin for 2 h, at a concentration of 10
ng/ml, 1 mg/ml chondroitin sulfate, and 0.01% Tween 20 in 10 mM MOPS containing 5 mM calcium acetate, pH 7.0 (probing
buffer). The blots were washed twice with 10 mM MOPS
containing 5 mM calcium acetate, pH 7.0, and then counted for
I-heparin binding. Binding data was analyzed using
weighted, nonlinear least-squares curve fitting (McPherson, 1985).
Small amounts of heparin (0.05% of total counts) bound to
nitrocellulose filters dotted either with buffer or with 1 mM bovine serum albumin.
Peptides were sequenced using an Applied Biosystems model 477 sequencer, with an on-line model 120 PTH amino acid analyzer (Biopolymers Laboratory, MIT).
Figure 1:
SDS-PAGE of
CNBr-digested heparinase I. Heparinase I was digested with CNBr as
described under ``Materials and Methods.'' The CNBr-digested
heparinase I fragments were run on a 15% SDS-PAGE gel, blotted onto
nitrocellulose membranes, and hybridized with I-heparin.
The membranes were then washed and counted for
I
incorporation. Lane 1, molecular size standards; lane
2, F. heparinum heparinase I; lane 3,
CNBr-digested heparinase I. See text and Table 1for
details.
It is important to point out that an unavoidable limitation of the technique described here is that when ligand-protein binding is probed on nitrocellulose (with the protein being denatured), only the primary (non-folded) structures that bind to the ligand can be detected. In order to further investigate the heparin binding region of heparinase I, we carried out proteolytic digests of heparinase I as described below.
Figure 2: Tryptic digest of heparinase I and heparin competition. Heparinase I was digested with trypsin essentially as described by Sasisekharan et al.(1993). Following trypsin digestion, the tryptic peptides were separated by RPHPLC with a 10-min isocratic run, followed by 0 to 80% acetonitrile (in 0.1% trifluoroacetic acid) for 120 min. Tryptic peptides were monitored at 210 and 277 nm (in mAU). A, control digest; B, digest with heparin. Note peak at 34 min (td4), peak at 43 min (td9), peaks eluting between 52 and 58 min (not numbered, shown by arrows) at 77 min (td39), peak at 71 min (td45), and peak at 84 min (td50) are all marked in chromatogram A. Arrows in B show the changes in the peak profiles when compared to the control. Refer to Table 1in Sasisekharan et al.(1993) for peptide sequences (tdLx in the table corresponds to td50).
The combined heparin binding experiments, with CNBr and tryptic digests of heparinase I, point to the residues 195-221 as being primarily involved in heparin binding. As mentioned earlier, this region contains two potential Cardin-Weintraub heparin binding consensus sequences (residues 197-204 and 207-212) and a calcium binding loop of the EF-hand structural domain (residues 206-220). Importantly, as this region contains multiple lysines it is likely to be degraded to very short peptides (di- and tripeptides) by trypsin. Thus, it was not expected to show up on the tryptic digest chromatogram, and its potential heparin binding properties might therefore have gone undetected in these experiments. To investigate the heparin-binding properties of these residues, a peptide corresponding to this region was synthesized and characterized as described below.
Figure 3:
Effects of HBP-I on heparinase I activity.
Heparin (0.2 mg/ml) was incubated with heparinase I in 5 mM calcium acetate, 100 mM MOPS buffer, pH 7.0, for 18 h as
described under ``Materials and Methods.'' The reaction was
then subjected to anion-exchange HPLC using a POROS Q/M (4.6 mm
100 mm) column (PerSeptive BioSystems) with a salt gradient of
0-1 M NaCl in 5 min and monitored at 232 nm. A shows the product profile of heparin degradation by heparinase I
in the absence of HBP-I; B and C show the product
profile of heparin degradation in the presence of HBP-I (1:2 and 1:10
heparinase I to HBP-I). Note the decrease of tetrasaccharide 3
(retention time of about 5 min; shown by arrow in B and C). However, when the digest is carried out in the
presence of a control peptide, there is no change in the product
profile or tetrasaccharide 3 (shown by arrow in D).
