The S-layer glycoprotein from H. volcanii presents novel N-linked oligosaccharides

PARENTE, J.E.; GIMENEZ, M.I; PAGGI, R.A; DE CASTRO, R; COUTO, A.S

Mendoza

Congreso; 48 Reunión Anual de la SAIB; 2012

SAIB

THE S-LAYER GLYCOPROTE IN FROM Haloferax volcanii PRESENTS NOVEL N-LINKED OLIGOSACCHARIDES Parente, J.E.1 ;Gimenez, M.I.2; Paggi, R.A.2; De Castro, R.2; Couto, A.S1. 1CIHIDECAR, Depto Q.Orgánica, FCEN-UBA, 2Inst. de Inv.Biológicas, UNMdP-CONICET, Argentina. E-mail:acouto@qo.fcen.uba.ar The S-layer glycoprotein from has been used as a model for determining the steps involved in haloarchaeal protein N-glycosylation. This protein displays a known pentasaccharide at positions N13 and N83. The analysis of the glycans present in this glycoprotein is of increasing interest as post-translational modifications are known to be involved in the proper folding of proteins specially needed to survive in adverse physical environmental conditions. In this work, membrane fractions of H26 were fractionated by SDS-PAGE. The gel band corresponding to the S-layer glycoprotein was excised and digested with peptide:N-glycosidase F. The released oligosaccharides were separated and analyzed by HPAEC-PAD and byMALDI-TOFMS. Mass spectrum performed in the negative ion mode, presented a main ion attributed to a NAcGlc-NAcGlc(Hex)-SQ-Hex structure bearing different number of methyl substituents. However, when the analysis was performed in the positive ion mode, a main peak attributed to NAcGlc-NAcGlc(Hex)-(SQ-Hex) was detected. MALDI-LID-MS/MS analysis of the main ions confirmed the assigned structures. Interestingly, MS/MS analysis in the negative ion mode was essential to determine the different structures. As far as we know, these high molecular weight oligosaccharides have not been reported in any haloarchaeal glycoprotein so far.