The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3.
The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3.
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DOI：
10.1016/j.matbio.2009.07.005
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发表时间：
2009-10
期刊：
影响因子：
6.9
通讯作者：
Nagase, Hideaki
中科院分区：
文献类型：
--
作者：
Troeberg, Linda;Fushimi, Kazunari;Scilabra, Simone D.;Nakamura, Hiroyuki;Dive, Vincent;Thogersen, Ida B.;Enghild, Jan J.;Nagase, Hideaki
我们研究了金属蛋白酶组织抑制因子(TIMP)-3与具有凝血酶反应蛋白基序的ADAMTS(ADAMTS)-4和ADAMTS-5的亲和力是否受这些酶的非催化辅助结构域的影响。为此，我们首先建立了一种新的纯化重组标记TIMP-3及其抑制N-末端结构域(N-TIMP-3)的方法，即用氯酸钠处理HEK293细胞以阻止硫酸乙酰肝素蛋白多糖介导的TIMP-3内化。测定了TIMP-3和N-TIMP-3与特定的基质金属蛋白酶的亲和力，以及缺乏连续C末端结构域的ADAMTS-4和-5的形式。TIMP-3和N-TIMP-3对多种基质金属蛋白酶的亲和力与先前报道的大肠杆菌表达的N-TIMP-3的亲和力相似。N-TIMP-3对ADAMTS-4和-5的抑制作用强于全长TIMP-3。酶的C末端结构域增强了与N-TIMP-3的相互作用，并在较小程度上增强了与全长抑制剂的相互作用。例如，N-TIMP-3对全长ADAMTS-5的KI值是单独催化和去整合素结构域的7.5倍。我们认为酶的C-末端结构域影响活性部位周围的结构，有利于与TIMP-3的相互作用。
We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better Ki value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.
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影响因子：
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作者：
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DOI：
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