Autocatalytic maturation of the prohormone convertase PC2.
PC2 is a member of the eukaryotic family of subtilisin-like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH2-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation occurs to generate a 75-kDa propolypeptide. A combination of site-directed mutagenesis and a cell-free translation/translocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa polypeptide underwent slow cleavage after the sequence Arg-Lys-Lys-Arg84 to generate a 68-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp142 was changed to Asn (PC2M4). This latter observation suggested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indicates that cleavage could occur through an intermolecular reaction. The results also demonstrate that the novel Xenopus egg extract translation/translocation system represents a powerful cell-free method for studying proteolytic processing of propolypeptides.
Matthews
G
G
School of Biochemistry, University of Birmingham, United Kingdom.
Shennan
K I
KI
Seal
A J
AJ
Taylor
N A
NA
Colman
A
A
Docherty
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D045706:Proprotein Convertase 2; D014176:Protein Biosynthesis; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D013381:Subtilisins; D045706:Proprotein Convertase 2