(Received for publication, July 17, 1996, and in revised form, December 20, 1996)
From the Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, 2199 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada
The binding properties of the COOH-terminal
hemopexin-like domain (C domain) of human gelatinase A (matrix
metalloproteinase-2, 72-kDa gelatinase) were investigated to determine
whether the C domain has binding affinity for extracellular matrix and
basement membrane components. Recombinant C domain (rC domain)
(Gly417-Cys631) was expressed in
Escherichia coli, and the purified protein, identified
using two antipeptide antibodies, was determined by electrospray mass
spectrometry to have a mass of 25,925 Da, within 0.1 Da of that
predicted. As assessed by microwell substrate binding assays and by
column affinity chromatography, the matrix proteins laminin, denatured
type I collagen, elastin, SPARC (secreted protein that is acidic and
rich in cysteine), tenascin, and MatrigelTM were not bound
by the rC domain. Unlike the hemopexin-like domains of collagenase and
stromelysin, the rC domain also did not bind native type I collagen.
Nor were native or denatured types II, IV, V, and X collagen, or the
NC1 domain of type VII collagen bound. However, binding to heparin and
fibronectin (Kd, 1.1 × 106
M) could be disrupted by 0.58-0.76 and 0.3 M
NaCl, respectively. Using nonoverlapping chymotrypsin-generated
fragments of fibronectin, binding sites for the rC domain were found on
both the 40-kDa heparin binding and the 120-kDa cell binding
fibronectin domains (Kd values, ~4-6 × 10
7 M). The Ca2+ ion, but not the
potential structural Zn2+ ion, were found to be essential
for maintaining the binding properties of the protein. The apo-form of
the rC domain did not bind heparin, and both ethylenediaminetetraacetic
acid and the specific Ca2+ ion chelator
1,2-bis(2-aminophenoxy)
ethane-N,N,N
,N
-tetraacetic acid, but not the
Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form
of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+
ion in the rC domain. In contrast, reduction with 65 mM
dithiothreitol did not interfere with heparin binding, further
emphasizing the crucial structural role played by the Ca2+
ion. Together, these data demonstrate for the first time that the
hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important
in maintaining the structure and function of the domain.
The matrix metalloproteinases (MMPs)1
constitute a family of proteolytic enzymes that together can degrade
all components of the extracellular matrix and basement membranes, with
each MMP having a distinct substrate preference (1, 2). MMP activity plays a major role during physiological and pathological processes, including embryogenesis, metastasis (3, 4), and inflammatory diseases
(5, 6). Most soluble MMPs are secreted as proenzymes and share
homologous primary and tertiary structures organized into distinct
structural domains, with some differences in domain composition and
number (6). These functionally and structurally defined domains include
the NH2-terminal zymogen domain containing the conserved
PRCGXPD motif involved in enzyme latency (7) and a Zn2+ and
Ca2+ ion binding catalytic domain. As with other
proteinases, the specificity of peptide bond cleavage is determined by
the S and S subsite defining amino acid residues (8). Equally
important are discrete substrate binding domains, or smaller functional modules, located outside of the active site, which form specialized exosites (9) to target proteolytic activity in tissues and are
essential for cleavage of some substrates. Immediately adjacent to the
catalytic site in gelatinase A (MMP-2, 72-kDa gelatinase) and
gelatinase B (MMP-9, 92-kDa gelatinase) is a fibronectin type II-like
module triple repeat (10, 11), which forms a collagen binding domain
(CBD) with strong affinity for elastin and denatured types I (12, 13),
IV, and V collagens, and native types I, V, and X collagens (9,
13),2 proteins degraded by the gelatinases.
Following the catalytic domain is a variably long linker, which in
collagenase-1 (MMP-1) may be important for triple helicase activity
(14). The linker connects to the COOH-terminal domain (C domain),
comprising four hemopexin-like modules possessed by all MMPs except
matrilysin (15). Three-dimensional structure analysis of the C domain
of gelatinase A (16, 17) and collagenase-1 (18) has revealed a
four-bladed
-propeller structure with a central Ca2+
ion, a potential Zn2+ ion binding site, and either a
Ca2+-Cl
(17) or a
Na+-Cl
(16) ion pair in the central
channel.
