Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose.
Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.
Human Health Therapeutics,  National Research Council Canada, Ottawa, Ontario,  Canada K1A 0R6.
Evgeny
E
Vinogradov
Lise
L
Deschatelets
Marc
M
Lamoureux
Girishchandra B
GB
Patel
Tammy-Lynn
TL
Tremblay
Anna
A
Robotham
Marie-France
MF
Goneau
Cathy
C
Cummings-Lorbetskie
David C
DC
Watson
Jean-Robert
JR
Brisson
John F
JF
Kelly
Michel
M
Gilbert
D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D005643:Fucose / Q000031:analogs & derivatives* / Q000378:metabolism*; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D020539:Sequence Analysis, Protein; D013822:Thermoplasma / Q000737:chemistry* / Q000378:metabolism
D019843:Archaeal Proteins; D008562:Membrane Glycoproteins; D009005:Monosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; D005643:Fucose