Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.
The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.
Benjannet
S
S
J.A. DeSève Laboratories of Molecular Neuroendocrinology, Clinical Research Institute of Montreal, University of Montreal, Que, Canada.
Mamarbachi
A M
AM
Hamelin
J
J
Savaria
D
D
Munzer
J S
JS
Chrétien
M
M
Seidah
N G
NG
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006706:Homeostasis; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones / Q000378:metabolism*; D045706:Proprotein Convertase 2; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D004792:Enzyme Precursors; D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones; D001224:Aspartic Acid; D014443:Tyrosine; D013381:Subtilisins; D045706:Proprotein Convertase 2