STRUCTURE OF THE O-DEACETYLATED GLUCURONOXYLOMANNAN FROM CRYPTOCOCCUS-NEOFORMANS CAP70 AS DETERMINED BY 2D NMR-SPECTROSCOPY

Cryptococcus neoformans, an opportunistic pathogen, is the fourth lead ing cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure o f glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was invest igated by chemical analysis and 2D NMR spectroscopy, The assignment of H-1 and C-13 chemical shifts for the O-deacetylated polysaccharide wa s accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enha nced HSQC spectra. The sequence and linkage positions of glycosyl resi dues were determined by NOESY and ROESY spectra. Two repeating polysac charide components were identified as having the following structures in approximately equal proportions: [GRAPHICS] It is not known if thes e repeating units comprise a single or two separate polymer chains. Pe ntasaccharide 2 has been known to be the major GXM polymer of B-3501 a nd other serotype D isolates. Hexasaccharide 1 is identified for the f irst time although it has subsequently been identified in other C. neo formans 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 ma nnose:xylose:glucuronic acid:O-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 c onfirmed 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 seroty pe D isolate like its parent strain.