Even though the primary and tertiary structures of the MMP C domains share extensive homology, they possess a range of different properties that are MMP-specific. The C domain of gelatinase B binds the natural tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) (11, 19, 20) whereas the C domain of gelatinase A is involved in binding to TIMP-2 (21) and to cell membranes (9, 22) on concanavalin A (ConA) activated normal cells (23) and tumor cells (24, 25). In addition, latent gelatinase A, in complex with TIMP-2, is activated on interaction with active membrane type MMPs (MT-MMPs) (26). Deletion mutants have demonstrated the requirement of the C domain of gelatinase A for cell binding and activation of the latent enzyme on cell surfaces (27), whereas the C domain of the collagenases mediates substrate specificity for native, triple helical type I collagen. In the absence of the C domain, the catalytic domain alone of collagenase-1 (28, 29) and neutrophil collagenase (30, 31) cleaves synthetic peptides and casein, but not native collagen. Indeed, the C domain alone of collagenase-1 binds native type I collagen (32-34), as can the C domain of stromelysin-1 (33, 34), even though collagen is not cleaved by stromelysin-1. In contrast, neither the activity nor the substrate specificity of the stromelysins is dramatically modified by the removal of the C domain (35-38). Likewise, the gelatinases degrade gelatin and native type IV collagen in a manner that appears independent of the presence of the C domain (39).
The gelatinases also efficiently degrade fibronectin, native types V, VII, and X collagen, and elastin (40-42). However, the importance of the C domain for these activities is unknown. Indeed, other than for elastin, native types I and V collagen, and gelatins that are bound by the CBD (12, 13, 42), exosites conferring specificity for these substrates have yet to be identified. The gelatinases may depend on the C domain for binding fibronectin or native type VII and X collagens as a requisite for efficient catalysis and triple helicase activity, but this has not been directly tested. In the present study we investigated the potential binding properties of a recombinant C domain of human gelatinase A for a number of extracellular matrix and basement membrane components. We found that the C domain exhibits strong binding properties for fibronectin and heparin and that the binding is dependent on the structural Ca2+ but not Zn2+ ion in the C domain.
Human types IV and V collagen
(non-pepsin-treated) were from Becton Dickinson and Life Sciences; type
X collagen and elastin were from Sigma; fibronectin
fragments (40 and 120 kDa) and laminin were from Life Sciences;
MatrigelTM was obtained from Beckton Dickinson; tenascin
was from Chemicon and Life Sciences; Affi-Gel heparin, Affi-Gel 10, and
10-DG columns were from Bio-Rad; chelating Sepharose 6B,
gelatin-Sepharose 4B, heparin-Sepharose CL-6B, and CM-Sepharose Fast
Flow were from Pharmacia Biotech Inc.; G-10 Sephadex, 1,2-bis(2-amino
phenoxy)-ethane-N,N,N,N
-tetraacetic acid
(BAPTA), and keyhole limpet hemocyanin were from Sigma; and Freund's
complete and incomplete adjuvants were from Calbiochem. Human
gelatinase A cDNA was a kind gift from Dr. K. Tryggvason (Karolinska Institute, Stockholm, Sweden) (43) secreted protein that is
acidic and rich in cysteine (SPARC) was kindly provided by Dr. J. Sodek
(University of Toronto, Toronto, Ontario Canada); the NC1 domain of
type VII collagen was generously provided by Dr. P. Rousselle (Centre
National de la Recherche Scientifique, Lyon, France); and type II
collagen was a gift from Dr. J. Mort (Shriners Hospital, Montreal,
Quebec, Canada).
Acid-soluble native type I collagen was prepared from rat tail tendons by extraction with 0.5 M acetic acid and purified by differential precipitation with 1.7 M NaCl (44). Fibronectin was affinity purified from bovine serum by binding to gelatin-Sepharose 4B and elution with a 0-10% Me2SO gradient (45). The Me2SO was then removed from the fibronectin using a 10-DG column. Purity was confirmed by SDS-PAGE analysis under reducing and nonreducing conditions.
Polyclonal Antipeptide AntibodiesRabbit polyclonal
His6 and
72ex12 antipeptide antibodies were raised
against a peptide present in the polyHis fusion tag and from part of a
surface exposed
-strand of hemopexin module III of the gelatinase A
C domain, respectively. Synthetic peptides were purified by high
performance liquid chromatography before coupling to keyhole limpet
hemocyanin (46). After emulsifying in Freund's complete adjuvant 1 mg
of each peptide was injected subcutaneously into two rabbits each.
Three to four biweekly boosts of peptide in incomplete Freund's
adjuvant were administered intramuscularly. Whole serum was
affinity-purified against peptide coupled to Affi-Gel 10 and
quantitated by enzyme-linked immunosorbent assay.
The 5- and 3
-boundaries of the cDNA encoding the
C domain and linker of gelatinase A
(Gly417-Cys631) were defined by the 5
-boundary
of exon 9 and the natural stop codon in exon 13, respectively. The
cDNA was polymerase chain reaction-amplified from the full-length
cDNA of human gelatinase A using the primers
5
-GCTAGCTAGCGGGGCCTCTCCTGACATT-3
and
5
-CTTAAGCTTCAGCAGCCTAGCCAGTC-3
, which added an NheI site
and a BamHI site in the 5
- and 3
-extensions, respectively.
