Knäuper V, López-Otin C, Smith B, Knight G, Murphy G
Strangeways Research Laboratory, Department of Cell and Molecular Biology, Worts' Causeway, Cambridge, United Kingdom.
J Biol Chem. 1996 Jan 19;271(3):1544-50. doi: 10.1074/jbc.271.3.1544.
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 procollagenase-3 and characterized the enzyme biochemically. The purified procollagenase-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 proenzyme 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 procollagenase-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.
一种新型基质金属蛋白酶——胶原酶-3（MMP-13）的互补DNA（cDNA）已从一个乳腺肿瘤文库中分离出来（弗雷耶，J.M.P.，迪茨-伊察，I.，巴尔宾，M.，桑切斯，L.M.，布拉斯科，R.，托利维亚，J.，以及洛佩斯-奥廷，C.（1994年）《生物化学杂志》269卷，第16766 - 16773页），并且有人提出这种酶在肿瘤进展中可能发挥作用。为了确定胶原酶-3在结缔组织更新中的可能作用，我们表达并纯化了重组人原胶原酶-3，并对该酶进行了生化特性分析。纯化后的原胶原酶-3显示为糖基化形式，分子量为60,000，其N端序列为LPLPSGGD，这与cDNA预测的序列一致。该酶原被对氨基苯汞乙酸盐或基质溶解素激活，产生一种分子量为50,000的中间形式，其N端序列为L58EVTGK。进一步的加工导致Glu84 - Tyr85肽键断裂，生成最终的活性酶（分子量48,000）。胰蛋白酶对原胶原酶-3的激活也产生了一个Tyr85的N端，但很明显C端结构域迅速丢失，因此胶原分解活性降低。对胶原酶-3底物特异性的分析表明，可溶性II型胶原优先被水解，而该酶在切割I型或III型胶原时效率低5至6倍。纤维状I型胶原的切割效率分别与成纤维细胞胶原酶和中性粒细胞胶原酶（MMP-1和MMP-8）相当。与这些胶原酶不同的是，明胶以及肽底物Mea-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2和Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2也能被有效水解，这正如从胶原酶-3（MMP-13）与明胶酶A和B的活性位点序列相似性所预测的那样。活性胶原酶-3以1:1的化学计量比被金属蛋白酶组织抑制剂TIMP-1、TIMP-2和TIMP-3抑制。这些结果表明，在体内胶原酶-3可能在结缔组织基质成分的更新中发挥重要作用。
