Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium.
The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N-glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser-, in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N-glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.
Zeitler
R
R
Lehrstuhl für Biochemie I, Universität Regensburg, Universit atsstrasse 31, 93040 Regensburg, Germany.
Hochmuth
E
E
Deutzmann
R
R
Sumper
M
M
D019843:Archaeal Proteins / Q000235:genetics; D002240:Carbohydrate Sequence; D002470:Cell Survival / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism; D019617:Halobacterium salinarum / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D015183:Restriction Mapping; D017421:Sequence Analysis
D019843:Archaeal Proteins; D008562:Membrane Glycoproteins; D009005:Monosaccharides; D009844:Oligosaccharides; D016695:Glycosyltransferases