The purified 688-base pair product was digested with NheI
and BamHI and ligated into the expression vector pGYMX (13),
which expresses recombinant protein with a short
NH2-terminal fusion tag comprising an initiation
methionine, a His6 tag, and a factor Xa
cleavage site (13). Sequencing (47) of the plasmid, pGYMX9-13,
confirmed the fidelity and reading frame of the cDNA.
E. coli strains were screened for recombinant protein expression after growth in 1% (w/v) tryptone (Becton Dickinson), 0.8% (w/v) yeast extract (Becton Dickinson), 0.5% (w/v) NaCl, and 100 µg/ml ampicillin, pH 7.4. Log phase seed cultures were used to inoculate either 35- or 60-liter Chemap fermentor cultures, which were then grown under controlled constant temperature and aeration conditions for 24 h. Collected cells were washed and then lysed, inclusion bodies were dissolved in guanidinium-HCl, and recombinant protein was refolded prior to purification using procedures slightly modified (48) from those previously demonstrated (13) to produce correctly folded, disulfide cross-linked monomeric recombinant protein. In brief, disulfide bond exchange was performed in 0.1 M sodium borate, pH 10, under highly aerated conditions for 2 h, and the guanidinium was then slowly removed by step dialysis to refold the protein.
Purification of the Recombinant C DomainRefolded protein was loaded on a Zn2+-charged chelating Sepharose 6B Column (Vt, 20 ml). After extensive washes with chromatography buffer (100 mM sodium dibasic phosphate buffer, 0.5 M NaCl, pH 8.0), nonspecifically bound bacterial proteins were eluted with a step pH gradient (pH 8.0-6.0) in 1.0 M NaCl. Recombinant protein was then eluted with a 0-400 mM imidazole gradient developed over 100 ml, and the pooled fractions buffer was exchanged to 50 mM Tris-HCl, pH 7.4, by gel filtration prior to snap freezing in liquid N2.
SDS-PAGE and EnzymographyHeat-denatured protein samples were separated under reducing (65 mM DTT) or nonreducing conditions by SDS-PAGE according to the method of Laemmli (49) using 15% polyacrylamide gels. Protein bands were stained with Coomassie Brilliant Blue R-250. Samples analyzed by enzymography were electrophoresed nonreduced on 10% polyacrylamide gels copolymerized with 1 mg/ml gelatin according to the method of Overall and Limeback (50).
Immunoblot AnalysisTo confirm the identity of the purified
protein, reduced and nonreduced recombinant protein was blotted onto a
polyvinylidene difluoride membrane (Millipore), reacted with
affinity-purified His6 or
72ex12 antibodies, and then
detected with peroxidase-conjugated goat anti-rabbit antibody and
enhanced chemiluminescence reagents (Amersham Corp.) on Kodak SB-5 film
(Eastman Kodak Co.).
The mass of the rC domain was measured by electrospray mass spectrometry using a PESCIEX API 300 spectrometer after sample injection on a C18 high performance liquid chromatography column at 50 µl/min. The Zn2+ ion content of the rC domain was measured three times by inductive coupled plasma mass spectrometry using 8.6-10 mg protein per analysis on a Sola (Finnigan-MAT) spectrometer.
Microwell Substrate Binding AssayBinding of the rC domain
to extracellular matrix proteins and substrates of gelatinase A was
determined using an enzyme-linked immunosorbent type assay (13).
Proteins tested were soluble fibronectin, laminin, SPARC, tenascin,
native and denatured types I, II, IV, V, and X collagens, the NC1
domain of type VII collagen, and reconstituted basement membrane
MatrigelTM. Denatured collagens were prepared by heat
denaturation at 56 °C for 30 min. Ovalbumin was used as a negative
control. To map C domain binding sites on fibronectin, nonoverlapping
40- and 120-kDa chymotrypsin fragments of fibronectin were used.
