ROLES OF THE SIGNAL PEPTIDE AND MATURE DOMAINS IN THE SECRETION AND MATURATION OF THE NEUTRAL METALLOPROTEASE FROM STREPTOMYCES-CACAOI

The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesize d as a prepro-Npr precursor form consisting of a secretory signal pept ide, a propeptide and the mature metalloprotease. The maturation of Np r occurs extracellularly via an autoproteolytic processing of the secr eted pro-Npr. The Integrity of the propeptide is essential for the for mation of mature active Npr but not for its secretion [Chang, Chang an d Lee (1994) J. Biol. Chem. 269, 3548-3554]. In this study we investig ated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations a t the positively charged region (mutant IR6LE), the central hydrophobi c region (mutants GI19EL and G19N), the boundary of the hydrophobic co re-cleavage region (mutant P30L) and at the residues adjacent to the s ignal peptidase cleavage site (mutant YA33SM). All these lesions delay ed the export of Npr to the growth medium and also resulted in a 2-10- fold decrease in Npr export. The most severe effect was noted in mutan ts GI19EL and P30L. When these signal peptide mutations were fused sep arately with the propeptide lacking the Npr mature domain, the secreto ry defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Np r signal peptide seems to have more constraints on the hydrophobic cor e region and at the proline residue within the boundary of the hydroph obic core-cleavage site. Deletion mutations within the C-terminal matu re protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our pre vious findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but no t in the maturation of Npr. On the contrary, the integrity of mature d omain and propeptide is not critical for secretion of the Npr derivati ve but is essential for the formation of a functional Npr. Therefore t he secretion and maturation of Npr are dependent on the integrity of t he signal peptide, propeptide and mature protease domains, and the rol es of these domains in this regard are functionally distinct.