Identification of Genes Involved in the Biosynthesis of the Third and Fourth Sugars of the Methanococcus maripaludis Archaellin N-Linked Tetrasaccharide

Abstract

c N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-␤-ManNAc3NAmA6Thr-1,4-␤-GlcNAc3NAcA-1,3-␤-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-Omethyl-␣-L-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes-mmp1084, mmp1085, mmp1086, and mmp1087-were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a ⌬mmp1086⌬mmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in ⌬mmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the ⌬mmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…Both genes have a predicted FeS/ SAM-binding site at the N-terminal region and a DUF4008 domain in the C-terminal region. Deletion of either gene alone did not lead to any change in glycan composition, as determined by mass spectrometry (127). Moreover, the possibility that one gene product could compensate for the loss of the other, given their sequence similarity, was discarded, as the glycan structure of a mutant carrying deletions of both mmp1086 and mmp1087 was identical to that of the parent strain.…”

“…Two of these genes, mmp1084 and mmp1085, demonstrated effects on the N-linked glycan and were designated aglU and aglV, respectively (127). Deletion of either gene resulted in a minor decrease in the apparent molecular mass of the archaellin.…”

“…The epimerase activity of MMP0357 then results in the generation of the third sugar precursor, which is further acted upon by AglXYZ to add the acetamidino functional group (125). AglU adds the threonine to sugar 3, likely only after the fourth sugar has been added by the AglL GT (127). AglV adds a methyl group to sugar 4 (127).…”

Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

“…These include AglXYZ (MMP1081 to MMP1083), responsible for the acetamidino group modification at C-3 of the third sugar (18), AglU (MMP1084), a threonine transferase that modifies the third sugar at C-6, and AglV (MMP1085), a methyltransferase for the methyl group at C-5 in the fourth sugar (19). MMP0350 was also identified as a putative acetyltransferase involved in biosynthesis of the second sugar (27).…”

“…Though N-linked glycosylation is widespread in Archaea, the study of N-linked glycosylation pathways by both genetic and structural methodologies is concentrated on a few model species (4), including the thermoacidophile Sulfolobus acidocaldarius (5-9), the halophile Haloferax volcanii (10)(11)(12)(13)(14), and the methanogens Methanococcus voltae (15,16) and Methanococcus maripaludis (17)(18)(19)(20)(21)(22)(23). In general, the archaeal N-linked glycosylation pathway contains aspects of both the eukaryotic and bacterial systems, with additional domain-specific traits that include unique glycan structures, a variety of linking sugars, and unusual dolichol carri- Citation Siu S, Robotham A, Logan SM, Kelly JF, Uchida K, Aizawa S-I, Jarrell KF.…”