Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y
Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan.
J Biol Chem. 1997 Jan 24;272(4):2446-51. doi: 10.1074/jbc.272.4.2446.
Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.
膜型1基质金属蛋白酶（MT1-MMP）在癌细胞膜上表达，并激活MMP-2（明胶酶A）的酶原。我们最近分离出了与金属蛋白酶组织抑制剂2（TIMP-2）复合的MT1-MMP，并通过明胶酶谱法证明MT1-MMP具有明胶分解活性（今井启、大内悦子、青木隆、野村浩、藤井洋、佐藤浩、关树、冈田洋（1996年）《癌症研究》56卷，2707 - 2710页）。在本研究中，我们进一步将缺乏跨膜结构域的MT1-MMP缺失突变体（DeltaMT1）和从人乳腺癌细胞系（MDA-MB-231细胞）分泌的天然MT1-MMP纯化至同质，并检测了它们的底物特异性。这两种蛋白酶均具有活性，无需任何激活处理，并且能将I型（豚鼠）、II型（牛）和III型（人）胶原蛋白消化成特征性的3/4和1/4片段。I型胶原蛋白的切割位点，对于α1（I）链是Gly775-Ile776键，对于α2（I）链是Gly775-Leu776键和Gly781-Ile782键。DeltaMT1水解I型胶原蛋白的优先程度比II型或III型胶原蛋白高6.5倍或4倍，而MMP-1（组织胶原酶）消化III型胶原蛋白比另外两种胶原蛋白更有效。对DeltaMT1和MMP-1活性的定量分析表明，DeltaMT1切割I型胶原蛋白的效率低5 - 7.1倍。另一方面，DeltaMT1的明胶分解活性比MMP-1高8倍。DeltaMT1还能消化软骨蛋白聚糖、纤连蛋白、玻连蛋白和层粘连蛋白-1以及α1-蛋白酶抑制剂和α2-巨球蛋白。DeltaMT1与MMP-2共同孵育时，其对I型胶原蛋白的活性会协同增加。这些结果表明，MT1-MMP是一种与间质胶原酶具有相同底物特异性的细胞外基质降解酶，并提示MT1-MMP在细胞外基质的病理生理消化中通过直接切割底物和激活前MMP-2发挥双重作用。
