Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.
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 approximately 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 approximately 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, approximately 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 alpha-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.
Xu
Xiaoping
X
Departments of Periodontics and Biochemistry MC 7894, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Wang
Yao
Y
Lauer-Fields
Janelle L
JL
Fields
Gregg B
GB
Steffensen
Bjorn
B
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology; D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009368:Neoplasm Transplantation; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins; D013379:Substrate Specificity
D024042:Collagen Type I; D010455:Peptides; D011994:Recombinant Proteins; D005780:Gelatin; D020778:Matrix Metalloproteinase 2