AglC and AglK are involved in biosynthesis and attachment of diacetylated glucuronic acid to the N-glycan in Methanococcus voltae

Abstract

Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the Methanococcus voltae N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidate M. voltae genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative M. voltae glycosyl transferase genes, designated aglC (for archaeal glycosylation) and aglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in M. voltae and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.

FIG. 1.

Current model of N-glycosylation of flagellin and S-layer proteins in M. voltae. Steps 1 to 3 diagram the assembly of the trisaccharide via glycosyl transferases onto a lipid carrier at the cytoplasmic face of the cytoplasmic membrane. Step 4 represents the translocation of the glycan to the exterior of the membrane via a flippase enzyme. Finally, step 5 shows the attachment of the complete glycan to the target protein via an STT3 oligosaccharyl transferase. AglH (Mv1751), AglA (Mv151), and AglB (Mv1749) have been previously reported to carry out steps 1, 3, and 5, respectively (8, 28). This study reports the finding that AglC (Mv990) and AglK (Mv991) carry out step 2 in glycan assembly.

FIG. 2.

Schematic of the M. voltae genomic regions of interest. Genes in boldface were targeted in this study.

FIG. 3.

Southern blot analysis confirming linear gene replacement. On the left are the Southern blot results of each deletion (the gene is indicated under each blot with the restriction enzyme used). M, λ-HindIII marker representing 23, 9, 6.5, 4.3, 2.3, 2, and 0.5 kb; wt, genomic DNA from wild-type M. voltae; Δ, genomic DNA from the indicated M. voltae deletion strain. On the right are schematics of the genome regions probed, with expected DNA fragment sizes. The targeted genes are dark gray; the pur^r cassette is represented by black arrows (promoters), a light-gray box (the puromycin gene), and a white block with X inside (the terminator); and the DIG-labeled probe is hatched. HindIII sites are marked by vertical arrows, while XhoI sites are marked by vertical round-topped markers where relevant.

FIG. 4.

Nano-LC-MS analysis of the FlaB2 tryptic glycopeptide T53-78 from M. voltae strains PS and PS*. (a) Amino acid sequence of T53-78Y showing the two sites of N-linked glycosylation (underlined). (b) MS/MS spectrum of the quadruply protonated T53-78 glycopeptide ion at m/z 1,094.5 from M. voltae PS flagellin. The corresponding LC-MS spectrum for this glycopeptide is presented in the inset. The lower half of the MS/MS spectrum is dominated by oxonium ions for β-ManpNAcA6Thr (▴, m/z 319) and GlcpNAc3NAcA (•, m/z 259) and their dehydration products. Larger oxonium ions composed of disaccharides (m/z 462.2 and m/z 577.2), as well as the entire trisaccharide glycan (m/z 780.3), were also observed (▪, GlcNAc). The high-mass region of this spectrum is dominated by fragment ions arising from the sequential loss of the components from the trisaccharides attached to both N-linked sites. Rel. int., relative intensity. (c) MS/MS spectrum of the quadruply protonated T53-78 glycopeptide ion at m/z 1,215.0 from M. voltae PS* flagellin. The corresponding LC-MS spectrum (inset) reveals a more complex glycoform profile due to the extension of the two N-linked glycans with either 220-Da (solid pentagon) or 262-Da (solid diamond) moieties (m/z 1,204.5, two 220-Da residues; m/z 1,215.0, one 220- and one 262-Da residue; and m/z 1,225.5, two 262-Da residues). The MS/MS spectrum indicates that the trisaccharide observed in the original M. voltae PS strain is modified here with one additional residue (either 220 or 262 Da) linked to the β-ManpNAcA6Thr (solid triangle) residue. Finally, though less intense than the glycan-related fragment ions, a good b fragment ion series was observed in this MS/MS spectrum, confirming the identity of the peptide (some of the b ions detected are annotated in the spectrum).

FIG. 5.

Immunoblot of M. voltae flagellins FlaB1/FlaB2 resolved on a 15% acrylamide gel. Lane 1, wild-type M. voltae (32 kDa); lane 2, Mv990 mutant (aglC); lane 3, Mv991 mutant (aglK); lane 4; M. voltae FlaB2 expressed in E. coli with and without (arrow) signal peptide (24 and 22 kDa, respectively); lane 5, Mv891 mutant; lane 6, wild-type M. voltae (32 kDa).

FIG. 6.

Electron micrographs of M. voltae wild type and mutants. (A) Wild-type M. voltae. (B) Mv990 mutant (aglC). (C) Mv991 mutant (aglK). (D) Mv891 mutant. Scale bar, 1 μm.

FIG. 7.

Cotranscriptional analysis of Mv990 and Mv991. The schematic at the top represents the genome region examined and PCR primer set names. *, The 991 primer set used Mv991 RNA, the Connect primer set used wild-type RNA, and the 990 primer set used Mv990 RNA. The RNA was added to the master mix and set on ice for 30 min, followed by 95°C denaturing of the reverse transcriptase.

FIG. 8.

Nano-LC-MS analysis of S-layer tryptic peptides T75-91 and T92-127 from M. voltae strain PS* and corresponding aglC and aglK mutant strains. The amino acid sequence for each peptide is presented above panels a and d, and the N-linked site of glycosylation is underlined. The relevant glycopeptide and peptide ions are underlined in all six spectra. The MS spectra show the multiply charged ions (2⁺ and 3⁺) for the T75-91 tryptic glycopeptide from the PS* strain (a), the doubly protonated ion at m/z 898.4 corresponding to the unmodified T75-91 peptide from the aglC mutant (b), the same peptide ion in the aglK mutant (c), the multiply protonated glycopeptide ions (3⁺ and 4⁺) for the T92-127 tryptic glycopeptide from the PS* strain (d), the triply protonated ion at m/z 1,255.4 corresponding to the T92-127 peptide from the aglC mutant (e), and the same peptide ion in the aglK mutant (f). Rel. int., relative intensity.