The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-β-GlcNAc3NGaAN-4-β-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide β-GlcNAc3NAcAN-4-β-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-β-Ribf-3-α-Rha2OMe-3-α-Rha-2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Post-translational modification, archaea, O-glycosylation, N-glycosylation, S-layer protein

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,4,3	l?GroA	?	73.9	64.7
2,4,2	Ac
2,4,4,2	Ac
2,4,4	aDGlcpN	98.0	54.5	71.9	70.7	73.7	61.3
2,4,6	NH2
2,4	bDGlcpN3NA	102.4	55.4	55.2	73.2	76.7	178.4
2	bDGlcp1N	80.4	72.8	76.2	79.6	77.5	61.3
	x?Asn


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,4,3	l?GroA	-	4.09	3.63-3.78
2,4,2	Ac
2,4,4,2	Ac
2,4,4	aDGlcpN	5.18	3.88	3.63	3.52	3.61	3.76-3.82
2,4,6	NH2
2,4	bDGlcpN3NA	4.80	3.87	4.35	4.14	4.09	-
2	bDGlcp1N	4.99	3.41	3.68	3.61	3.58	3.62-3.82
	x?Asn


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,4,3	l?GroA		73.9/4.09	64.7/3.63-3.78
2,4,2	Ac
2,4,4,2	Ac
2,4,4	aDGlcpN	98.0/5.18	54.5/3.88	71.9/3.63	70.7/3.52	73.7/3.61	61.3/3.76-3.82
2,4,6	NH2
2,4	bDGlcpN3NA	102.4/4.80	55.4/3.87	55.2/4.35	73.2/4.14	76.7/4.09	
2	bDGlcp1N	80.4/4.99	72.8/3.41	76.2/3.68	79.6/3.61	77.5/3.58	61.3/3.62-3.82
	x?Asn

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](O[C@@H]2[C@@H](C(N)=O)O[C@@H](O[C@@H]3[C@@H](CO)O[C@@H](NC(CC(N)=O)C(=O)O)[C@H](O)[C@H]3O)[C@H](NC(C)=O)[C@H]2NC(=O)C(O)CO)O[C@H](CO)[C@@H](O)[C@@H]1O

800.725 g/mol (C₂₉H₄₈N₆O₂₀)

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-β-GlcNAc3NGaAN-4-β-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide β-GlcNAc3NAcAN-4-β-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-β-Ribf-3-α-Rha2OMe-3-α-Rha-2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Post-translational modification, archaea, O-glycosylation, N-glycosylation, S-layer protein

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,4,2	Ac
2,4,6	NH2
2,4	bDGlcpN3NA	102.6	55.1	55.9	70.9	76.8	178.8
2	bDGlcp1N	80.5	72.8	76.2	79.6	77.5	61.3
	x?Asn


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,4,2	Ac
2,4,6	NH2
2,4	bDGlcpN3NA	4.76	3.85	4.05	3.70	4.04	-
2	bDGlcp1N	4.94	3.41	3.68	3.61	3.58	3.62-3.82
	x?Asn


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,4,2	Ac
2,4,6	NH2
2,4	bDGlcpN3NA	102.6/4.76	55.1/3.85	55.9/4.05	70.9/3.70	76.8/4.04	
2	bDGlcp1N	80.5/4.94	72.8/3.41	76.2/3.68	79.6/3.61	77.5/3.58	61.3/3.62-3.82
	x?Asn

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](CO)O[C@@H](NC(CC(N)=O)C(=O)O)[C@H](O)[C@H]2O)O[C@H](C(N)=O)[C@@H](O)[C@@H]1N

509.469 g/mol (C₁₈H₃₁N₅O₁₂)

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-β-GlcNAc3NGaAN-4-β-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide β-GlcNAc3NAcAN-4-β-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-β-Ribf-3-α-Rha2OMe-3-α-Rha-2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Post-translational modification, archaea, O-glycosylation, N-glycosylation, S-layer protein

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,3,3	bDRibf	108.3	81.7	71.5	84.1	63.9
2,3,2	Me	59.2
2,3	aLRhap	99.7	80.9	78.8	72.8	70.4	17.8
2	aLRhap	103.2	71.1	79.5	72.6	70.6	17.8
	aLRhap	100.8	79.2	71.1	73.5	70.6	17.9


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,3,3	bDRibf	5.36	4.20	4.35	4.07	3.68-3.87
2,3,2	Me	3.45
2,3	aLRhap	5.23	3.83	3.96	3.46	3.87	1.29
2	aLRhap	4.98	4.18	3.87	3.58	3.78	1.29
	aLRhap	5.13	4.10	3.93	3.52	3.76	1.32


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,3,3	bDRibf	108.3/5.36	81.7/4.20	71.5/4.35	84.1/4.07	63.9/3.68-3.87
2,3,2	Me	59.2/3.45
2,3	aLRhap	99.7/5.23	80.9/3.83	78.8/3.96	72.8/3.46	70.4/3.87	17.8/1.29
2	aLRhap	103.2/4.98	71.1/4.18	79.5/3.87	72.6/3.58	70.6/3.78	17.8/1.29
	aLRhap	100.8/5.13	79.2/4.10	71.1/3.93	73.5/3.52	70.6/3.76	17.9/1.32

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O[C@@H]2[C@@H](O)[C@H](C)O[C@@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@@H](O[C@H]4[C@H]([*])O[C@@H](C)[C@H](O)[C@H]4O)[C@@H]3O)[C@@H]2OC)O[C@H](CO)[C@H]1O

584.568 g/mol (C₂₄H₄₀R₂O₁₆, R = next and previous repeats, not counted in mol weight)