Proteins (0.5 µg) in 15 mM
Na2CO3, 35 mM NaHCO3,
and 0.02% (w/v) NaN3, pH 9.6, were coated on 96-well
microtiter plates for 18 h at 4 °C. Consistent and equal
coating of test substrates in this assay has been previously confirmed
(13). Wells were then blocked with 2.5% (w/v) ovalbumin, and serially
diluted rC domain was added (1024 to 4 pmol/100 µl (10.24 µM to 40 nM) in 20 mM Tris-HCl,
pH 7.4, for 1 h. Extensive washes with phosphate-buffered saline,
0.02% Tween 20 (v/v) followed, and the bound rC domain was then
quantitated using either the His6 or the
72ex12
antibodies followed by incubation with goat anti-rabbit alkaline
phosphatase-conjugated secondary antibody and p-nitrophenyl
phosphate disodium as substrate. Assays were performed at least in
duplicate per plate, and data were only compared for experiments on the
same plate. All experiments were repeated six times, except for type II
and X collagens, the type VII collagen NC1 domain, SPARC, and tenascin,
which were performed in quadruplicate. Specificity was confirmed by
comparing the binding of the rC domain with another recombinant domain
from gelatinase A, the fibronectin type II-like triple repeat
previously designated the collagen binding domain (13). The rCBD
contains the same fusion tag and was expressed in the same E. coli strain used for rC domain expression.
Binding properties of the rC domain were also determined by affinity chromatography. Minicolumns containing either gelatin-Sepharose 4B, elastin/Sephadex G-10, heparin-Sepharose CL-6B, fibronectin coupled to gelatin-Sepharose 4B, or fibronectin coupled to Affi-Gel 10 (Vt, 100, 200, 100, 70, and 50 µl, respectively) were used in 50 mM Tris-HCl with or without 0.15 M NaCl, pH 7.4, chromatography buffer as appropriate (51). To ensure saturation of binding sites on the affinity matrix, a standardized quantity (50 µg, 2 nmol) of the rC domain was loaded onto the columns, which would result in excess protein being recovered in the unbound and wash fractions. To confirm binding to affinity matrices, any rC domain recovered in the unbound material was reapplied to another column of the affinity matrix, and binding was compared. Binding of the rC domain was typically assessed by step elution with NaCl to 1.0 M followed by a step gradient of Me2SO (1-10%) in chromatography buffer. Chromatography fractions were analyzed by SDS-PAGE using 15% gels. Affinity chromatography experiments were performed at least six times for each matrix. FPLC was also performed using both heparin-Sepharose and Affi-Gel heparin 1.0-ml columns. After sample loading, a 0-1.0 M NaCl gradient was developed over 10 ml at 1.0 ml/min.
Chelation ExperimentsTo assess the role of the structural divalent cations on the binding of the rC domain to heparin and fibronectin, chelators (EDTA, 1,10-phenanthroline, EGTA, and BAPTA) were used to attempt elution from these affinity matrices. To confirm the elution by chelators, columns were then eluted with 1.0 M NaCl to recover any remaining bound rC domain. The apo rC domain was prepared before Affi-Gel heparin chromatography by chelation of structural Ca2+ ions for 1 h by 50 or 100 mM EDTA followed by desalting on a Sephadex G-10 spun column.
To
investigate the properties of the hemopexin-like C domain of human
gelatinase A, recombinant protein encoded by exons 9-13 of gelatinase
A (Gly417-Cys631) was expressed in E. coli. The purified rC domain electrophoresed as a single band on
15% SDS-PAGE gels with an apparent molecular mass of ~26.5 kDa under
reducing conditions (Fig. 1A). The absence of
intermolecular disulfide linked multimeric forms of the recombinant protein was evident following electrophoresis under nonreducing conditions (Fig. 1A). The downshift in apparent molecular
mass (0.8 kDa) for nonreduced samples indicated the presence of an intact disulfide bond between Cys440 and Cys631
(20) within the protein. The precise mass of the rC domain was measured
to be 25,925.0 Da by electrospray mass spectrometry, within 0.1 Da of
the predicted mass of a NH2-terminal methionine-processed form of the recombinant protein (25, 924.9 Da), confirming the fidelity
of correct expression. Immunoreactivity with two affinity purified
antipeptide antibodies,
72ex12 (Fig. 1B) and
His6 (data not shown), further verified the identity of
the purified protein. Importantly, essentially no disulfide
cross-linked multimeric forms were observed on the immunoblots even
when the ECL reaction was allowed to proceed well beyond the linear
range.
Characterization of rC Domain Binding to Fibronectin, Laminin, SPARC, and Tenascin
Binding of the rC domain to basement membrane
components was quantitated by the microwell substrate binding assay.
Although the rC domain did not bind MatrigelTM and laminin
(Fig. 2A) or SPARC and tenascin (not shown),
saturable binding to fibronectin in either NaCl-free 50 mM
Tris buffer or phosphate-buffered saline was found, with an apparent
Kd of 1.1 × 106 M. A
recombinant collagen binding domain (13) from gelatinase A with the
identical fusion tag did not bind fibronectin (Fig. 2B),
further confirming the specificity of the rC domain interaction. Using
nonoverlapping chymotrypsin-generated fragments of fibronectin, binding
sites for the rC domain were found on both the 40-kDa heparin binding
and the 120-kDa cell binding fibronectin fragments with a similar but
slightly stronger (apparent Kd values, 4 × 10
7 and 6 × 10
7 M,
respectively) affinity than for intact fibronectin (Fig.
