Biosynthesis, glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I
Biosynthesis, glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I
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DOI：
10.1074/jbc.m211872200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Wisniewski, KE
中科院分区：
文献类型：
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作者：
Golabek, AA;Kida, E;Wisniewski, KE
人三肽基肽酶I（TPP I，CLN 2蛋白）是一种溶酶体丝氨酸蛋白酶，其从小多肽的游离N末端去除三肽，并且还显示出较小的内切蛋白酶活性。由于各种自然发生的。突变时，TPP I活性的遗传性缺乏引起致命的溶酶体储存障碍，典型的晚期婴儿神经元蜡样质脂褐质沉积症（CLN 2）。在本研究中，我们分析了生物合成，糖基化，运输，和蛋白水解处理这种酶在稳定转染的中国仓鼠卵巢细胞，以及成熟的内吞酶原在CLN 2成淋巴细胞，成纤维细胞，和N2 a细胞。人TPP I最初被鉴定为类似于68 kDa的单一前体多肽，其在几小时内被转化为类似于48 kDa的成熟酶。影响细胞内酸性隔室的pH的化合物，那些干扰细胞内囊泡运输以及通过温度阻断或3-甲基腺嘌呤抑制晚期内体和溶酶体之间的融合的化合物，阻碍了TPP I酶原转化为成熟形式，这表明该过程发生在溶酶体隔室中。用N-糖苷酶F和内切糖苷酶H消化免疫沉淀的TPP I酶原以及用衣霉素处理细胞使TPP I酶原的分子量降低了约10 kDa，这表明TPP I中所有五个潜在的N-糖基化位点都被利用。成熟的TPP I被发现是部分耐endo H处理，因此，它的一些N-连接的寡糖是复杂/杂合型。不同种类的蛋白酶抑制剂和活性位点Ser（475）突变对培养细胞中人TPP I成熟的影响的分析表明，尽管TPP I酶原能够在体外自激活，但对AEBSF敏感的丝氨酸蛋白酶参与了酶原在体内成熟、活性形式的加工。
Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring. mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of similar to68 kDa, which, within a few hours, was converted to the mature enzyme of similar to48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by similar to10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.