PolysacDB Glucurunoxylomannan 1001
From DrugPedia: A Wikipedia for Drug discovery
Revision as of 06:02, 8 April 2010
Carbohydrate Name
Glucurunoxylomannan
Carbohydrate Class
Miscellaneous
Source Microbe
Cryptococcus neoformans serotype A
Basic Structure
A linear α(1-->3)-linked mannan backbone singly substituted with nonreducing β(1-->2) xylose and β(1-->2) glucuronic acid side branches ; O-acetyl groups are present at C-6 of the mannosyl residues
Proposed functions
Antiphagocytic ; an important virulence factor
Antigenic Nature used to produce antibodies
glycoconjugates.
Carrier Name
Pseudomonas aeruginosa exoprotein A (rEPA), tetanus toxoid
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
Antibodies
Polysera
Antibody type and class
IgM
Assay System
Double immunodiffusion, ELISA
Cross-reactivity
Polsera cross-reacted with capsular polysaccharides of C neoformans serotype A and D
Proposed epitopes
O-acetyl groups, glucuronyl residues
Proposed Utility
The conjugate vaccines prepared through hydroxyl activation of glucurunoxylomannan are sufficiently immunogenic and appear to be suitable for clinical evaluation
