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

JULIANA E. PARENTE ; MARÍA I. GIMENEZ; ROBERTO A. PAGGI,; ROSANA DE CASTRO ; ALICIA S. COUTO

Mendoza

Congreso; XLVIII reunion anual de SAIB; 2012

Soc. argentina deInvestigaciones en Bioquimica y Biologia Molecular

The S-layer glycoprotein from Haloferax volcanii presents novel N-linked oligosaccharides Juliana E. Parente1, María I. Gimenez2, Roberto A. Paggi2, Rosana De Castro2 and Alicia S. Couto1 1CIHIDECAR, Depto Química Orgánica, FCEN-UBA, 2Inst. de Investigaciones Biológicas, UNMdP-CONICET, Argentina. The S-layer glycoprotein from H. volcanii has been extensively used as a model for determining the steps involved in haloarchaeal protein N-glycosylation. This protein displays a pentasaccharide of known composition at positions N13 and N83. Up to date, there have not been any reports on the occurrence of other oligosaccharides associated to this glycoprotein. The analysis of the glycans present in this glycoprotein is of increasing interest as post-translational modifications are known to be involved in the properly folding of proteins specially needed to survive in adverse physical environmental conditions. In this work, membrane fractions of H. volcanii 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. The sample was further analyzed by MALDI-TOF MS. 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)6 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.