Insight into the secretion and maturation pathways of the subtilisin-like extracellular protease from the haloalkaliphilic archaeon Natrialba magadii

RUIZ, D. M.; GIMÉNEZ M.I.; DE CASTRO R.E.

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiologia General; 2009

The Twin arginine translocation (Tat) pathway is unique in its ability to translocate folded proteins across and into the cytoplasmic membrane and is extensively used by Haloarchaea. The alkaliphilic haloarchaeon <i>Natrialba magadii</i> (optimum growth in 20% NaCl, pH 12) produces an extracellular serine protease (Nep) that has been biochemically characterized in our laboratory. The gene encoding Nep was cloned, sequenced and recombinant active enzyme was produced in <i>Haloferax volcanii</i> cells. The polypeptide deduced from the gene showed that Nep is closely related to proteases of the subtilisin family and has a N-terminal prepropeptide containing the Tat consensus motif which is absent in the mature enzyme. Subtilisin-like proteases are synthesized as inactive precursors having at the N-terminus a signal peptide (prepeptide) followed by a propeptide which prevents protease activity. Once translocated by the Sec pathway (general protein secretion pathway), the prepeptide is cleaved by a signal peptidase and the propeptide is processed autocatalytically to render active enzyme. To date, the mechanisms of secretion and maturation of extracellular proteases remains unclear in halophilic archaea. We have shown that the precursor of Nep synthesized in <i>E. coli</i> can be trans-activated in vitro by the mature enzyme. The aim of this work was to confirm the mechanism of secretion and get insight into the maturation process of the extracellular protease Nep.         The coding sequence of nep was PCR-amplified, cloned into pET24b and used as template for site-directed mutagenesis to generate modified versions of the enzyme: 1. in the signal peptide of Nep (GRRSVL) the arginine residues were substituted by lysines (RR/KK); 2. the serine residue of the active site was replaced by alanine (S/A). Each version of nep gene (wt, RR/KK and S/A) was subcloned into the shuttle vector pJAM and transformed into the neutrophilic haloarchaeon <i>H. volcanii</i>. Secretion and maturation of Nep were analysed by Western blotting and protease activity determination on casein contaning-agar plates and by the azocasein assay. Nep(RR/KK), was only immunodetected in the cellular fraction while Nep(wt) and Nep(S/A) were visualized in the cells as well as in the culture medium, although the amount of Nep(wt) accumulated in the extracellular fraction was higher than that for Nep(S/A). Interestingly, Nep(S/A) displayed a higher apparent molecular mass than the two other forms. When the protease activity was measured, cells expressing Nep(wt) produced the highest level of activity in the culture medium while Nep(RR/KK) showed  lower levels of total activity which was associated to the cells. As expected, Nep(S/A) did not display protease activity in any fraction. Altogether, these results confirm experimentally that Nep is secreted by a Tat-dependent pathway, and that protease maturation is an autocatalytic process. Supported by UNMDP, ANPCyT and CONICET.