ProGlyProt ID AC120 Organism Information Organism Name Sulfolobus acidocaldarius (DSM 639) Domain Archaea Classification Family: Sulfolobaceae Order: Sulfolobales Class: Thermoprotei or Crenarchaeota Division or phylum: "Crenarchaeota" Taxonomic ID (NCBI) 2285 Genome Sequence (s) GeneBank CP000077.1 EMBL CP000077 Gene Information Gene Name cbsA(Saci_1858) NCBI Gene ID 3474781 GenBank Gene Sequence 3474781 Protein Information Protein Name Cytochrome b558/566 subunit A (b type hemoprotein) UniProtKB/SwissProt ID O54088 NCBI RefSeq YP_256457.1 EMBL-CDS AAY81164.1 UniProtKB Sequence >sp|O54088|CBSA_SULAC Cytochrome b558/566 subunit A OS=Sulfolobus acidocaldarius GN=cbsA PE=1 SV=1 MSLKIKSKITIGVLLIIFLLSIIFTLENVSLAQTSPQISVYKVVGSADLSNPGSAGYWSQ IPWTNISLTANIPMAPTSGLTHYLLVKAAWNGSWIFILEEWQAPEPAFNAWSTAVAGIYP NASGPGLFRMIELTPGTTYSLERNYTNYVSIINGKEETGRIVFNYSGITLPAPNNTEITV MSNGTILLWHSPRPVEDLLYNDGMFYGYYVNSTWYYPDRAAIMWYLGSGVPTKDDMNIGG KYPGQQFDGITFKDAGGSLAQSGGSANIWMWVSGATWNNSTYDPAFKSNIWQNESLTGLS YVDSGNHGFAVPLYTNNTNMYEVDTAGIWYTPVASEGLNGSLFFIWTGAKYENGSWVVEF ARPLSVPLDYQPFMPNITVGKTYYVAFAVWQGRLGETLFDKSITSSFLSLELVTTPPTST TTSTSPVTTISSAIPPVTLYVTIIGVVVALVALVILYVVFRR Sequence length 462 AA Subcellular Location Integral membrane protein Function Membrane residing ectoenzyme. Due to the likely pseudoperiplasmic location, cytochrome c-like function is proposed analogous to the situation in Paracoccus denitrificans linking pseudoperiplasmic redox metabolism to membrane-residing electron transport systems. Cytochrome b558/566 is significantly up-regulated under certain organotrophic growth conditions in combination with low oxygen tension. Protein Structure PDB ID Glycosylation Status Glycosylation Type N (Asn) linked (O linked residues not yet identified) Experimentally Validated Glycosite(s) in Full Length Protein N144, N164 Experimentally Validated Glycosite(s ) in Mature Protein N144, N164 Glycosite(s) Annotated Protein Sequence >sp|O54088|CBSA_SULAC Cytochrome b558/566 subunit A OS=Sulfolobus acidocaldarius GN=cbsA PE=1 SV=1 MSLKIKSKITIGVLLIIFLLSIIFTLENVSLAQTSPQISVYKVVGSADLSNPGSAGYWSQ IPWTNISLTANIPMAPTSGLTHYLLVKAAWNGSWIFILEEWQAPEPAFNAWSTAVAGIYP NASGPGLFRMIELTPGTTYSLERN*(144)YTNYVSIINGKEETGRIVFN*(164)YSGITLPAPNNTEITV MSNGTILLWHSPRPVEDLLYNDGMFYGYYVNSTWYYPDRAAIMWYLGSGVPTKDDMNIGG KYPGQQFDGITFKDAGGSLAQSGGSANIWMWVSGATWNNSTYDPAFKSNIWQNESLTGLS YVDSGNHGFAVPLYTNNTNMYEVDTAGIWYTPVASEGLNGSLFFIWTGAKYENGSWVVEF ARPLSVPLDYQPFMPNITVGKTYYVAFAVWQGRLGETLFDKSITSSFLSLELVTTPPTST TTSTSPVTTISSAIPPVTLYVTIIGVVVALVALVILYVVFRR Sequence Around Glycosites (21 AA) TPGTTYSLERNYTNYVSIING GKEETGRIVFNYSGITLPAPN Glycosite Sequence Logo seqlogo Glycosite Sequence Logo Technique(s) used for Glycosylation Detection Chemical deglycosylation (with TFMS) and mass shift on SDS-PAGE. Technique(s) used for Glycosylated Residue(s) Detection MALDI-MS, 1H-NMR spectroscopy of trypsin/pronase digested, anion-exchange HPLC purified glycan linked dipeptides. Protein Glycosylation- Implication The increased glycosylation of Cytochrome b558/566 subunit A has been suggested to have a role in the protection of enzyme against the harsh acidic conditions (pH 2 - 2.5) of S. acidocaldarius, in its thermoacidophilic habitat. Upon glycosylation with a low pKa sulfonate carrying glycans the