The roles of propeptide in maturation and secretion of Npr protease from Streptomyces.
The cloned npr gene of Streptomyces cacaoi encodes a 60-kDa protein (prepro-Npr) consisting of a typical secretory signal peptide, a propeptide (22 kDa), and the 35-kDa mature metalloprotease (Npr). The maturation of Npr occurs extracellularly via an autocatalytic cleavage of the secreted intermediate pro-Npr (Chang, P.C., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 3952-3958). In this study, we investigated the roles of the propeptide in the maturation and secretion of Npr. Partial deletion of the propeptide region while leaving the signal peptide and the mature Npr sequence intact all led to abolishment of Npr activity and caused concomitant slight and transient accumulation of low molecular weight forms of Npr or pro-Npr derivatives extracellularly. The intact propeptide and its truncated form alone could be secreted into the medium if their NH2 termini were directly fused with the signal peptide sequence of Npr. However, similar fusion of the mature protease domain to the signal peptide without the propeptide sequence completely abolished the Npr production intracellularly and extracellularly. All these results demonstrate that the propeptide plays an important role in maturation and secretion of Npr protease, as in the case of alpha-lytic protease and subtilisin. In addition, our data suggest that an intact propeptide region is essential for the formation of mature active Npr, but not for the secretion of Npr and its derivatives. This distinguishes the maturation and secretion of S. cacaoi Npr from those of other propeptide-containing bacterial serine proteases and thermolysin-like protease.
Chang
S C
SC
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
Chang
P C
PC
Lee
Y H
YH
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000096:biosynthesis; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C085836:Npr protease