3).
To further investigate the binding of the rC domain to fibronectin, the
recombinant protein was applied to minicolumns of fibronectin coupled
to gelatin-Sepharose columns in chromatography buffer containing 0.15 M NaCl. After adding the rC domain to saturation, as
determined by the eventual detection of the recombinant protein in the
unbound fraction (Fig. 4A, U), the
bound rC domain was step eluted with 0.3-0.5 M NaCl in 50 mM Tris-HCl, pH 7.4. No further rC domain was recovered
with 1.0 M NaCl or when Me2SO was used to elute
the gelatin-bound fibronectin (not shown). That the rC domain did not
bind to the gelatin-Sepharose was confirmed in control experiments
(Fig. 4B). Identical elution profiles were obtained when the
rC domain was loaded in chromatography buffer with NaCl omitted (data
not shown). To ensure that the small amount of unbound rC domain did
not represent a misfolded form of the protein, this material was
reapplied to another fibronectin affinity column. Fibronectin binding
was again demonstrated (data not shown), confirming that the unbound rC
domain was the result of overloading after saturation of fibronectin
binding sites.
rC Domain Interaction with Type I, II, IV, V, VII, and X Collagens and Elastin
Although the C domains of collagenase-1 and
stromelysin-1 bind native type I collagen, there was no binding of the
gelatinase A rC domain to either native or denatured type I collagen in
the microwell substrate binding assay compared with the negative
control ovalbumin (Fig. 5A). The rC domain
also did not bind gelatin-Sepharose (Fig. 4B), being
quantitatively recovered in the unbound and wash fractions with no
protein recovered in any elutes. In contrast, the rCBD from gelatinase
A, used as a positive control for collagen interaction, showed
avid binding to both forms of type I collagen (Fig. 5A).
To determine whether the gelatinase A C domain contributes to the
binding specificity for other substrates of the enzyme, the rC domain
was tested for interaction with types IV and V collagen and elastin. As
for type I collagen, the rC domain did not bind these collagens in
either their native or denatured forms (Fig. 5B). Native and
denatured types II and X collagen and the NC1 domain of type VII
collagen were also not bound by the rC domain (not shown). In
confirmation of these data using the His6 antibody, identical results were also obtained with the
72ex12 antibody (not
shown). rC domain binding to elastin was assessed using 6 mg of
insoluble elastin mixed with 100 µl of Sephadex G-10. All the loaded
rC domain protein was recovered in the unbound and first wash
fractions, and none was eluted with NaCl or Me2SO (data not
shown). This indicated that the rC domain does not contribute to the
elastin binding properties of the enzyme that are localized to the
fibronectin type II-like CBD of gelatinase A (13) and shown to be
essential for elastinolysis by gelatinase B (42).
Heparin-Sepharose minicolumns
and FPLC columns were used to investigate the binding properties of the
C domain of gelatinase A to the heparan sulfate component of basement
membranes and cell surface proteoglycans. The bound rC domain was step
eluted off heparin-Sepharose minicolumns with 0.5 M NaCl
(Fig. 6A) and at 0.58 M NaCl on a
NaCl gradient developed on a 1.0-ml heparin-Sepharose FPLC column (data
not shown). Taking into consideration the calculated lag time for
protein elution off the 1.0-ml column, the results of the minicolumn
and FPLC were in accordance. Binding to Affi-Gel heparin was
consistently stronger, requiring 0.76 M NaCl for peak elution (Fig. 7A). Heparin specificity was
confirmed by the absence of binding to the negatively charged
CM-Sepharose and that the unbound overloaded protein from the
minicolumns could bind heparin-Sepharose on subsequent chromatography
(data not shown).
To compare the heparin binding properties of the rC domain with
gelatinase A, active enzyme was obtained from confluent cell cultures
treated for 18 h with 20 µg/ml ConA (23) and then
chromatographed over heparin-Sepharose minicolumns. Similar to the rC
domain and confirming our previous studies (23, 51), the activated
gelatinase A was also eluted off the column by 0.5 M NaCl
(Fig. 6B). Previously we have shown that the gelatinase A
CBD has a low affinity heparin binding site (13), and our recent
mutagenesis studies3 have identified
Lys357 as an essential residue in this site. Therefore, to
compare the relative importance of this and other possible heparin
binding sites on gelatinase A, active enzyme was loaded onto
heparin-Sepharose, and competition was attempted with >20-fold molar
excess rC domain added before elution was continued. Since only a small
amount of the bound enzyme could be competed off the column by the rC domain, with the bulk of the enzyme being recovered in the 0.5 M NaCl elute (Fig. 6C), this indicated that
other heparin binding sites on gelatinase A remained associated with
the heparin. Since the previously described "mini-gelatinase,"
representing a ConA-induced processed form of gelatinase A lacking the
C domain (23, 52) (Fig. 6, B and C,
C-Gs), was predominantly but not quantitatively recovered
in the unbound and wash fractions, this reveals the importance of the C
domain heparin binding site relative to the other heparin binding sites
on human gelatinase A.
