IIB   20738
INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The rhomboid protease RhoII from Haloferax volcanii is involved in regulation of protein glycosylation
Autor/es:
GIMÉNEZ M.I.; PAGGI RA; PARENTE, J.; COUTO A.; DE CASTRO R.E.
Lugar:
Mar del Plata
Reunión:
Congreso; VIII Congreso de Microbiología General SAMIGE; 2012
Institución organizadora:
SAMIGE
Resumen:
The rhomboid protease RhoII from Haloferax volcanii is involved in regulation of protein glycosylation Giménez M.I.;  Paggi RA;  Parente, J.;  Couto A.;  De Castro R.E. Rhomboids are intramembrane serine proteases conserved in the three domains of life. Even though they share topological traits, their roles in different organisms, when known, are very diverse. Particularly, nothing is known on the biology of these proteases in Archaea.   Haloarchaeal genomes encode for 2-5 rhomboid protease homologs, one of these with an N-terminal AN1-Zn finger domain which is conserved in all haloarchaea. The model haloarchaeon H. volcanii has two potential rhomboids, including one Zn finger-homolog, which was denoted by us as RhoII. In order to identify RhoII substrates, the SDS-PAGE profile of membrane fractions of a rhoII strain (MIG1) was compared to that of the wild type. Interestingly two glycoproteins (250 and 100 kDa) were enriched in the null strain which were identified by MALDI-TOF MS as S-layer glycoprotein and a putative periplasmic dipeptides/oligopeptides ABC transporter substrate-binding protein, respectively. In addition, cell motility which relies on a glycoprotein flagellum, was also reduced in MIG1. These observations prompted us to speculate that RhoII could be implicated in protein glycosylation regulation in H. volcanii.    The aim of this work was to investigate the effect of rhoII deletion on protein glycosylation in H. volcanii, using the S-layer glycoprotein as a model.  Membranes of the wild and MIG1strains were analyzed by SDS-PAGE and protein bands corresponding to the S-layer protein were excised of the gel and digested with PNGase F. The released oligosaccharides from each sample were separated and compared by HPAEC-PAD. Interestingly, although the pattern was similar, the mutant strain presented accumulation of oligosaccharides migrating with lower retention times. Samples were analyzed by MALDI-TOF MS. In the negative ion mode, a main ion attributed to a NacGlc-NAcGlc(Hex)-SQ-Hex structure (m/z 1031.7) was detected in both samples.  However when the analysis was performed in the positive ion mode the wild type strain showed a main peak at m/z 3254.8 attributed to NacGlc-NAcGlc(Hex)-(SQ-Hex)6. MALDI-LID-MS/MS analysis of the main ions confirmed the assigned structures. As far as we know, these high molecular weight oligosaccharides have not been reported in haloarchaeal glycoproteins so far.   These results suggest that rhomboids are involved in the regulation of the archaeal S-layer glycosylation events (probably by processing of a key enzyme) and also contribute to the basic knowledge on protein glycosylation in prokaryotic cells.  This work was supported by ANPCyT, CONICET, UNMdP and UBA.