Recombinant E-peptides of pro-IGF-I have mitogenic activity

In mammals, the post-translational proteolytic modification of many pro-hor mones generates multiple peptides with similar or distinct biological activ ities. The production of mature insulin-like growth factor I (IGF-I) involv es the cleavage of an E-peptide from pro-IGF-I. Although the IGF-I E-peptid es are conserved among vertebrate species, their fate and biological roles have not been identified. To test whether the E-peptides possess any biolog ical activity, three recombinant E-peptides of pro-IGF-I, namely Ea-2-, Ea- 3- and Ea-4-peptides of rainbow trout, Oncorhynchus mykiss, were produced i n vitro with a His-tag expression system and partially purified with an aff inity Ni++ column The mitogenic activity of each E-peptide was determined b y (1) the stimulation of total DNA content increase as measured by a fluoro metric method and/or (2) stimulation of [H-3]-thymidine incorporation into DNA. Recombinant Ea-4-peptide elicited a dose-related increase in both mito genic assays in NIH3T3 cells. To further test the specificity of the mitoge nic activity of Ea-4-peptide, three other cell lines were used: retroviral transformed human embryonic kidney cells (293 GP), human mammary gland tumo r cells (MCF-7) and caprine mammary epithelial cells (CMEC). Similar levels of mitogenic activity were observed in all cell lines tested for Ea-4-pept ide. Mitogenic activity was also observed with recombinant Ea-2- and Ea-3-p eptides when assayed in NH3T3 cells. These results suggest that E-peptides of rainbow trout pro-IGF-I possess mitogenic activity in heterologous syste ms.