The structural
importance of the Cys440-Cys631 disulfide bond
for heparin binding was examined after treatment of the rC domain with
65 or 130 mM DTT for 60 min prior to heparin-Sepharose
chromatography. All minicolumn solutions also included freshly prepared
65 mM DTT to ensure that reducing conditions were
maintained throughout chromatography. As shown in Fig.
8A, there was no change in the heparin-Sepharose elution profile of the reduced rC domain compared with unreduced samples (see Fig. 6A). When more precisely
analyzed by Affi-Gel heparin FPLC, the reduced rC domain (130 mM DTT; Fig. 7B) was found to possess
essentially identical elution properties as the unreduced protein (Fig.
7A) at 22 °C.
The recently published three-dimensional structures of the collagenase
(18) and gelatinase A C domains (16, 17) show a divalent ion, modeled
as a Ca2+ ion, in the central channel of the four-bladed
-propeller structure, together with either a
Na+-Cl
(16) or
Ca2+-Cl
(17) ion pair. In addition, a
potential Zn2+ ion was also modeled in hemopexin module IV
(16). In support of an important structural and/or functional role for
the Ca2+ ions and a possible Zn2+ ion for
binding of the rC domain to matrix components, the apo-rC domain was
found to have lost binding potential for Affi-Gel heparin (Fig.
7C) and heparin-Sepharose (not shown) on FPLC. The loss of
binding properties on removal of the structural Ca2+ was
also confirmed by the elution of the heparin-Sepharose-bound holo-rC
domain by 50 mM EDTA on minicolumns (Fig. 8B).
Elution was quantitative, with no further protein recovered by 100 mM EDTA or in the subsequent 0.5 M NaCl
elution. Lower concentrations of EDTA (5, 10, and 20 mM)
did not elute the bound rC domain (data not shown), suggesting a tight
coordination of the divalent cations with the protein. Moreover, the rC
domain treated with 15 mM EDTA prior to FPLC over Affi-Gel
heparin retained heparin binding and elution properties identical to
that of the holo protein (data not shown).
Since EDTA
is a chelator of several divalent cations, including Ca2+
and Zn2+, more specific chelators were used to identify the
important structural ions in the rC domain. The well characterized
Zn2+ ion chelator 1,10-phenanthroline had no apparent
effect on the binding of the rC domain to heparin, with all bound
protein being recovered with the final 0.5 M NaCl elution
(Fig. 9A). Thus, either no Zn2+
ions are normally ligated to the C domain, or if present, then chelation by 1,10 phenanthroline does not alter the binding properties of the protein. Moreover, inductive coupled plasma mass spectrometry analysis of the rC domain failed to detect a biologically significant Zn2+ ion content in the protein (8.5 × 106 mol of Zn2+/mol of rC domain).
Surprisingly, 100 mM EGTA was ineffective in eluting the rC domain from heparin-Sepharose (Fig. 9B). This was confirmed by Affi-Gel heparin FPLC analysis in which elution was achieved only at 180-200 mM EGTA (not shown). Since EGTA is a Ca2+ and Mg2+ ion chelator, this result was paradoxical. However, several Ca2+ binding proteins are known to directly bind EGTA (53) due to the high electronegativity of the chemical. Therefore, to confirm the identity of the central Ca2+ ion, a newer and one of the most highly specific chelators of Ca2+ ions, BAPTA, was used. Low concentrations of BAPTA were ineffective in eluting rC domain (1 and 5 mM), but at 20 mM BAPTA (Fig. 9C) the bound rC domain was quantitatively eluted from the heparin-Sepharose. No further protein was recovered with 0.5 M NaCl (cf. 1,10-phenanthroline and EGTA; Fig. 9, A and B), confirming the importance and strength of association of the Ca2+ ion.
