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. 2001 May;69(5):3398-409.
doi: 10.1128/IAI.69.5.3398-3409.2001.

VSports最新版本 - Molecular basis for immunoglobulin M specificity to epitopes in Cryptococcus neoformans polysaccharide that elicit protective and nonprotective antibodies

A Nakouzi  1 , P Valadon, J Nosanchuk, N Green, A Casadevall
Affiliations

Molecular basis for immunoglobulin M specificity to epitopes in Cryptococcus neoformans polysaccharide that elicit protective and nonprotective antibodies

A Nakouzi et al. Infect Immun. 2001 May.

Abstract

The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V(H)) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V(H) binding cavity to the corresponding residues in the 13F1 V(H) altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues. VSports手机版.

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Figures

FIG. 1
FIG. 1
VH and VL amino acid sequences for MAbs 2D10, 12A1, and 13F1 and the 12A1 VH mutants. Boxes indicate the amino acid residues shared by the three MAbs which produce a punctate binding pattern.
FIG. 2
FIG. 2
Three-dimensional representation of the Ag binding site of MAb 12A1 with van der Waals surface. The model is based on the 2H1 structure in complex with the peptide PA1 (47). CDR surfaces are shown colored pale blue (VH) and light green (VL). The amino acid positions of MAb 12A1 which were mutagenized are shown in yellow, except for position 80 in magenta, located in the VH framework, and positions 33 and 57 corresponding to the double mutation. Amino acids at these positions for MAbs 2H1, 12F1, and 13F1, respectively, are as follows: 31, S, N, and S; 33, F, F, and Y; 50, M, M, and A; 53, S, I, and N; 56, D, N, G; 57, K, N, and S; 80, L, D, and Y; and 103, A, F, and Y.
FIG. 3
FIG. 3
IF patterns produced by parental MAbs 12A1 (a) and 13F1 (b) and variants 12A1VH-12A1VL (c), M50A (d), D80Y (e), and the F33Y N57Y double mutant (f). Magnification, ×250.
FIG. 4
FIG. 4
Scanning electron micrographs of yeast cells coated with MAbs 12A1VH-12A1VL (a), 13F1 (b), F33Y (c), and the F33Y N57Y double mutant (d). Bar, 10 μm. For each Ab at least 15 to 20 cells were imaged, and representative yeast cells are shown.
FIG. 5
FIG. 5
Competition ELISA between Abs 12A1, 13F1, 12A1VH-12A1VL, and the F33Y N57Y double mutant with the IgG3 MAb 3E5. At increasing concentrations of MAbs 12A1 and 12A1VH-12A1VL the binding of MAb 3E5 is inhibited. In contrast, no significant inhibition of MAb 3E5 binding is produced by either MAb 13F1 or the F33Y N57Y double mutant. AP-GAM-IgG3, alkaline phosphatase-conjugated anti-mouse IgG3.
FIG. 6
FIG. 6
Zeta potentials of C. neoformans cells coated with MAbs 12A1 and 13F1 and the various 12A1→13F1 mutants. ∗, the zeta potential of the N56G variant was significantly lower (P = 0.017) than that of the parent MAb 12A1. Error bars indicate standard deviations.
FIG. 7
FIG. 7
Reactivities of MAbs 12A1 and 13F1 and various 12A1→13F1 mutants with the anti-idiotypic MAb 7B8 as determined by ELISA.
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References

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