Processing of Wild-type and Mutant Proinsulin-like Growth Factor-IA by Subtilisin-related Proprotein Convertases*

Abstract

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.