Title : Biochemical characterization of human
collagenase-3
Abstract :
- The cDNA of a novel matrix metalloproteinase , collagenase-3 ( MMP-13 ) has been isolated from a breast tumor library (Freije, J. M. P., Dicz-Itza, I. , Balbin , M., Sanchez, L. M., Blasco, R., Tolivia, J., and López-Otin , C. (1994) J. Biol
- Chem
- 269, 16766-16773), and a potential role in tumor progression has been proposed for this enzyme
- In order to establish the possible role of collagenase-3 in connective tissue turnover, we have expressed and purified recombinant human pro collagenase-3 and characterized the enzyme biochemically
- The purified pro collagenase-3 was shown to be glycosylated and displayed a M(r) of 60,000, the N-terminal sequence being LPLPSGGD, which is consistent with the cDNA-predicted sequence
- The pro enzyme was activated by p-aminophenylmercuric acetate or stromelysin, yielding an intermediate form of M(r) 50,000, which displayed the N-terminal sequence L58EVTGK
- Further processing resulted in cleavage of the Glu84-Tyr85 peptide bond to the final active enzyme (M(r) 48,000)
- Trypsin activation of pro collagenase-3 also generated a Tyr85 N terminus , but it was evident that the C-terminal domain was rapidly lost, and hence the collagenolytic activity diminished
- Analysis of the substrate specificity of collagenase-3 revealed that soluble type II collagen was preferentially hydrolyzed, while the enzyme was 5 or 6 times less efficient at cleaving type I or III collagen
- Fibrillar type I collagen was cleaved with comparable efficiency to the fibroblast and neutrophil collagenases ( MMP-1 and MMP-8 ), respectively
- Unlike these collagenases, gelatin and the peptide substrates Mea-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Mca- Pro- Cha- Gly-Nva-His-Ala-Dpa-NH2 were efficiently hydrolyzed as well, as would be predicted from the similarities between the active site sequence of collagenase-3 ( MMP-13 ) and the gelatinases A and B. Active collagenase-3 was inhibited in a 1:1 stoichiometric fashion by the tissue inhibitors of metalloproteinases, TIMP-1 , TIMP-2 , and TIMP-3
- These results suggest that in vivo collagenase-3 could play a significant role in the turnover of connective tissue matrix constituents
Output (sent_index, trigger,
protein,
sugar,
site):
- 5. glycosylated, , collagenase-3, -, -
Output(Part-Of) (sent_index,
protein,
site):
- 11. collagenase-3, sequence
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):