Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[β-GalA3OMe4OAc5CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

glycan, Post-translational modification, glycoprotein, archaea, nuclear magnetic resonance (NMR), mass spectrometry (MS), glycobiology, archaellin, S-layer protein, thermophile

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7
3,2,4,3	Me	58.7
3,2,4,4	Ac	173.7	21.4
3,2,4	bDSugp	102.2	71.9	79.1	72.4	81.8	176.9	19.1
3,2	aDGalpA	96.8	68.7	71.4	82.1	73.0	174.8
3,4,2,2	Ac	174.8-175.7	23.1
3,4,2,3	Me	56.8
3,4,2	aDGalpNA	99.1	49.8	76.0	66.4	74.7	176.2
3,4,3,3	Me	58.4
3,4,3,6	Me	56.9
3,4,3	aXDDmanHepp	99.7	66.8	81.6	67.4	70.6	81.5	60.9
3,4	bDManp	102.6	77.0	74.2	70.1	78.3	61.8
3,6	NH2
3	aDGalpA	95.1	72.1	68.2	79.9	72.0	174.0
2	Ac	174.8-175.7	23.1
	bDGalpN1N	79.4	50.7	78.9	65.5	77.6	62.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7
3,2,4,3	Me	3.47
3,2,4,4	Ac	-	2.10
3,2,4	bDSugp	4.79	3.61	3.79	5.67	?	-	1.57
3,2	aDGalpA	5.46	4.12	3.96	4.57	4.76	-
3,4,2,2	Ac	-	1.94-2.04
3,4,2,3	Me	3.45
3,4,2	aDGalpNA	5.49	4.21	4.02	4.63	4.89	-
3,4,3,3	Me	3.38
3,4,3,6	Me	3.37
3,4,3	aXDDmanHepp	5.56	4.16	3.80	3.53	3.94	3.79	3.71-3.85
3,4	bDManp	4.99	4.31	3.73	3.91	3.34	3.77-3.92
3,6	NH2
3	aDGalpA	5.33	3.99	4.03	4.47	4.23	-
2	Ac	-	1.94-2.04
	bDGalpN1N	5.12	4.08	3.93	4.30	3.78	3.78-3.78


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7
3,2,4,3	Me	58.7/3.47
3,2,4,4	Ac		21.4/2.10
3,2,4	bDSugp	102.2/4.79	71.9/3.61	79.1/3.79	72.4/5.67	81.8/?		19.1/1.57
3,2	aDGalpA	96.8/5.46	68.7/4.12	71.4/3.96	82.1/4.57	73.0/4.76	
3,4,2,2	Ac		23.1/1.94-2.04
3,4,2,3	Me	56.8/3.45
3,4,2	aDGalpNA	99.1/5.49	49.8/4.21	76.0/4.02	66.4/4.63	74.7/4.89	
3,4,3,3	Me	58.4/3.38
3,4,3,6	Me	56.9/3.37
3,4,3	aXDDmanHepp	99.7/5.56	66.8/4.16	81.6/3.80	67.4/3.53	70.6/3.94	81.5/3.79	60.9/3.71-3.85
3,4	bDManp	102.6/4.99	77.0/4.31	74.2/3.73	70.1/3.91	78.3/3.34	61.8/3.77-3.92
3,6	NH2
3	aDGalpA	95.1/5.33	72.1/3.99	68.2/4.03	79.9/4.47	72.0/4.23	
2	Ac		23.1/1.94-2.04
	bDGalpN1N	79.4/5.12	50.7/4.08	78.9/3.93	65.5/4.30	77.6/3.78	62.3/3.78-3.78

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CO[C@H]1[C@H](O)[C@@H]([C@@H](CO)OC)O[C@H](O[C@@H]2[C@H](O[C@H]3O[C@H](C(=O)O)[C@H](O)[C@H](OC)[C@H]3NC(C)=O)[C@H](O[C@H]3[C@@H](C(N)=O)O[C@H](O[C@H]4[C@@H](O)[C@@H](CO)O[C@@H](N)[C@@H]4NC(C)=O)[C@H](O[C@H]4O[C@H](C(=O)O)[C@H](O[C@@H]5O[C@](C)(C(=O)O)[C@H](OC(C)=O)[C@H](OC)[C@H]5O)[C@H](O)[C@H]4O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O

1431.27 g/mol (C₅₄H₈₆N₄O₄₀)