Glycosaminoglycans modulate activation, activity, and stability of tripeptidyl-peptidase I in vitro and in vivo
Glycosaminoglycans modulate activation, activity, and stability of tripeptidyl-peptidase I in vitro and in vivo
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DOI：
10.1074/jbc.m412047200
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发表时间：
2005-03-04
影响因子：
4.8
通讯作者：
Kida, E
中科院分区：
文献类型：
--
作者：
Golabek, AA;Walus, M;Kida, E
三肽基肽酶I（TPP I，CLN2蛋白）是一种溶酶体外肽酶，其顺序地从多肽的N末端去除三肽，并显示出较小的内切蛋白酶活性。TPP I突变导致经典的晚期婴儿神经元蜡样脂褐质沉积症，一种神经退行性溶酶体贮积病。TPP I酶原在另一种蛋白酶的协助下在溶酶体中转化为成熟酶，并且能够通过单分子机制在体外酸性pH下自动活化。由于在体外溶酶体报告的pH值下的自活化产生了无活性的酶，因此我们打算确定生理相关因素是否可以改变该过程，使其在体内也是合理的。在这里，我们报告说，高离子强度和糖胺聚糖（GAGs）增加产量（离子强度）或产量和速率（GAGs）的激活，提高降解释放的TPP我prosegment片段，并切换有效的自动激活TPP我的酶原向酸性较低的pH值（pH值6.0）。尽管离子强度和糖胺聚糖也抑制体外和活细胞中的TPP I活性，但抑制程度（20%至60%）似乎具有相当有限的功能意义。重要的是，与GAG的结合改善了TPP I的热稳定性，并在体外（在肝素存在下，在pH 7.4下成熟酶的t（1/2）增加了约8倍）和体内（在巴弗洛霉素A1处理后，缺乏GAG的细胞中TPP I的损失比对照细胞高2倍）保护酶免受碱性pH诱导的变性。这些研究结果阐明了一个有效的生理相关的机制，TPP我的监管GAG和表明，通过自动激活的活性酶的生成不仅可以在体外，但在体内以及完成。
Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t(1/ 2) of mature enzyme at pH 7.4 increased by similar to 8-fold in the presence of heparin) and in vivo (similar to 2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.