
<ArticleTitle>Molecular cloning, expression and modelling of cat allergen, <prot>cystatin</prot> (<prot>Fel d 3</prot>), a cysteine protease inhibitor.</ArticleTitle>
<AbstractText>BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, <prot>Fel d 1</prot> and <prot>albumin</prot>, have been cloned and sequenced. IgE antibodies to <prot>Fel d 1</prot> and <prot>albumin</prot> do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding <prot>cystatin</prot>, was expressed in E. coli. The three dimensional structure of cat <prot>cystatin</prot> was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human <prot>cystatin A</prot>, respectively. The cat <prot>cystatin</prot> sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed <prot>cystatin</prot> clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, <prot>Can f 1</prot> and <prot>Can f 2</prot>, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat <prot>cystatin</prot>. The three-dimensional structure of cat <prot>cystatin</prot> was modelled on human <prot>cystatin</prot> structures. CONCLUSION: A newly identified allergen, <prot>cystatin</prot> (<prot>Fel d 3</prot>), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.</AbstractText>