The rC Domain also Binds Fibronectin in a Ca2+ Ion-dependent MannerTo determine whether the
structural Ca2+ ion has a similar influence on the binding
properties of the C domain for fibronectin, minicolumns of fibronectin
coupled to gelatin-Sepharose were loaded with the rC domain. Both 50 mM EDTA (Fig. 10A) and 20 mM BAPTA (Fig. 10B) quantitatively dissociated
the rC domain from the fibronectin, with no further rC domain being
recovered with 1.0 M NaCl. Overall, these experiments
exclude the possibility of a structurally important Zn2+
ion in the rC domain, chemically identify the essential divalent cation
in the rC domain as a Ca2+ ion, indicate the strength of
the Ca2+ ion coordination with the protein, and reveal its
important role in defining the binding properties of the rC domain to
heparin and fibronectin.
Reported here are the fibronectin and heparin binding properties
of the COOH-terminal hemopexin-like domain of human gelatinase A, the
first such report for any of the MMPs. Fibronectin is a complex
mutidomain matrix glycoprotein with multiple binding sites for
extracellular matrix components and cell membrane proteins. Indeed, the
rC domain binds two nonoverlapping chymotrypsin-generated fragments of
fibronectin with similar Kd values
(107 M range). The display of binding sites
on fibronectin is conformation-dependent, with some cryptic
sites being exposed on denaturation, surface binding, or cleavage (54),
possibly explaining the slight difference in binding strength of the rC
domain to the intact protein (apparent Kd, 1.1 × 10
6 M) compared with the 40- and 120-kDa
fibronectin fragments. Fibronectin and its alternately spliced variants
also occur as matrix-bound and soluble forms in serum. Accordingly, the
exact binding stoichiometry and dynamics of the gelatinase A C domain
association with fibronectin in tissues is likely a complex
interaction. Thus, although these experiments directly revealed the
presence of multiple potential gelatinase A binding sites on the
fibronectin chains, these sites may not always be available for
concurrent occupancy due to structural differences between the intact
tertiary fold of the protein and the cleavage fragments or to steric
clashes, which may prevent multiple binding.
Fibronectin cleavage by gelatinase A results in prominent degradation fragments (40) that include the amino- and carboxyl-terminal fibrin binding domains and the larger central cell binding domain (54). Other fibronectin-degrading MMPs include the stromelysins, matrilysin, and metalloelastase (2), but as with gelatinase A, their fibronectin-degradative activities have received little study. With the exception of matrilysin, a fibronectin binding site might also be located on the C domain of these MMPs. Fibronectin binding may be a requisite for efficient catalysis, but previous C domain deletion mutant studies of gelatinase A (27, 55) or other MMPs (39, 55) did not investigate fibronectin binding or degradation. Fibronectin binding might also represent another means by which gelatinase A can bind cell surfaces. This may produce a reservoir of latent gelatinase A in proximity to the cell surface for eventual interaction with TIMP-2 and its receptor (56), poised for cis activation by MT-MMP-expressing cells (26, 57).
Gelatinase A also degrades native type IV basement membrane collagen and is considered pivotal for basement membrane remodeling during embryogenesis4 and in tumor cell invasion and metastasis (58). However, the rC domain did not bind basement membrane components either individually (type IV collagen, tenascin, laminin, and SPARC) or in combination as reconstituted basement membrane (MatrigelTM) or to the NC1 domain of anchoring fibril type VII collagen. This is consistent with the findings of others that C domain deletion mutants of gelatinase A did not bind (55) but still degraded (27) native type IV collagen and laminin. The rC domain also did not bind native type I, V, or X collagens, which are degraded by gelatinase A, or other collagens such as type II, which are not. Thus, the C domain of gelatinase A differs from those of the collagenases and stromelysins, which bind native type I collagen (32, 35-37, 55) and which, for the collagenases, are absolutely essential for collagenolytic activity (28-31). This indicates that the molecular mechanism involved in the triple helicase activity of gelatinase A for native type I, IV, V, and X collagens is fundamentally different from that of the collagenases. Indeed, a C domain deletion mutant of gelatinase A showed only slight alteration in type IV collagen cleavages (27). Alternatively, the collagen binding properties of the CBD of gelatinase A may compensate for the absence of collagen tethering by the C domain to potentiate triple helicase activity. Nonetheless, this does not explain the mechanism of type IV collagenolytic activity, since the isolated CBD does not bind native type IV collagen (13).
The heparin binding properties of gelatinase A have previously been
described (51, 59) and shown to be important for enzyme activation
(59). A gelatinase A heparin binding site has been previously located
within the CBD (13).3 The heparin binding site located in
the C domain is specific, since the rC domain did not bind and was only
slightly retarded by interaction with the negatively charged
CM-Sepharose. Although no match to the canonical heparin binding
consensus sequences (XBBXBX and
XBBBXXBX; B, basic amino acids;
X, undefined) (60) is found in the C domain, a site that
matches the XBBXBX site in reverse
(SKNKKT) and a sequence with high similarity (VKKKMDG) are
located in the lysine- and arginine-rich hemopexin-like module III.
