CLONING AND SEQUENCING OF THE GENE THAT CODES FOR THE KLEBSIELLA-PNEUMONIAE GROEL-LIKE PROTEIN ASSOCIATED WITH ANKYLOSING-SPONDYLITIS

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the s acroiliac joints and vertebral column of unknown aetiology, but strong ly related to the presence of the HLA-B27 antigen. The participation o f bacterial infections as triggering factors have also been suggested. We have associated the 60kDa heat shock protein of Klebsiella pneumon iae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitop es involved in the recognition. In order to know the amino acid sequen ce of HSP60Kp, and to be able to analyse in the future the relevant ep itopes, we amplified by PCR and cloned the gene coding for this protei n into the Smal site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniq ues. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this seque nce was compared to the antigenic analysis of the reported sequences o f Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a so ftware to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) resi dues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 o f Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, w hich indicates B cell epitopes, showed three similar strongly antigeni c regions in the three proteins. (C) 1998 Academic Press.