Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain
Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain
复制标题
DOI：
10.1074/jbc.271.47.30174
复制
发表时间：
1996-11-22
影响因子：
4.8
通讯作者：
Zucker, S
中科院分区：
文献类型：
--
作者：
Cao, JA;Rehemtulla, A;Zucker, S
膜型基质金属蛋白酶 1 (MT-MMP1) 是基质金属蛋白酶家族的一个新的 63 kDa 成员，是一种膜锚定酶和明胶酶 A 的激活剂。除了 C 端疏水性跨膜结构域外，MT-MMP1 在其前肽和催化结构域之间插入了 11 个氨基酸，并用配对的碱性氨基酸裂解酶的 RRKR 识别基序加密，弗林。在本报告中，我们研究了高尔基体相关弗林蛋白酶对 MT-MMP1 的 RRKR 基序的裂解是否类似于用 Stromelysin-3 观察到的类似酶激活机制。将突变形式的 MT-MMP1 与明胶酶原 A 和/或弗林蛋白酶的 cDNA 共转染至 COS-1 细胞中。使用特异性抗体进行免疫沉淀和免疫印迹来表征细胞蛋白，虽然弗林蛋白酶容易切割缺乏跨膜结构域的可溶性 MT-MMP1 (Delta MT-MMP1)，但没有 RRKR 基本基序的可溶性基质溶素-1/Delta MT-MMP1 嵌合体对弗林蛋白酶诱导的切割具有抵抗力。用野生型 MT-MMP1 cDNA 和弗林蛋白酶 cDNA 共转染的 COS-1 细胞在 SDS 聚丙烯酰胺凝胶电泳上显示出 63 kDa 蛋白（潜伏酶），而不是预期的较低分子量激活酶。用α1-蛋白酶抑制剂(Pittsburgh)(一种弗林蛋白酶抑制剂)抑制弗林蛋白酶活性并不影响共转染MT-MMP1和前明胶酶A cDNA的COS-1细胞中的前明胶酶A激活机制。此外，通过定点诱变用丙氨酸残基取代 MT-MMP1 的 RRKR 基序，产生了相同的 63 kDa 蛋白，且不损失前明胶酶 A 激活功能。这些数据表明，弗林蛋白酶诱导的 MT-MMP1 激活并不是明胶酶原 A 激活的先决条件，细胞结合的 MT-MMP1 激活机制仍有待阐明。
Membrane type matrix metalloproteinase 1 (MT-MMP1), a novel 63-kDa member of the matrix metalloproteinase family, is a membrane-anchored enzyme and an activator for gelatinase A. In addition to its C-terminal hydrophobic transmembrane domain, MT-MMP1 has an insertion of 11 amino acids between its propeptide and catalytic domain encrypted with a RRKR recognition motif for the paired basic amino acid cleaving enzyme, furin. In this report, we investigated whether the cleavage of the RRKR motif of MT-MMP1 by Golgi-associated furin is analogous to a similar enzyme activation mechanism observed with stromelysin-3. Mutant forms of MT-MMP1 were cotransfected into COS-1 cells with cDNAs for pro-gelatinase A and/or furin. Immunoprecipitation and immunoblotting using specific antibodies were employed to characterize cell proteins, Whereas furin readily cleaved soluble MT-MMP1 lacking the transmembrane domain (Delta MT-MMP1), a soluble stromelysin-1/Delta MT-MMP1 chimera without the RRKR basic motif was resistant to furin-induced cleavage. COS-1 cells cotransfected with wild type MT-MMP1 cDNA and furin cDNA demonstrated a 63-kDa protein (latent enzyme) on SDS-polyacrylamide gel electrophoresis rather than the anticipated lower molecular weight activated enzyme. Inhibition of furin activity with alpha 1-protease inhibitor(Pittsburgh) (a furin inhibitor) did not affect the pro-gelatinase A activation mechanism in COS-1 cells cotransfected with MT-MMP1 and pro-gelatinase A cDNAs. Furthermore, substitution of the RRKR motif of MT-MMP1 with alanine residues by site-directed mutagenesis resulted in the same 63-kDa protein without loss of pro-gelatinase A activation function. These data indicate that furin-induced activation of MT-MMP1 is not a prerequisite for pro-gelatinase A activation, The mechanism of activation of cell-bound MT-MMP1 remains to be elucidated.