However, competition experiments using an antipeptide antibody raised
against this sequence (
72ex12) had no effect on the binding of the
rC domain to heparin (data not shown). These and other positively
charged clusters within hemopexin-like modules III and IV are now being
studied by site directed mutagenesis to identify the binding sites for
heparin and the negatively charged COOH-terminal peptide of
TIMP-2.5 Thus, heparan sulfate binding to
the C domain may alter TIMP-2 interaction, which could in turn modulate
cell surface activation of gelatinase A.
The Ca2+ ion dependence of the C domain interaction with
fibronectin and heparan sulfate proteoglycans points to a new potential therapeutic target for gelatinase A. Confirmation of the importance and
identity of the structural Ca2+ ions was shown in the apo
form of the rC domain by disruption of the structural properties
important for ligand binding. Chelation with EDTA and BAPTA, a new
derivative of EGTA that is a highly specific Ca2+ chelator
(61) but which has greatly improved rate constants and pH insensitivity
over EGTA (61, 62), also eluted the bound holo-rC domain from heparin.
The unexpected results obtained with EGTA may be due to direct
association of EGTA with the protein (53) or the neutral pH used. At pH
7, EGTA occurs as a dianion, which results in a reduced ability to bind
Ca2+ ions by 2 to 3 orders of magnitude compared with pH
8.6 (61). Together, these effects minimize the effective chelating
capacity of the free EGTA for the heptacoordinated Ca2+
ion. Therefore, the chelation results obtained with BAPTA were important in clearly establishing the identity and importance of the
Ca2+ ions in the rC domain. The chelation experiments
further revealed the tight coordination of the Ca2+ ions in
the protein, supporting the conclusion that the Ca2+ ions
play an essential stabilizing role in the domain, possibly acting like
a hub to centrally pin the four -blades together. Last, since
inductive coupled plasma mass spectrometry analysis did not measure any
Zn2+ ions in the rC domain and 1,10-phenanthroline did not
alter the binding properties of the rC domain, these data failed to
confirm the presence of a structural Zn2+ ion in the C
domain of gelatinase A. Together, these data indicate that the
Zn2+ ion modeled in a potential binding site on module IV
of the C domain was likely an artifact of crystallization in 150 mM Zn2+ acetate (16).
The central Ca2+ ion was also more effective than the
disulfide bond in maintaining the structural integrity of the rC domain at 22 °C. Of note, these chromatography experiments were performed entirely under reduced conditions and not with reduced and alkylated protein. The introduction of blocking groups or a charged moiety during
alkylation after reduction and protein denaturation by 6 M
guanadinium or by heat can alter the structural properties of the
protein distinct from those due to disulfide bond reduction alone.
Indeed, reduced and alkylated rCBD from gelatinase A does not bind
gelatin (13, 63), whereas rCBD protein chromatographed and analyzed
under continuous reducing but nondenaturing conditions retains gelatin
interaction equal to nonreduced rCBD (13). Thus, these experiments
indicate that the integrity of the four-bladed -propeller structure
of the rC domain was not markedly perturbed at 22 °C by reduction of
the disulfide bond but was altered sufficiently by chelation and loss
of the structural Ca2+ ions to disrupt the heparin and
fibronectin binding sites.
The widespread and near constitutive expression of gelatinase A
indicates that the most important level of regulation of gelatinase A
activity may be cellular activation and inhibition by TIMPs rather than
by regulated transcription (1, 23, 65). In this regard, we have
previously proposed that latent gelatinase A may bind native type I
collagen through the CBD (13) and so may remain localized in tissues,
poised for proteolysis of gelatin on collagenase cleavage of the native
collagen and trans activation by other MT-MMP-expressing
cells. A similar reservoir of enzyme may also be important for
elastinolysis, since binding to elastin also occurs through the CBD of
gelatinases (13, 42).2 The binding of rC domain to
fibronectin and potentially to heparan sulfate proteoglycans reveals
additional extracellular or pericellular matrix components that may
serve as anchors to sequester gelatinase A in tissues and to the cell
surface. This may render the enzyme readily accessible to MT-MMPs for
activation and to substrates, such as fibronectin and proteoglycan core
proteins, on activation for cleavage. Together with the potential for
the CBD to bind pericellular collagen,3 this also provides
a novel additional mechanism for cell surface interaction that may
complement those involving v
3-integrin, MT-MMPs, and TIMP-2 receptors.
We thank Dr. Stephen Withers for access to the electrospray mass spectrometer, Dr. Herman Ziltner for helpful discussions designing the antipeptide antibodies, and Dr. B. Steffensen for providing the rCBD protein.