Duguay S J, Jin Y, Stein J, Duguay A N, Gardner P, Steiner D F
Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 1998 Jul 17;273(29):18443-51. doi: 10.1074/jbc.273.29.18443.
Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.
胰岛素样生长因子2（IGF-2）在大多数胚胎组织中表达，是妊娠期正常发育所必需的。出生后，IGF-2在大多数组织中的表达消失，但该基因在肿瘤发生过程中常被重新激活。肿瘤分泌高分子量形式的IGF-2，这是前胰岛素样生长因子2异常翻译后加工的结果。作为理解高分子量IGF-2肽如何促进肿瘤进展的第一步，我们对人胚胎细胞系中IGF-2的生物合成进行了表征。我们发现，前胰岛素样生长因子2最初可以形成两种二硫键异构体，它们在体内重排为单一构象。N-乙酰半乳糖胺添加到Ser71、Thr72、Thr75和Thr139上可能发生在顺式高尔基体中。唾液酸的添加始于反式高尔基体，但IGF-2肽必须到达反式高尔基体网络才能完成寡糖成熟。内切蛋白水解与寡糖成熟同时或稍晚发生。仅在Arg104处观察到切割，导致IGF-2-(1-104)和游离E肽的分泌。蛋白水解需要P1（Arg104）和P4（Arg101）位置的碱性残基，被弗林蛋白酶抑制剂完全阻断，并通过与弗林蛋白酶、PACE4、PC6A、PC6B和LPC共表达而增强。这些数据表明，枯草杆菌蛋白酶相关前体蛋白转化酶家族的成员介导了前胰岛素样生长因子2在Arg104处的加工。在这个表达系统中，我们没有检测到正常血清中最丰富的IGF-2肽、成熟IGF-2和IGF-2-(1-87)，这表明新的内切蛋白酶负责产生这些产物。
