Cryptococcus neofonnams an opportunistic pathogen, is the fourth leading cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure of glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was investigated by chemical analysis and 2D NMR spectroscopy. The assignment of 1H and 13C chemical shifts for the O-deacetylated polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enhanced HSQC spectra. The sequence and linkage positions of glycosyl residues were determined by NOESY and ROESY spectra. Two repeating polysaccharide components were identified as having the following structures in approximately equal proportions: -3)[bDXylp(1-4),bDGlcpA(1-2)]aDManp(1-3)aDManp(1-3)[bDXylp(1-2)]aDManp(1- and -3)[bDGlcpA(1-2)]aDManp(1-3)aDManp(1-3)[bDXylp(1-2)]aDManp(1-. It is not known if these repeating units comprise a single or two separate polymer chains. Pentasaccharide 2 has been known to be the major GXM polymer of B-3501 and other serotype D isolates. Hexasaccharide 1 is identified for the first time although it has subsequently been identified in other C. neoformans isolates. The presence of 1 in the GXM of Cap70 is consistent with the extra xylose found relative to that in isolate B-3501. The mannose: xylose:glucuronic acid;0-acetyl molar ratio of Cap70 GXM is 3.00:1.73;0.78:1.75, while the same ratio for B-3501 and other serotype D isolates is approximately 3.00:1.00:0.80:1.75. Methylation analysis confirmed that the GXM of Cap70 contains unsubstituted, monosubstituted (2-linked), and disubstituted (2- and 4-linked) mannose in a ratio of 0.87:1.75:0.38. Dot blot immunoassay indicates that Cap70 is a serotype D isolate like its parent strain.

NMR, polysaccharide, capsular polysaccharide, capsule, 2D NMR, Cryptococcus, Cryptococcus neoformans, Glucuronoxylomannan

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2	bDGlcpA	104.28	74.97	77.84	74.06	79.27	?
3,3	aDManp	102.63	79.87	79.80	68.64	75.88	?
3	aDManp	104.54	72.20	81.25	68.56	75.79	?
2	bDXylp	105.63	75.12	78.07	71.68	67.54
	aDManp	103.54	80.61	78.49	69.55	75.68	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	bDGlcpA	4.475	3.370	3.476	3.566	3.648	-
3,3	aDManp	5.212	4.268	4.084	3.808	3.820	3.807-3.879
3	aDManp	5.173	4.210	3.956	3.781	3.971	?
2	bDXylp	4.369	3.302	3.414	3.638	3.254-3.985
	aDManp	5.173	4.210	4.034	3.674	3.988	3.760-3.840


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	bDGlcpA	104.28/4.475	74.97/3.370	77.84/3.476	74.06/3.566	79.27/3.648	
3,3	aDManp	102.63/5.212	79.87/4.268	79.80/4.084	68.64/3.808	75.88/3.820	?/3.807-3.879
3	aDManp	104.54/5.173	72.20/4.210	81.25/3.956	68.56/3.781	75.79/3.971	?/?
2	bDXylp	105.63/4.369	75.12/3.302	78.07/3.414	71.68/3.638	67.54/3.254-3.985
	aDManp	103.54/5.173	80.61/4.210	78.49/4.034	69.55/3.674	75.68/3.988	?/3.760-3.840

The spectrum also has 4 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

794.662 g/mol (C₂₉H₄₆R₂O₂₅, R = next and previous repeats, not counted in mol weight)