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	103.82	74.97	77.84	74.00	73.19	?
3,3,4	bDXylp	106.36	76.05	78.26	72.23	67.59
3,3	aDManp	102.27	79.27	76.43	77.47	74.90	62.00
3	aDManp	104.56	72.20	81.22	68.18	75.81	?
2	bDXylp	105.714	75.00	78.069	71.658	67.710
	aDManp	102.8	80.10	78.57	69.89	75.73	63.60


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	bDGlcpA	4.472	3.397	3.478	3.589	3.643	-
3,3,4	bDXylp	4.382	3.270	3.458	3.578	3.306-3.891
3,3	aDManp	5.194	4.270	4.217	4.018	3.197	3.923
3	aDManp	5.193	4.197	3.972	3.842	3.907	?
2	bDXylp	4.270	3.328	3.408	3.666	3.174-4.060
	aDManp	5.164	4.121	3.995	3.644	3.946	3.752-3.860


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	bDGlcpA	103.82/4.472	74.97/3.397	77.84/3.478	74.00/3.589	73.19/3.643	
3,3,4	bDXylp	106.36/4.382	76.05/3.270	78.26/3.458	72.23/3.578	67.59/3.306-3.891
3,3	aDManp	102.27/5.194	79.27/4.270	76.43/4.217	77.47/4.018	74.90/3.197	62.00/3.923
3	aDManp	104.56/5.193	72.20/4.197	81.22/3.972	68.18/3.842	75.81/3.907	?/?
2	bDXylp	105.714/4.270	75.00/3.328	78.069/3.408	71.658/3.666	67.710/3.174-4.060
	aDManp	102.8/5.164	80.10/4.121	78.57/3.995	69.89/3.644	75.73/3.946	63.60/3.752-3.860

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

926.777 g/mol (C₃₄H₅₄R₂O₂₉, R = next and previous repeats, not counted in mol weight)