Determination of isoforms, N-linked glycan structure and disulfide bond linkages of the major cat allergen Fel d1 by a mass spectrometric approach.
The domestic cat (Felis domesticus) is an important source of indoor allergens, the major allergen being Fel d1 (formerly cat allergen 1). Fel d1 is responsible for cat allergy and has also been established to cause cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked polypeptide chains, a light alpha-chain and a heavy beta-chain containing an N-linked oligosaccharide. In this study a variety of endoproteinase digestions of the native allergen in combination with HPLC and matrix-assisted laser desorption mass spectrometry was used to determine the position of the disulfide bridges and to demonstrate that the peptide chains are linked in an anti parallel way. Enzymatic digestion of the reduced and alkylated peptides located the N-glycan to Asn33. Moreover, Fel d1 is found to be partially truncated and to exist in several isoforms. Sequential degradation of the glycosylated peptide with specific glycosidases monitored by mass spectrometry, shows that the glycan is a heterogeneous triantennary complex type structure. The heterogeneity is caused by terminal sialic acid and a fucose residue attached to a beta-galactose residue.
Kristensen
A K
AK
Department of Molecular Biology, Odense University, Denmark.
Schou
C
C
Roepstorff
P
P
D000478:Alkylation; D000485:Allergens / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D007536:Isomerism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D004220:Disulfides; D006023:Glycoproteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases; C059296:Fel d 1 protein, Felis domesticus