Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asnâlinked highly branched hexasaccharide chain containing 6âsulfoquinovose

Zähringer, Ulrich; Moll, Hermann; Hettmann, Thomas; Knirel, Yuriy A.; Schäfer, Günter

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asnâlinked highly branched hexasaccharide chain containing 6âsulfoquinovose

2000

Zähringer, Ulrich | Moll, Hermann | Hettmann, Thomas | Knirel, Yuriy A. | Schäfer, Günter

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membraneâbound bâtype hemoprotein [Hettmann, T., Schmidt, C. L., Anemüller, S., Zähringer, U., Moll, H., Petersen, A. & Schäfer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified cytochrome b558/566 was characterized by MALDI MS as a 64âkDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively Oâmannosylated with monosaccharides and Nâglycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:Nâglycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6âdeoxyâ6âsulfoglucose (6âsulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asnâlinked hexasaccharide chain, from which an Nâglycosylated TyrâAsn dipeptide was purified by gel chromatography and anionâexchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asnâlinked hexasaccharide chain of cytochrome b558/566.

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