Insulin‐Like Growth Factor‐II is a Substrate for Dipeptidylpeptidase I (Cathepsin C)

Abstract

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP-I) caused the release of trichloroacetic-acid-soluble radioactivity from rat 1251-insulin-like growth factor-I1 . This activity could be blocked by dipeptide inhibitors of DAP-I, and was enhanced by chloride. Treatment of unlabeled rat IGF-I1 with DAP-I converted approximately 50% of the IGF-I1 to a species with a slightly shorter elution time on reverse-phase HPLC, whereas treatment of human IGF-I1 caused complete conversion to the species with the shorter elution time. Rat IGF-I1 purified from the rat BRL 3A cell line is a mixture of two molecules beginning with Ala-Tyr-Arg-Pro-Ser-and Tyr-Arg-Bid. Chem. 256, 6859 -68651 while human IGF-I1 begins with Ala-Tyr-Arg-Pro-Ser-. Determination of the N-terminal amino acid sequence of human IGF-11 before and after digestion with DAP-I showed that DAP-I cleaved Ala-Tyr, terminating at Arg-Pro-; the rat IGF-I1 species beginning with Tyr-Arg-Pro-Serwas resistant to digestion. In order to compare DAP-I-treated IGF-I1 with native IGF-I1 for binding to IGF receptors and IGF-binding proteins and in a bioassay, rat and human IGF-I1 were treated with DAP-I and the digested and undigested species were isolated by reverse-phase HPLC. The IGF-IVmannose 6-phosphate receptor was purified from rat placental membranes, the IGF-I receptor was solubilized from human placental membranes and IGF-binding proteins were partially purified from adult and three-day-old rat sera by sequential gel filtration on Sephadex G-200 (pH 8.0) and Sephadex G-50 (acid pH). The dosehesponse curves of the two IGF-I1 species were indistinguishable in radioreceptor assays utilizing the IGF-IYmannose 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 native IGF-I1 species were also equipotent in stimulating ['Hlthymidine incorporation into DNA in the human osteosarcoma cell line, MG-63. We conclude that DAP-I cleaves an N-terminal dipeptide from IGF-I1 and that this does not result in a change in the biological activity of the molecule.