In chromatogram A, I represents the disaccharide, II represents the three tetrasaccharides (1-3), and III represents the hexasaccharide.
Figure 4: Effect of calcium on heparinase I activity. Plot of the relative reaction rate as a function of calcium concentration. Heparin concentration was fixed at 25 mg/ml, and the calcium concentration was varied in the assay. Heparinase I (0.5 µg) was desalted and added to the reaction mixture.
Figure 5: Affinity co-electrophoresis of heparinase I. Autoradiographs of gels in which radiolabeled heparin was electrophoresed through zones containing heparinase I in increasing concentrations (in µg) from left to right. A (left) represents the ACE gel of native heparinase I in the absence of calcium. B (right) represents the ACE gel of modified heparinase I (the cysteines blocked by IAA) also in the absence of calcium.
To further investigate a functional role of the heparin binding domain, site-directed mutagenesis was carried out. Histidine 203, as a basic residue, was targeted for site-directed mutagenesis, because it is known to play a role in both heparin binding as well as an acid/base catalyst in some proteins (Fersht, 1985; Talpas and Lee, 1993; Fan et al., 1994). Furthermore, it has been suggested that histidine may act as a nucleophilic residue in the elimination mechanism of lyases (Greiling et al., 1975). H203A and H203D mutations were introduced, and mutant r-heparinases I were expressed in E. coli. While the H203A mutant had no detectable enzymatic activity, the H203D mutant showed a 30-fold decrease in activity. The kinetic parameters for (-L)r-heparinase I and for the mutants are shown in Table 3. These results strongly indicate that histidine 203 is an essential amino acid for enzyme activity. In addition, the results point to the fact that the heparin binding region around residue 203 is in close proximity to the scissile bond during catalysis.
For enzymatic reactions the interplay between binding and catalysis determines both specificity and rate of reaction. This interplay is especially interesting for eliminases/lyases that degrade complex acidic polysaccharides like pectin and heparin, which are highly heterogeneous polymers. In the case of the pectate lyases, while the three-dimensional structures of the enzymes are available, there is limited information and understanding of the residues and the domains of the enzyme involved in catalysis (Yoder et al., 1993a). Little is known of the role of binding in catalysis for the eliminases in general, and the current study addresses this issue.
Heparin binding to heparinase I (K 60
nM) was demonstrated using ACE. This is about 1 order of
magnitude higher than the dissociation constants determined by the same
technique for binding of heparin to bFGF (2.2 nM) and AT III
(11 nM) (Lee and Lander, 1991). This also is consistent with
heparinase I eluting from a heparin affinity column at 200 mM NaCl, while bFGF and other heparin binding growth factors
typically elute around 1-1.5 M NaCl (Folkman and
Klagsbrun, 1987). When ACE was performed with modified heparinase I
(where the cysteines were blocked with IAA), there was no alteration on
the heparin retardation, indicating that cysteine 135 does not
influence heparin binding, but affects only enzyme catalysis.
From equilibrium dialysis, it was found that calcium binds to heparin at a stoichiometry of about 1 calcium ion/tetrasaccharide (Boyd et al., 1980; Hunter et al., 1988), and fiber diffraction studies show that calcium causes a significant change in the helical structure of heparin, heparan sulfate, and other glycosaminoglycans (Nieduszynski, 1985). This suggests that calcium, by binding to specific regions of heparin, alters heparin conformation and hence activates heparinase I. Furthermore, pectate lyase, an enzyme from the same family of acidic polysaccharide eliminases as heparinase I, is also activated by calcium (Yoder et al., 1993b). The recently solved crystal structures of three pectate lyases showed that the substrate binding cleft was embedded with one or more calcium ions (at the calcium binding loop of EF-hand domains), indicating a direct interaction between calcium and the enzyme (Pickersgill et al., 1994; Yoder et al., 1993a, 1993b). Interestingly, the putative calcium binding sequence in heparinase I is contained in the heparin binding site, which appears to be close to the active site cysteine 135 in the correctly folded active protein. Therefore, the effect of calcium on heparinase I and other acidic polysaccharide lyases, is possibly mediated through a conformational change of the substrate upon calcium binding, which is further stabilized by direct interaction between enzyme and calcium at the active site.
