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 peptide but analysis of IGF-I cDNAs predicts two pro-hormone precursors (proIGF-IA and proIGF-IB) with distinct C-terminal E domains. The function of these precursors, and the E peptides generated on cleavage to mature IGF-I, is unknown, largely because of a lack of tools for distinguishing precursors from constituent peptides. We used a synthetic Ea peptide to develop monoclonal antibodies (MAbs) which can recognize the carboxy-terminal sequence of proIGF-IA. These were characterized using proIGF-IA generated by transfected HEK293 cells. The anti-proIGF-IA MAbs immunoprecipitated two peptides (19--21 and 14 kDa) which were also recognized by MAbs to mature IGF-I. The proIGF-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 bands to be glycosylated proIGF-IA and Ea peptide respectively. Using these antibodies, we have identified proIGF-IA secreted from the IM9 B-lymphocyte cell line. This work paves the way for studies on proIGF-IA and Ea peptide regulation and function.
Wilson
H E
HE
Endocrine Sciences Research Group, University of Manchester, UK.
Westwood
M
M
White
A
A
Clayton
P E
PE
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D012333:RNA, Messenger; C053766:insulin-like growth factor 1A prohormone (91-103); D007334:Insulin-Like Growth Factor I