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-ac etylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type) . This asparagine of the linkage unit is located within the N-terminal sequ ence 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 meth od csg mutants were created which replace the serine residue of the consens us sequence by valine, leucine, and asparagine. Unexpectedly, this eliminat ion of the consensus sequence did not prevent N-glycosylation. All respecti ve 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-glycosylated via a G lc(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 t hese results we postulate the existence of two different N-glycosyltransfer ases in H.halobium, one of which does not use the typical consensus sequenc e Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.