Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe(88)-Gln(89) and Phe(91)-Glu(92) bonds of the catalytic domain
Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe(88)-Gln(89) and Phe(91)-Glu(92) bonds of the catalytic domain
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DOI：
10.1074/jbc.272.41.25628
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发表时间：
1997-10-10
影响因子：
4.8
通讯作者：
Caughey, GH
中科院分区：
文献类型：
--
作者：
Fang, KC;Raymond, WW;Caughey, GH
在先前的工作中，我们发现金属明胶酶是从狗肥大细胞瘤细胞分泌的，并直接被肥大细胞α-糜蛋白酶激活。目前的工作确定的蛋白酶作为犬同源物的明胶酶B（92-kDa明胶酶，MMP-9），确定的网站切割的α-糜蛋白酶，并探讨了调节明胶酶表达肥大细胞瘤细胞。为了获得编码肥大细胞瘤明胶酶B的完整序列的cDNA，从BR肥大细胞瘤文库中分离编码明胶酶的2.3-β-淀粉酶克隆。测序的cDNA预测了一个704个氨基酸的蛋白质，与人β-胶原酶B 80%相同。被认为对活性位点潜伏期至关重要的区域，如含Cys的前肽序列PRCGVPD和催化结构域序列HEFGHALGLDHSS，是完全保守的。用纯化的狗α-糜蛋白酶切割明胶酶B，产生类似于84-kDa的产物，其含有两个NH 2-末端氨基酸序列，QTFEGDLKXH和EGDLKXHHND，分别对应于cDNA预测序列的残基89-98和92-101。因此，α-糜酶在Phe（88）-Gln（89）和Phe（91）-Glu（92）键处切割明胶酶B的催化结构域。与BR细胞一样，犬肥大细胞瘤细胞的C2系组成性分泌α-糜蛋白酶激活的β-弹性蛋白酶B。相比之下，不产生糜酶的C1细胞仅响应于12-O-十四烷酰基佛波醇-13-乙酸酯而分泌明胶酶B（其保留在其预制体中）。而BR细胞的12-O-十四酰基佛波醇-13-乙酸酯刺激产生明胶酶B mRNA表达的类似15倍增加，地塞米松下调其表达的类似5倍。因此，细胞外刺激可以调节肥大细胞表达的肥大细胞黄体酮酶B的量。这些数据进一步支持肥大细胞α-糜蛋白酶在涉及明胶酶B介导的基质蛋白降解的组织重塑中的作用。
In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2.3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an similar to 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe(88)-Gln(89) and Phe(91)-Glu(92) bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its preform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a similar to 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by similar to 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.