CIHIDECAR   12529
CENTRO DE INVESTIGACIONES EN HIDRATOS DE CARBONO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The rhomboid protease RhoII from Haloferax volcanii is involved in regulation of protein glycosylation.
Autor/es:
M. I. GIMENEZ; R. A. PAGGI; J. E. PARENTE; A. S. COUTO ; R. E. DE CASTRO
Lugar:
Mar del Plata
Reunión:
Congreso; VIII Congreso Argentino de la Soc. Argentina de Microbiología General (SAMIGE; 2012
Resumen:
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 DrhoII 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.