The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan.
Most monoclonal antibodies (mAbs) to Cryptococcus neoformans glucuronoxylomannan (GXM) antigen (Ag) are grouped as Class II based on usage of V(H)7183, Vkappa5.1, J(H)2, and Jkappa1 gene elements. Comparative analysis of 43 Class II mAbs revealed conservation of I51, G54, and D61 in heavy-chain variable region (V(H)) complementarity determining region 2 (CDR2), and R95 and D96 in CDR3. Furthermore, position 100b (Kabat numbering scheme) in CDR3 always had an aromatic amino acid (aa) and F was found at position 100c in 96% of mAbs. The function of these conserved residues for binding to GXM and peptide mimetics, and idiotype (Id) structure, was investigated using site-directed mutagenesis. In addition, we mutated W36 and V37 in the second framework. Mutations W36A, Y100bA, and F100cA interfered with antibody (Ab) secretion, but not assembly, and cytoplasmic Ab bound to GXM and Id mAbs. In contrast, mutations V37A, I51A, G54A, and D61A did not affect assembly, secretion, or binding to GXM. Mutating the R95-D96 motif in CDR3 to DR, DD, RR, RA, AD, KD, HD, RE, RN or AA revealed that the positive charge at position 95 was essential for binding GXM, whereas the negative charge at position 96 could be substituted for a non-charged aa. Our results: (1) extend the concept that CDR3 diversity is essential for Ag and Id specificity to a polysaccharide-binding Ab; (2) show that aa conservation in CDRs does not imply a requirement for Ag binding; (3) establish a role for W36 in secretion; and (4) demonstrate that aa motifs used for binding GXM and peptide mimetics can differ.
Nakouzi
Antonio
A
Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Casadevall
Arturo
A
D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D017124:Conserved Sequence; D003455:Cryptococcus neoformans / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000502:physiology; D011134:Polysaccharides / Q000276:immunology*; D020539:Sequence Analysis, Protein
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D011134:Polysaccharides; C027478:glucuronoxylomannan