The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1)
| Autor: | Dieter Moosmayer, Peter Angel, Ralph Müller, Elke Gerlach, Rüdiger Vallon |
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| Rok vydání: | 1997 |
| Předmět: |
Mutant
Gelatinase A Sf9 Matrix metalloproteinase Biology Matrix Metalloproteinase Inhibitors Spodoptera Biochemistry Catalysis medicine Animals Humans Protease Inhibitors Amino Acid Sequence Collagenases Cells Cultured Glycoproteins Binding Sites Hemopexin Tissue Inhibitor of Metalloproteinases Trypsin Recombinant Proteins Protein Structure Tertiary Enzyme Activation Collagenase Matrix Metalloproteinase 1 medicine.drug Binding domain |
| Zdroj: | European journal of biochemistry. 244(1) |
| ISSN: | 0014-2956 |
| Popis: | Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active-site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)-MMP-1 proteins are able to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1. |
| Databáze: | OpenAIRE |
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