The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process.
Recombinant proPC2 purified from the medium of CHO cells overexpressing both the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino terminal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme revealed a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was inhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the specific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences between the proPC2 conversion reaction and the hydrolysis of synthetic substrates by mature PC2. In support of this idea, proPC2 conversion was not calcium dependent and was unaffected by 5 mM EDTA. The rate of conversion of proPC2 remained similar with a 10-fold difference in zymogen concentration, implicating an intramolecular rather than intermolecular mechanism of activation. Interestingly, the rate of proPC2 conversion was extremely pH dependent, occurring most extensively between pHs 4.0 and 4.9. Taken together, our results suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium levels, but by the decreasing pH gradient along the secretory pathway.
Lamango
N S
NS
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana, 70112, USA.
Apletalina
E
E
Liu
J
J
Lindberg
I
I
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology; D051859:Neuroendocrine Secretory Protein 7B2; D010446:Peptide Fragments / Q000494:pharmacology; D010907:Pituitary Hormones / Q000235:genetics / Q000276:immunology / Q000502:physiology; D045706:Proprotein Convertase 2; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection