Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues - . Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Gl Ma GlcNA plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya. 1. Introduction In many Archaea the surface layer (S-layer) proteins are the sole cell wall component [1]. These S-layer proteins assemble into a natural 2-D crystal structure with very strong self interactions. In Archaea, which do not possess other cell wall components, the S-layer has to maintain the cell integrity and stabilize as well as to protect the cell against mechanical and osmotic stresses or extreme pH conditions. It is also predicted that the S-layer has to maintain or even determine the cell shape [2–6]. In Sulfolobus spp. the S-layer is composed of two proteins: a small protein of approximately 45？kD, SlaB, and a large protein, SlaA, of approximately 120？kDa. SlaB is an integral membrane protein and its strong interaction with SlaA, which covers the whole cell surface, tethers the S-layer to the membrane [8, 9]. Taking into account the harsh growth condition of the thermoacidophilic Sulfolobus spp. (pH 2-3 and 75– C), the S-layer proteins will play an important role in maintaining cell integrity and must be adapted to be functional under these conditions. One possible posttranslational modification proteins can undergo is glycosylation, which has a major effect on stability and half-life [10]. Indeed, all archaeal S-layer proteins which have been structurally studied to date, have been found to carry N-glycans [11–17]. Although Eukarya, Bacteria, and Archaea all share certain characteristics of
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