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 a llergens, the major allergen being Feld1 (formerly cat allergen 1). Fe ld1 is responsible for cat allergy and has also been establishedto cau se cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked po lypeptide chains, a light alpha-chain and a heavy beta-chain containin g an N-linked oligosaccharide. In this study a variety of endoproteina se digestions of the native allergen in combination with HPLC and matr ix-assisted laser desorption mass spectrometry was used to determine t he position of the disulfide bridges and to demonstrate that the pepti de chains are linked in an anti parallel way. Enzymatic digestion of t he reduced and alkylated peptides located the N-glycan to Asn(33). Mor eover, Feld1 is found to be partially truncated and to exist in severa l isoforms. Sequential degradation of the glycosylated peptide with sp ecific glycosidases monitored by mass spectrometry, shows that the gly can is a heterogenous tri-antennary complex type structure. The hetero geneity is caused by terminal sialic acid and a fucose residue attache d to a beta-galactose residue.