Processing of α4 integrin by the proprotein convertases:: histidine at position P6 regulates cleavage
Processing of α4 integrin by the proprotein convertases:: histidine at position P6 regulates cleavage
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DOI：
10.1042/bj20021630
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发表时间：
2003-07-15
影响因子：
4.1
通讯作者：
Seidah, NG
中科院分区：
文献类型：
--
作者：
Bergeron, E;Basak, A;Seidah, NG
前蛋白转化酶 (PC) 参与 H-592 VISKR597 向下箭头 ST 位点处整联蛋白 α4 亚基的有限蛋白水解（其中下划线残基表示对 PC 介导的裂解很重要的带正电荷的氨基酸，并表示裂解位点），因为这种裂解受到丝氨酸蛋白酶抑制剂 α1 PDX（at-抗胰蛋白酶波特兰）的抑制。 LoVo（缺乏弗林蛋白酶的人结肠癌）细胞中 α4 与每种转化酶的共表达表明，弗林蛋白酶和前蛋白转化酶 5A (PC5A) 是最好的前 α4 转化酶。一致地，在过表达任一酶的稳定转染子中，人淋巴母细胞 CEM-T4 细胞中内源性 pro-α4 的加工大大增强。在许多白细胞系中，弗林蛋白酶的表达与内源加工效率密切相关，表明弗林蛋白酶是候选的原a4转化酶。突变分析表明，用丙氨酸 (R597A) 替换 P1 Arg(597) 可消除裂解，而 P6 突变体 H592R 甚至可以更好地被中国仓鼠卵巢 CHO-K1 细胞的内源转化酶加工。使用合成肽的体外动力学研究证实了 P6 处带正电荷的残基的重要性，并表明弗林蛋白酶和 PC5A 分别在酸性和中性 pH 值下对野生型 α4 的加工效果最好。在存在或不存在弱碱 NH4Cl 的情况下，对 pro-alpha4 及其 H592R 和 H592K 突变体进行生物合成分析，结果表明 P6 组氨酸残基使其通过弗林蛋白酶进行的加工对细胞 pH 值敏感。这表明 pro-alpha4 裂解优先发生在酸性区室中。总之，虽然公认的弗林蛋白酶加工基序是 Arg-Xaa-(Lys/Arg)-Arg down arrow ，但我们的数据进一步扩展它，在前体中包含 P6 处的调节组氨酸残基，而 P4 处缺乏碱性残基。
The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H-592 VISKR597 down arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1 PDX (at-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-a4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH4Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg down arrow , our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.