PT  - JOURNAL ARTICLE
AU  - Benjamin H. Meyer
AU  - Ben A. Wagstaff
AU  - Panagiotis S. Adam
AU  - Sonja-Verena Albers
AU  - Helge C. Dorfmueller
TI  - N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation
AID  - 10.1101/2021.01.19.427365
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.01.19.427365
4099  - http://biorxiv.org/content/early/2021/02/10/2021.01.19.427365.short
4100  - http://biorxiv.org/content/early/2021/02/10/2021.01.19.427365.full
AB  - Protein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.First identification and characterization of a thermostable β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.A highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.Agl24-like homologs are broadly distributed among Archaea.The eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.Competing Interest StatementThe authors have declared no competing interest.Aglarchaeal glycosylation enzymeAlgAsparagine linked glycosylationCAZyCarbohydrate-Active enZymes databaseDoldolicholDol-Pdolichol-phosphateDPANNarchaeal superphylum containing Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota, Woesearchaeota and PacearchaeotaGlcglucoseGlcAglucuronic acidGlcNAcN-acetylglucosamineGFPgreen fluorescent proteinGTglycosyltransferaseLLOlipid-linked oligosaccharideMALDI-MSmatrix-assisted laser desorption ionization mass spectrometryOSToligosaccharyltransferaseSDS-PAGEsodium dodecyl sulfate–polyacrylamide gel electrophoresisTACKarchaeal superphylum containing Thaumarchaeota, Aigarchaeota, Crenarchaeota and KorarchaeotaTMDtransmembrane domainUDPuridine diphosphateUndundecaprenol