Monoclonal antibodies to the carboxy-terminal Ea sequence of pro-insulin-like growth factor-Ia (proIGF-IA) recognize proIGF-IA secreted by IM9 B-lymphocytes

Insulin-like growth factor-I (IGF-I) circulates in human serum as a 7 kDa p eptide but analysis of IGF-I cDNAs predicts two pro-hormone precursors (pro lGF-IA and prolGF-IB) with distinct C-terminal E domains. The function of t hese precursors, and the E peptides generated on cleavage to mature IGF-I, is unknown, largely because of a lack of tools for distinguishing precursor s from constituent peptides. We used a synthetic Ea peptide to develop mono clonal antibodies (MAbs) which can recognize the carboxy-terminal sequence of prolGF-IA. These were characterized using prolGF-IA generated by transfe cted HEK293 cells. The anti-prolGF-IA MAbs immunoprecipitated two peptides (19-21 and 14 kDa) which were also recognized by MAbs to mature IGF-I. The prolGF-IA MAbs could also detect peptides of 9 and 4 kDa predicted to be Ea peptides. Treatment with N-glycosidase proved the 19-21 kDa and 9 kDa band s to be glycosylated prolGF-IA and Ea peptide respectively, Using these ant ibodies, we have identified prolGF-IA secreted from the IM9 B-lymphocyte ce ll line. This work paves the way for studies on prolGF-IA and Ea peptide re gulation and function. (C) 2001 Harcourt Publishers Ltd.