Kolkenbrock H, Hecker-Kia A, Orgel D, Ulbrich N, Will H
Deutsches Rheumaforschungszentrum Berlin, Germany.
Biol Chem. 1997 Feb;378(2):71-6. doi: 10.1515/bchm.1997.378.2.71.
C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.
C 端截短的膜型 2 基质金属蛋白酶（MT2-MMP1-269），包含前结构域和催化结构域，在大肠杆菌中表达为可溶性蛋白。与可分离为 31 kDa 蛋白的相应形式的 MT1-MMP 不同，MT2-MMP1-269 相对不稳定，刚分离的制剂在 SDS-PAGE 中就显示出几种代表 MT2-MMP1-269（33 kDa）的蛋白以及四种 N 端截短形式，其 N 端分别为甲硫氨酸 32（30 kDa）、异亮氨酸 37（30 kDa）、亮氨酸 84（24 kDa）和亮氨酸 93（22 kDa），即催化结构域。冷冻制剂解冻后，33 kDa 和 30 kDa 的前体形式在 SDS-PAGE 中不再可检测到，仅剩下 24 kDa 和 22 kDa 的形式。MT2-MMP 的催化结构域通过将 72 kDa 的前明胶酶 A 切割产生 67 kDa 的明胶酶 A，从而激活前明胶酶 A 以及前明胶酶 A/TMP-2 复合物，然后 67 kDa 的明胶酶 A 转化为 62 kDa 的明胶酶 A。62 kDa 形式对合成底物 N-(2,4)-二硝基苯基-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg 的活性约为 67 kDa 形式的两倍。游离和复合的明胶酶 A 形式之间未发现活性有显著差异。前明胶酶 A/TIMP-2 复合物的激活分两步进行：首先，MT2-MMP 被前明胶酶 A/TIMP-2/MT2-MMP 复合物抑制，从而产生三元复合物前明胶酶 A/TIMP-2/MT-2MMP。然后该三元复合物被过量的 MT2-MMP 激活。我们的结果提示了一种由膜型 MMP 激活介导的细胞外明胶酶 A 活性空间调控机制；游离的明胶酶 A 释放到细胞外空间，而与 MT-MMP 结合的明胶酶 A/TIMP-2 仍锚定在细胞表面。
