Identification of the extracellular matrix (ECM) binding motifs of tissue inhibitor of metalloproteinases (TIMP)-3 and effective transfer to TIMP-1
Identification of the extracellular matrix (ECM) binding motifs of tissue inhibitor of metalloproteinases (TIMP)-3 and effective transfer to TIMP-1
复制标题
DOI：
10.1074/jbc.m610490200
复制
发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Murphy, Gillian
中科院分区：
文献类型：
--
作者：
Lee, Meng-Huee;Atkinson, Susan;Murphy, Gillian
金属蛋白酶组织抑制剂（Tissue inhibitor of metalloproteinases，TIMPs）是基质金属蛋白酶家族中锌依赖性内肽酶的内源性抑制剂。存在四种哺乳动物TIMP（TIMP-1至TIMP-4），但仅TIMP-3被隔离至细胞外基质（ECM）。TIMP-3与ECM结合的分子基础至今尚未完全研究。在这份报告中，我们确定了独特的氨基酸结构，构成了TIMP-3中ECM结合基序的基础。通过系统地交换TIMP的亚结构域和TIMP-3的穷举突变，我们已经确定了直接负责ECM协会的表面残基。与公认的观点相反，我们发现TIMP-3通过其N-和C-末端结构域与ECM相互作用。参与ECM结合的氨基酸本质上都是碱性的：N-末端结构域的Lys-26、Lys-27、Lys-30、Lys-76和C-末端结构域的Arg-163、Lys-165。用谷氨酸（E）和谷氨酰胺（Q）（K26/27/30/76 E + R163/K165 Q）替换这些残基导致缺乏ECM粘附能力的可溶性TIMP-3。使用源自TIMP-3的ECM结合基序，我们还创建了能够ECM缔合的TIMP-1突变体（K26/27/30 + K76移植物）。这是TIMPs被有意地呈现为可溶性或ECM结合的第一个实例。以可溶性或ECM结合形式制备TIMP的能力也为未来的TIMP研究开辟了新的途径。
Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent endopeptidases of the matrix metalloproteinase families. There are four mammalian TIMPs (TIMP-1 to -4) but only TIMP-3 is sequestered to the extracellular matrix (ECM). The molecular basis for the TIMP-3:ECM association has never been fully investigated until now. In this report, we identify the unique amino acid configuration that constitutes the basis of the ECM binding motif in TIMP-3. By systematically exchanging the subdomains of the TIMPs and exhaustive mutation of TIMP-3, we have identified the surface residues directly responsible for ECM association. Contrary to the accepted view, we have found that TIMP-3 interacts with the ECM via both its N- and C-terminal domains. The amino acids involved in ECM binding are all basic in nature: Lys-26, Lys-27, Lys-30, Lys-76 of the N-terminal domain and Arg-163, Lys-165 of the C-terminal domain. Replacement of these residues with glutamate (E) and glutamine (Q) (K26/27/30/76E + R163/K165Q) resulted in a soluble TIMP-3 devoid of ECM-adhering ability. Using the ECM binding motif derived from TIMP-3, we have also created a TIMP-1 mutant (K26/27/30 + K76 transplant) capable of ECM association. This is the first instance of TIMPs being intentionally rendered soluble or ECM-bound. The ability to prepare TIMPs in soluble or ECM-bound forms also opens new avenues for future TIMP research.