Accurate and efficient cleavage of the human insulin proreceptor by the human proprotein-processing protease furin. Characterization and kinetic parameters using the purified, secreted soluble protease expressed by a recombinant baculovirus.
Accurate and efficient cleavage of the human insulin proreceptor by the human proprotein-processing protease furin. Characterization and kinetic parameters using the purified, secreted soluble protease expressed by a recombinant baculovirus.
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DOI：
10.1016/s0021-9258(18)47322-x
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发表时间：
1994-10
期刊：
影响因子：
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通讯作者：
Deborah;Bravo;Julja B. GleasonSn;Rosa I. Sanchez;Richard;Rothll;Robert;Fuller
中科院分区：
文献类型：
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作者：
Deborah;Bravo;Julja B. GleasonSn;Rosa I. Sanchez;Richard;Rothll;Robert;Fuller
胰岛素前体受体在晚期高尔基体隔室中的成熟需要在Arg-Lys-Arg-Arg加工位点处裂解，这表明涉及弗林蛋白酶，一种Kex 2家族加工酶的跨膜丝氨酸蛋白酶。通过重组杆状病毒感染昆虫细胞表达的基因工程分泌的可溶形式的人弗林蛋白酶（β-弗林蛋白酶）被纯化至接近同质。β-弗林蛋白酶表现出快速和有效的切割的两种亚型的人胰岛素前受体在培养的哺乳动物细胞表达preproreceptor cDNA的溶解提取物。前受体裂解发生在生理加工位点，通过该位点突变对纯化的β-弗林蛋白酶裂解的影响来判断。此外，纯化的β-弗林蛋白酶表现出相同的内源性胰岛素受体前体加工酶的前体裂解的特异性。弗林蛋白酶因此显示出胰岛素前体受体加工酶的预期特性，因为它（i）在正确的位点有效地切割前体受体;（ii）在加工变体前体受体方面表现出与内源酶相同的特异性;（iii）似乎定位于正确的分泌区室;和（iv）具有与胰岛素前体受体相同的广泛组织分布模式。
Maturation of the insulin proreceptor in a late Golgi compartment requires cleavage at an Arg-Lys-Arg-Arg processing site, suggesting involvement of furin, a transmembrane serine protease of the Kex2 family of processing enzymes. A genetically engineered secreted, soluble form of human furin (ss-furin), expressed by infection of insect cells with a recombinant baculovirus, was purified to near homogeneity. ss-Furin exhibited rapid and efficient cleavage of both isoforms of the human insulin proreceptor in solubilized extracts of cultured mammalian cells expressing preproreceptor cDNA. Proreceptor cleavage occurred at the physiological processing site as judged by the effects of mutations in this site on cleavage by purified ss-furin. Moreover, purified ss-furin exhibited specificity for proreceptor cleavage identical to that of the endogenous insulin proreceptor-processing enzyme. Furin thus displays the properties expected of an insulin proreceptor-processing enzyme in that it (i) cleaves the proreceptor efficiently and at the correct site; (ii) exhibits the same specificity in processing variant proreceptors as the endogenous enzyme; (iii) appears to be localized in the correct secretory compartment; and (iv) has the same broad pattern of tissue distribution as the insulin proreceptor.