Protein-polysaccharide interactions. A monoclonal antibody specific for the capsular polysaccharide of Cryptococcus neoformans.
Monoclonal antibodies that react with the capsular polysaccharide, termed glucuronoxylomannan (GXM), of Cryptococcus neoformans have potential roles in the diagnosis, monitoring of disease progress, and immunotherapy of cryptococcal GXM of all four serotypes. A molecular model of the Fab fragment of monoclonal antibody 439 was constructed from the amino acid sequence and a template antibody molecule, Fab 4-4-20. A tryptophan is present on the surface between light chain CDR3 and heavy chain CDR3 in the putative binding site. This tryptophan residue proved to be a reporter group, and a fluorescence study of Fab 439 was performed to analyze the interaction between cryptococcal GXM and Fab 439. Binding of the polysaccharide enhanced the intrinsic fluorescence and caused a blue shift in the emission maximum, indicating that the environment of a tryptophan changes from a polar to less polar environment. This is consistent with the loss of water from the binding site caused by the binding of antigen. This interpretation was confirmed by acrylamide quenching, which showed that 1 less tryptophan was exposed to solvent in the Fab-GXM complex than in free Fab. Fluorescence titration was used to determine binding and dissociation constants (KD). The apparent KD values for serotypes A-C were approximately the same; the KD for serotype D GXM was 5-11-fold greater. De-O-acetylation of serotype A GXM produced a 31-fold increase in the KD, indicating that the O-acetyl groups are important, but not essential, for binding. Carboxyl groups appear to be essential for strong binding because the KD for carboxyl-reduced GXM was so large that it could not be determined.
Otteson
E W
EW
Department of Microbiology, University of Nevada, Reno 89557.
Welch
W H
WH
Kozel
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011134:Polysaccharides; D011994:Recombinant Proteins; C027478:glucuronoxylomannan