TY - JOUR
T1 - Contributions of the MMP-2 collagen binding domain to gelatin cleavage
T2 - Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides
AU - Xu, Xiaoping
AU - Wang, Yao
AU - Lauer-Fields, Janelle L.
AU - Fields, Gregg B.
AU - Steffensen, Bjorn
N1 - Funding Information:
This work was supported by NIH grants DE12818 and DE14236 to B.S. and CA77402 and CA98799 to G.B.F. We gratefully acknowledge Dr S. Weintraub, University of Texas Health Science Center at San Antonio Institutional Mass Spectrometry Laboratory (supported by NIH grant CA54174), for performing the mass spectrometric analyses. Drs I. Collier and G. Goldberg, Washington University School of Medicine, St. Louis, MO generously allowed the use of the MMP-2 plasmid p186, and Dr M. Hidalgo, John Hopkins University School of Medicine, kindly provided mouse tumor tissues for the experiments.
PY - 2004/6
Y1 - 2004/6
N2 - Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the ∼20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by ∼70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, ∼65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen α-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.
AB - Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the ∼20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by ∼70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, ∼65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen α-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.
KW - BSA, bovine serum albumin
KW - CBD, collagen binding domain
KW - Collagen binding domain
KW - Collagens
KW - DMSO, dimethyl sulfoxide
KW - DTT, dithiothreitol
KW - ELISA, enzyme-linked immunoabsorbance assay
KW - Gelatinase A
KW - MMP
KW - MMP-2
KW - Matrix metalloproteinase
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U2 - 10.1016/j.matbio.2004.05.002
DO - 10.1016/j.matbio.2004.05.002
M3 - Article
C2 - 15296945
AN - SCOPUS:3843059079
VL - 23
SP - 171
EP - 181
JO - Collagen and Related Research
JF - Collagen and Related Research
SN - 0945-053X
IS - 3
ER -