FINE SPECIFICITY OF B-CELL EPITOPES ON FELIS-DOMESTICUS ALLERGEN-I (FEL D-I) - EFFECT OF REDUCTION AND ALKYLATION OR DEGLYCOSYLATION ON FELD-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 (Feli dae) species. Four essentially nonoverlapping epitopes on Fel d I, des ignated Fd1A to D, were defined by use of is anti Fel d I MoAbs in cro ss-inhibition radioimmunoassay. Only MoAbs directed against epitope Fd 1B bound to putative Fel d I homologues in hair and dander extracts fr om 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 fuco se, as well as glucosamine, galactose, and mannose. Binding of MoAbs a nd human IgG or IgE antibody to native reduced and alkylated or deglyc osylated Fel d I was compared by means of immunoprecipitation and immu noassay and the effects of these treatments on the structure of Fel d I were analyzed by sodium dodecylsulfate-polyacrylamide gel electropho resis. On reduction and alkylation, Fel d I dissociated into 14 kd and 3.2 kd peptides, and deglycosylation with trifluoromethane sulfonic a cid 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 c aused significant loss of secondary structure e, as judged by circular dichroism spectroscopy. Treatment with potassium hydroxide also cause d a marked loss in antigenic reactivity. In contrast, enzymatic deglyc osylation generated a 9 kd peptide, which showed strong reactivity wit h murine and human antibodies, comparable to native Fel d I. The resul ts 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 a re conformational and do not appear to involve oligosaccharide residue s.