Duguay S J, Milewski W M, Young B D, Nakayama K, Steiner D F
Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA. stdu.midway.uchicago.edu
J Biol Chem. 1997 Mar 7;272(10):6663-70. doi: 10.1074/jbc.272.10.6663.
Insulin-like growth factor I (IGF-I) is required for normal embryonic development and postnatal growth. Like most hormones and growth factors, IGF-I is synthesized as a proprotein that is converted to the mature form by endoproteolysis. Processing of pro-IGF-I to mature IGF-I occurs by cleavage within the unique pentabasic processing motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg77. We have previously shown that human embryonic kidney 293 cells process pro-IGF-IA at Arg71 to generate IGF-I-(1-70) and at Arg77 to produce IGF-I-(1-76). Cleavage at each of these sites requires upstream basic residues, indicating that subtilisin-related proprotein convertases (SPCs) may be involved. In order to investigate the identity of the endogenous enzymes involved in maturation of pro-IGF-IA, we have expressed wild-type and mutant pro-IGF-IA in 293 cells and in the furin-deficient Chinese hamster ovary cell line, RPE.40. We have also co-expressed these constructs with SPCs that are thought to play a role in processing precursor proteins in the constitutive pathway: furin, PACE4, PC6A, PC6B, and LPC. The results show that furin is most active at cleaving wild-type and mutant pro-IGF-IA and can cleave these precursors at multiple sites within the pentabasic motif. PC6A and LPC are less active than furin but cleave only at Arg71. PACE4 and PC6B have very little activity on pro-IGF-IA precursors. Wild-type pro-IGF-IA was correctly processed to mature IGF-I in 10 of 10 cell lines that were tested. Since furin, PC6A, and LPC are known to have a broad pattern of tissue distribution and we have demonstrated expression of LPC in RPE.40 cells, our results suggest that these SPCs may be responsible for the endogenous pro-IGF-IA processing activity observed in a wide variety of cell lines.
胰岛素样生长因子I（IGF-I）是正常胚胎发育和出生后生长所必需的。与大多数激素和生长因子一样，IGF-I作为前体蛋白合成，通过内切蛋白水解作用转化为成熟形式。前胰岛素样生长因子I（pro-IGF-I）加工成成熟的IGF-I是通过在独特的五碱基加工基序Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg77内切割来实现的。我们之前已经表明，人胚肾293细胞在Arg71处加工pro-IGF-IA以产生IGF-I-(1-70)，并在Arg77处加工以产生IGF-I-(1-76)。在这些位点的每一处切割都需要上游碱性残基，这表明枯草杆菌蛋白酶相关前体蛋白转化酶（SPCs）可能参与其中。为了研究参与pro-IGF-IA成熟的内源性酶的身份，我们在293细胞和缺乏弗林蛋白酶的中国仓鼠卵巢细胞系RPE.40中表达了野生型和突变型pro-IGF-IA。我们还将这些构建体与被认为在组成型途径中加工前体蛋白起作用的SPCs共表达：弗林蛋白酶、PACE4、PC6A、PC6B和LPC。结果表明，弗林蛋白酶在切割野生型和突变型pro-IGF-IA方面最具活性，并且可以在五碱基基序内的多个位点切割这些前体。PC6A和LPC的活性低于弗林蛋白酶，但仅在Arg71处切割。PACE4和PC6B对pro-IGF-IA前体几乎没有活性。在测试的10个细胞系中的10个中，野生型pro-IGF-IA被正确加工成成熟的IGF-I。由于已知弗林蛋白酶、PC6A和LPC具有广泛组织分布模式，并且我们已经证明LPC在RPE.40细胞中表达，我们的结果表明这些SPCs可能负责在多种细胞系中观察到的内源性pro-IGF-IA加工活性。
