Studies on the biochemical structure of the major cat allergen Felis domesticus I.
The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.
Duffort
O A
OA
Departamento de Investigación, Algeria e Immunología Abelló S.A., Madrid, Spain.
Carreira
J
J
Nitti
G
G
Polo
F
F
Lombardero
M
M
D000485:Allergens / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002415:Cats / Q000276:immunology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D007123:Immunoelectrophoresis, Two-Dimensional; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011852:Radioallergosorbent Test
D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins