Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles.
Polyclonal antibodies reactive with a cleavage site in human proinsulin (HPI) (C-peptide-A-chain junction) have been raised (rabbit, guinea pig) using a synthetic peptide antigen coupled with keyhole limpet hemocyanin. These antisera recognize native HPI and des-31,32-HPI equally well but react 20-50 times less well with des-64,65-HPI, the intermediate cleaved at the C-peptide-A-chain junction and lacking the Lys-Arg pair. The guinea pig antisera did not recognize insulin but reacted weakly with C peptide at high concentrations; the rabbit antisera reacted with neither insulin nor C peptide. Immunocytochemical studies with human islet tissue localized the immunoreactivity of these antisera to clathrin-coated (pro)secretory vesicles derived from the trans Golgi, indicating that cleavage of the C-peptide-A-chain junction of proinsulin occurs mainly, if not exclusively, in this compartment of the beta cell.
Steiner
D F
DF
Michael
J
J
Houghten
R
R
Mathieu
M
M
Gardner
P R
PR
Ravazzola
M
M
Orci
L
L
D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001665:Binding Sites; D002966:Clathrin / Q000378:metabolism; D003429:Cross Reactions; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D007515:Islets of Langerhans / Q000276:immunology / Q000378:metabolism; D011384:Proinsulin / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D002966:Clathrin; D011384:Proinsulin