Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion - Conversion of ovalbumin to a maspin-like molecule
Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion - Conversion of ovalbumin to a maspin-like molecule
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DOI：
10.1074/jbc.m302408200
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发表时间：
2003-08-22
影响因子：
4.8
通讯作者：
Twining, SS
中科院分区：
文献类型：
--
作者：
Ngamkitidechakul, C;Warejcka, DJ;Twining, SS
Maspin是一种ov-serpin，抑制肿瘤侵袭并诱导细胞与细胞外基质分子粘附。在这里，我们使用maspin/卵清蛋白嵌合蛋白和maspin反应位点环（RSL）肽的特点的作用，在maspin介导的功能的RSL。用卵白蛋白替换Maspin的RSL加上C-末端区域或RSL单独导致角膜基质细胞粘附于I型胶原、纤连蛋白和层粘连蛋白以及乳腺癌MDA-MB-231细胞粘附于纤连蛋白的刺激作用丧失。具有卵清蛋白作为C-末端区域的Maspin保留活性，表明maspin C-末端多肽不是必需的。R340 Q突变体保留了完整的maspin活性;然而，R340 A突变体失去了活性。这表明在推定的P1位点处的精氨酸侧链形成氢键而不是离子键。单独的RSL肽（P10-P5 '，氨基酸330-345）诱导乳腺癌细胞和角膜基质细胞的细胞-基质粘附并抑制癌细胞的侵袭。用maspin的RSL取代卵清蛋白的RSL将无活性的卵清蛋白转化为完全活性的分子。Maspin与乳腺癌细胞表面特异性结合，k（d）为367 +/- 67 nM，结合位点/细胞为32.0 +/- 2.2 x 10（6）。maspin RSL肽抑制结合，表明RSL参与maspin与细胞的结合。maspin RSL活性的充分性表明maspin调节细胞-基质粘附和肿瘤细胞侵袭的机制不涉及蛋白酶抑制的丝氨酸蛋白酶抑制剂机制。
Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide ( P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a k(d) of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.