CarboDB Glucurunoxylomannan 1031
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[edit] Carbohydrate Name
Glucurunoxylomannan
[edit] Carbohydrate Class
Miscellaneous
[edit] Source Microbe
Cryptococcus neoformans
[edit] Basic Structure
The GXM is a high-molecular-weight linear (1-->3)-α-D-mannan substituted with single (1-->2)-β-xylosyl and (1-->2)-β-glucuronosyl residues. 0 acetylation occurs on the C-6 of about half of the mannose residues. The molar ratio of xylose-mannose-glucuronic acid is 2:3:1 for serotype A
[edit] Proposed functions
The capsular glucuronoxylomannan (GXM) of C. neoformans is anantiphagocytic and tolerogenic polysaccharide and potentiates virulence
[edit] Antigenic Nature used to produce antibodies
Glycoconjugates
[edit] Carrier Name
Tetanus toxoid
[edit] Conjugation Method
GXM was derivatized by the following two methods. (i) In method 1, ADH [adipic acid dihydrazide] was introduced into GXM by the activation of carboxyl groups with EDAC. GXM (5 mg/ml of 0.2 M NaCl) was derivatized with 0.5 M ADH and 0.1 M EDAC at pH 4.85 for 3.5 h at room temperature, using a pH Stat. After extensive dialysis against 0.2 M NaCl, the reaction mixture was passed through a 2B-CL Sepharose column (1.5 by 30 cm) equilibrated in 0.2 M NaCl. The fractions containing GXM were pooled and concentrated to the original volume. (ii) In method 2, ADH was introduced into GXM by the activation of hydroxyl groups with CNBr. GXM (5 mg/ml of 0.2 M NaCl) was activated with an equal weight of CNBr at pH 10.5 for 6 min at 4C, using a pH Stat. An equal volume of 0.5 M NaHCO3 (pH 8.5) containing 0.5 M ADH was added. The reaction mixture was tumbled at 3 to 8C for 18 to 20 h, dialyzed against 0.2 M NaCl, and passed through a 2B-CL Sepharose column (1.5 by 30 cm). The fractions containing GXM were pooled and concentrated to the original volume. The reaction mixture, containing equal concentrations (3.0 to 7.5 mg/ml) of GXM-AH (derivatized by either method) and TT or rEPA in 0.2 M NaCl, was brought to pH 5.6 with 0.05 N HCl, and 0.05 to 0.1 M EDAC was added; the pH was maintained at 5.6 in a pH Stat for 1 to 3 h at 4C. The reaction mixture was dialyzed against 0.2 M NaCl at 3 to 8C and passed through a Sepharose 2B-CL column (1.5 by 30 cm) equilibrated in 0.2 M NaCl. The void volume fractions containing the GXM and the protein were pooled and stored in 0.01% thimerosal at 3 to 8C
[edit] Antibodies
Mab 18B7
[edit] Antibody type and class
IgG1
[edit] Assay System
Invivo protection assay
[edit] Cross-reactivity
This antibody cross-reacted with all 4 serotypes- A, B, C and D.
[edit] Proposed epitopes
N\A
[edit] Proposed Utility
This antibody opsonized Serotypes A and D and was shown to activate the complement pathway. This antibody is in pre-clinical development for treatment of Cryptococcus neoformans infections