Histidine residues are involved in
several enzymatic reactions via different mechanisms, which include
acid/base catalysis, electrophilic catalysis, and substrate binding
through electrostatic interactions (Munier et al., 1992). To
differentiate between a substrate binding and a catalytic role for
histidine 203 in heparinase I, this residue was replaced with alanine
(which can perform neither of the above functions) and with aspartic
acid (which can play a role in acid/base catalysis but is not known to
participate in heparin binding). The H203A mutation abolished enzyme
activity, while the H203D mutant retained residual activity, strongly
suggesting a catalytic role for this residue. ()It is
considered unlikely that a change in the binding site far away from the
scissile bond could produce such a dramatic change in enzyme activity.
It is possible that histidine acts as a nucleophilic residue in the
elimination mechanism, similar to that suggested for hyaluronate lyase
(Greiling et al., 1975). Alternately, the scissile bond
cleavage might occur through a proton relay mechanism involving
histidine 203 and another nucleophile in close proximity to this
residue during elimination reaction (Gacesa, 1987). The site-directed
mutation experiments taken together with the biochemical studies
provide compelling evidence for histidine 203 being in close proximity
to the scissile HI linkage during catalysis.
In a previous report, an active site residue (cysteine 135) was identified and characterized by chemical modification and site-directed mutagenesis (Sasisekharan et al., 1995). It was found that this active site has a net positive environment and may be close to the heparin binding region. From the results described in this work and from the earlier study (Sasisekharan et al., 1995), a molecular mechanism for the interplay between binding and catalysis is emerging; cysteine 135 is catalytically active, but is not a determinant for heparin binding. The heparin binding site (residues 195-221) drives enzymatic selectivity in terms of substrate size, and the histidine 203 from this region also assists the catalytic mechanism, possibly by acting as a secondary nucleophile or as an essential amino acid in a possible ``proton relay system.'' This site also contains the calcium co-ordination site, which bridges heparin to heparinase through calcium and may orient the substrate to the active site region. In conclusion, we propose that the heparin binding site (residues 195-221) and the basic residues (lysine 132, arginine 141) close to cysteine 135 together constitute a heparin binding domain in heparinase I. The heparin binding domain provides the necessary charge complementarity for very specific heparin binding on the one hand, while on the other it creates a basic environment for biasing the active site reactivity (Fig. 6).
Figure 6:
Schematic representation of the active
site of heparinase I. The figure shows a heparin tetrasaccharide (about
0.165 nm in length; Nieduszynski(1985)) bound to the 18-amino acid
heparin binding site. It is interesting to note that several secondary
structure prediction algorithms suggest that the 18-amino acid heparin
binding site contained the main -helix forming potential in
heparinase I, which is otherwise dominated by
-sheet forming
potential (Sasisekharan, 1991; Yoder et al., 1993a). It is
hypothesized that the core fold of heparinase I is dominated by
-sheets (as seen in pectate lyase; Yoder et al., 1993a)
and that the region around 195-221 forms a large loop region with
perhaps a partial
-helical secondary structure. Cysteine 135 and
histidine 203 are in close proximity to each other and to the hydrogen
(of the iduronate) marked by an arrow. Flanking these residues
(cysteine 135 and histidine 203) are the heparin binding consensus
sequences (lysine 198 to aspartic acid 204 and glutamic acid 207 to
aspartic acid 212), the calcium co-ordinating motif (valine 206 to
glycine 213), and lysine 132 and arginine 141 which together form the
heparin binding domain of heparinase I.