Duguay S J, Lai-Zhang J, Steiner D F
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
J Biol Chem. 1995 Jul 21;270(29):17566-74. doi: 10.1074/jbc.270.29.17566.
Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg77 and requires both Arg74 (P4) and Arg77 (P1). Cleavage at Arg77 does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg71 and requires both Lys68 (P4) and Arg71 (P1). Lys65 in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg71 since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.
胰岛素样生长因子I（IGF-I）是一种促有丝分裂肽，在大多数组织和细胞系中产生，在胚胎发育和出生后生长中起重要作用。IGF-I最初作为一种激素原前体合成，通过羧基末端E结构域的内切蛋白水解作用转化为成熟的IGF-I。原IGF-I向成熟IGF-I转化的调节是一种潜在机制，通过该机制可能调节这种生长因子的生物活性。IGF-I激素原的内切蛋白水解作用发生在独特的五碱性基序Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg处。最近，发现了一类在含有多个碱性残基的位点切割激素原前体的酶。本研究的目的是：1）确定五碱性原IGF-I加工位点中的哪些碱性残基对于正确切割是必需的；2）研究枯草杆菌蛋白酶相关的前蛋白转化酶1（SPC1/弗林蛋白酶）在原IGF-I加工中可能发挥的作用。我们已经表明，编码表位标记的原IGF-I的表达载体在人胚肾293细胞中指导成熟IGF-I-(1-70)、延伸IGF-I-(1-76)、原IGF-I和N-糖基化原IGF-I的合成和分泌。延伸IGF-I-(1-76)是通过在Arg77处切割产生的，需要Arg74（P4）和Arg77（P1）两者。在SPC1缺陷的细胞系RPE.40和LoVo中不会发生在Arg77处的切割，这表明该位点的加工是由SPC1介导的。成熟IGF-I-(1-70)是通过在Arg71处切割产生的，需要Lys68（P4）和Arg71（P1）两者。P7位置的Lys65对于有效切割很重要。由于在RPE.40和LoVo细胞中发生在Arg71处的切割，所以SPC1不是在Arg71处加工所必需的。这些数据表明存在一种对原IGF-I的Lys-X-X-Arg基序具有特异性的加工酶。