protein achieves a negative surface potential at the locus of the cytochrome. The glycosylation of a major internal loop region also may improve resistance against hydrolytic cleavage which is even better th Glycan Information Glycan Annotation Linkage: β-GlcNAc-Asn. 64-kDa glycoprotein expresses 17% glycosylation. Total sugar analysis revealed the relative proportions of approximately 7:2:2 for mannose, glucose and GlcNAc. N-linked hexasaccharide units (at least seven)- two residues each of Man and GlcNAc (one trisubstituted) and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose- a rare acidic sugar). β-D-Glcp-(1→4)-β-D-Quip(6--SO3-)-(1→3)-[α-D-Manp(1→4), α-D-Manp(1→6)] β-D-GlcpNAc-(1→ Technique(s) used for Glycan Identification Identification of oligosaccharides using Gel Chromatography and GLC-MS (PNGase F treatment followed by HPAEC analysis). Glycan structure is elucidated with the help of 1H-NMR spectrum from two-dimensional 1H, 1H correlation NMR Spectroscopy . Linkage analysis was carried out using 13C NMR Chemical Shift analysis. Protein Glycosylation linked (PGL) gene(s) OST Gene Name Saci_1274 OST NCBI Gene ID 3473075 OST GenBank Gene Sequence 3473075 OST Protein Name Conserved membrane protein OST UniProtKB/ SwissProt ID Q4J9B4 OST NCBI RefSeq YP_255909.1 OST EMBL-CDS AAY80616.1 OST UniProtKB Sequence >tr|Q4J9B4|Q4J9B4_SULAC Conserved membrane protein OS=Sulfolobus acidocaldarius GN=Saci_1274 PE=4 SV=1 MQSTSILARIDKFKFLDAIIIGSLALFSILIRIISITAFPQTINGFDSWYLFYNALLIVK AGGNWYAVPPDVHAWFPWGYFIELENTIGLPFLVALFSVPFYSIFGQNIVYTLTLVSPIV LDGIGVVAAFLAVESITNSRVGGYIAAAITAFTPSLTYKNILGSLPKTSWGGVFVLFTIY FLSLAIKKKKPLYGIPAGIMIFLANITWGGYTYIDISLAIAAFLIVLFNKNDEISAKTLT ISGITAAFLTSLSPNTIGFMSEVAHGLALLIIPLFLYLDLYLRRVLPKDIVDSKNIVIGA AIILLVSLVVLGSVAFKVQLIPSRYYAIINPFFQFTVPIDRTVAEYIPQSIAAMIQDFGI GLFLSIIGIYFLLTRKQDMAGIWLVVLGAASIYGTSEQPYLFNYTIYIVAALAGVAVAEL FSRFMERKIRIAPILMLTLIGVALLADAGIAVEASYAPQALINSSTSYLTTNYAWISALD WINQNTPNNAFILSWWDYGYWIHAVGNRTVIDENNTLNGTQIKLMAEMFLNNESFAVNVL ENDFHLYPYGNPNYTRPVYIVAYDAVTEYIVNNQYPVWFIGYPTNFPGTFIGYTTSLGDI AKAIGAMTTIAGYNTNSYVNTTYINETASYAAQYNSQLASIIANSLPMAWTPKTYNSLIG SMFIEAIQSLNQGPVQAPFSISLSQLLQSSSTSLYNPNALLPRVNLMYFKPVYIALFPLS VTNALGGEAIVYIMVYIYQFVMPNVIIPPTISTA OST EC Number (BRENDA) 2.4.1.119 OST Genome Context Q4J9B4 Characterized Accessory Gene(s) PGL Additional Links CAZy Literatures Reference(s) 1) Zahringer, U., Moll, H., Hettmann, T., Knirel, Y.A. and Schafer, G. (2000) Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose. Eur J Biochem, 267, 4144-4149. [PubMed: 10866817] 2) Hettmann, T., Schmidt, C.L., Anemuller, S., Zahringer, U., Moll, H., Petersen, A. and Schafer, G. (1998) Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bo Additional Comments Provides first extensive study and a structure for oligosaccharide attached to a crenarchaeotal glycoprotein. The protein attached hexasaccharide is unique in terms of the presence of a trisubstituted N acetylglucosamine residue and a rare acidic residue Qui6S (sulfonated sugar). Qui6S like sugar has previously been observed only in glycolipids of chloroplast and photosynthetic bacteria. Only other case where sufated sugar has been observed is H. salinaium. Unusally, only one type of g Year of Identification 1998 Year of Validation 2000