Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that β-galactofuranose (β-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

capsular polysaccharides, galactofuranose, O-acetyl, NMR-spectroscopy, Cryptococcus, Cryptococcus neoformans, galactoxylomannans

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6	aDGalp	99.4	69.5	70.7	70.7	70.0	67.7
3,4,2	bDXylp	104.9	73.9	76.8	70.4	66.2
3,4,3	aDManp	103.6	71.2	70.6	67.2	74.5	62.0
3,4	aDManp	101.0	79.6	76.5	67.4	74.4	61.3
3	bDGalp	105.5	72.2	73.3	78.5	75.9	61.6
	aDGalp	99.1	68.6	81.2	70.3	70.0	67.9


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6	aDGalp	4.99	3.86	3.77	3.91	4.26	3.70-3.96
3,4,2	bDXylp	4.41	3.32	3.47	3.65	3.32-4.05
3,4,3	aDManp	5.16	4.09	4.05	3.87	3.96	3.82-3.95
3,4	aDManp	4.99	4.17	4.09	3.91	4.09	3.84-3.87
3	bDGalp	4.68	3.64	3.77	4.10	3.87	3.75-3.75
	aDGalp	5.03	4.06	4.03	4.31	4.22	3.72-3.94


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6	aDGalp	99.4/4.99	69.5/3.86	70.7/3.77	70.7/3.91	70.0/4.26	67.7/3.70-3.96
3,4,2	bDXylp	104.9/4.41	73.9/3.32	76.8/3.47	70.4/3.65	66.2/3.32-4.05
3,4,3	aDManp	103.6/5.16	71.2/4.09	70.6/4.05	67.2/3.87	74.5/3.96	62.0/3.82-3.95
3,4	aDManp	101.0/4.99	79.6/4.17	76.5/4.09	67.4/3.91	74.4/4.09	61.3/3.84-3.87
3	bDGalp	105.5/4.68	72.2/3.64	73.3/3.77	78.5/4.10	75.9/3.87	61.6/3.75-3.75
	aDGalp	99.1/5.03	68.6/4.06	81.2/4.03	70.3/4.31	70.0/4.22	67.9/3.72-3.94

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@H](OC[C@H]2O[C@H](*)[C@H](O)[C@@H](O[C@@H]3O[C@H](CO)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]5O)[C@@H]4O[C@@H]4OC[C@@H](O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H]2O)[C@H](O)[C@@H](O)[C@H]1O

942.82 g/mol (C₃₅H₅₈R₂O₂₉, R = next and previous repeats, not counted in mol weight)