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Recombinant E-Peptides of Pro-IGF-I Have Mitogenic Activity

Biotechnology Center and Department of Molecular and Cell Biology (X.C.T., M.J.C., T.T.C.), and Department of Pathobiology (A.G.P., T.J.Y.), University of Connecticut, Storrs, Connecticut 06269-3149

Address all correspondence and requests for reprints to: Thomas T. Chin, University of Connecticut, Department of Molecular & Cell Biology, 75 Neagleville Road, V-44, Storrs, Connecticut 06269.

In mammals, the post-translational proteolytic modification of many pro-hormones generates multiple peptides with similar or distinct biological activities. The production of mature insulin-like growth factor I (IGF-I) involves the cleavage of an E-peptide from pro-IGF-I. Although the IGF-I E-peptides are conserved among vertebrate species, their fate and biological roles have not been identified. To test whether the E-peptides possess any biological 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 in vitro with a His-tag expression system and partially purified with an affinity Ni⁺⁺ column. The mitogenic activity of each E-peptide was determined by (1) the stimulation of total DNA content increase as measured by a fluorometric method and/or (2) stimulation of [³H]-thymidine incorporation into DNA. Recombinant Ea-4-peptide elicited a dose-related increase in both mitogenic assays in NIH3T3 cells. To further test the specificity of the mitogenic activity of Ea-4-peptide, three other cell lines were used: retroviral transformed human embryonic kidney cells (293GP), human mammary gland tumor cells (MCF-7) and caprine mammary epithelial cells (CMEC). Similar levels of mitogenic activity were observed in all cell lines tested for Ea-4-peptide. Mitogenic activity was also observed with recombinant Ea-2- and Ea-3-peptides when assayed in NIH3T3 cells. These results suggest that E-peptides of rainbow trout pro-IGF-I possess mitogenic activity in heterologous systems.

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