Fine specificity of B-cell epitopes on Felis domesticus allergen I (Fel d I): effect of reduction and alkylation or deglycosylation on Fel d I structure and antibody binding.
The repertoire of B-cell epitopes on the major cat allergen, Fel d I, was analyzed with monoclonal antibodies (MoAbs) in topographic mapping studies and in immunoassays with antigen derived from other cat (Felidae) species. Four essentially nonoverlapping epitopes on Fel d I, designated Fd1A to D, were defined by use of 15 anti Fel d I MoAbs in cross-inhibition radioimmunoassay. Only MoAbs directed against epitope Fd1B bound to putative Fel d I homologues in hair and dander extracts from seven other feline species (Panthera species, [n = 5], Leptailurus serval, and Leopardus pardalus). Quantitative monosaccharide analysis showed that Fel d I was a glycoprotein, containing high levels of fucose, as well as glucosamine, galactose, and mannose. Binding of MoAbs and human IgG or IgE antibody to native, reduced and alkylated or deglycosylated Fel d I was compared by means of immunoprecipitation and immunoassay, and the effects of these treatments on the structure of Fel d I were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. On reduction and alkylation, Fel d I dissociated into 14 kd and 3.2 kd peptides, and deglycosylation with trifluoromethane sulfonic acid produced a 12 to 14 kd peptide. These procedures resulted in a 100- to 1000-fold loss in murine or human antibody binding activity and caused significant loss of secondary structure, as judged by circular dichroism spectroscopy. Treatment with potassium hydroxide also caused a marked loss in antigenic reactivity. In contrast, enzymatic deglycosylation generated a 9 kd peptide, which showed strong reactivity with murine and human antibodies, comparable to native Fel d I. The results show that MoAbs define a broad repertoire of B-cell epitopes on Fel d I, one of which is expressed by other cat species. These epitopes are conformational and do not appear to involve oligosaccharide residues.
Vailes
L D
LD
Department of Medicine, University of Virginia, Charlottesville 22908.
Li
Y
Y
Bao
Y
Y
DeGroot
H
H
Aalberse
R C
RC
Chapman
M D
MD
D000478:Alkylation; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology*; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D010084:Oxidation-Reduction
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; C059296:Fel d 1 protein, Felis domesticus