INSULIN-LIKE GROWTH FACTOR-II IS A SUBSTRATE FOR DIPEPTIDYLPEPTIDASE-I (CATHEPSIN-C) - BIOLOGICAL PROPERTIES OF THE PRODUCT

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP -I) caused the release of trichloroacetic-acid-soluble radioactivity f rom rat I-125-insulin-like growth factor-II (IGF-II). This activity co uld be blocked by dipeptide inhibitors of DAP-I, and was enhanced by c hloride. Treatment of unlabeled rat IGF-II with DAP-I converted approx imately 50% of the IGF-II to a species with a slightly shorter elution time on reverse-phase HPLC, whereas treatment of human IGF-II caused complete conversion to the species with the shorter elution time. Rat IGF-II purified from the rat BRL 3A cell line is a mixture of two mole cules beginning with Ala-Tyr-Arg-Pro-Ser- and Tyr-Arg-Pro-Ser- [Marqua rdt, H., Todaro, G. J., Henderson, L. E. & Oroszlan, S. (1981) J. Biol . Chem. 256, 6859-6865] while human IGF-II begins with Ala-Tyr-Arg-Pro -Ser-. Determination of the N-terminal amino acid sequence of human IG F-II before and after digestion with DAP-I showed that DAPI cleaved Al a-Tyr, terminating at Arg-Pro-; the rat IGF-II species beginning with Tyr-Arg-Pro-Ser- was resistant to digestion. In order to compare DAP-I -treated IGF-II with native ICF-II for binding to IGF receptors and IG F-binding proteins and in a bioassay, rat and human IGF-II were treate d with DAP-I and the digested and undigested species were isolated by reverse-phase HPLC. The IGF-II/mannose 6-phosphate receptor was purifi ed from rat placental membranes, the IGF-I receptor. was solubilized f rom human placental membranes and IGF-binding proteins were partially purified from adult and three-day-old rat sera by sequential gel filtr ation on Sephadex G-200 (pH 8.0) and Sephadex G-50 (acid pH). The dose /response curves of the two IGF-II species were indistinguishable in r adioreceptor assays utilizing the IGF-II/mannose 6-phosphate receptor and the IGF-I receptor and in IGF competitive binding assays utilizing partially purified IGF-binding proteins. The DAP-I-digested and nativ e IGF-II species were also equipotent in stimulating [H-3]thymidine in corporation into DNA in the human osteosarcoma cell line, MG-63. We co nclude that DAP-I cleaves an N-terminal dipeptide from IGF-II and that this does not result in a change in the biological activity of the mo lecule.