Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose

Abstract

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type 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 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). 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 Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange 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 Asn-linked hexasaccharide chain of cytochrome b558/566.