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 sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (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 epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes, we amplified by PCR and cloned the gene coding for this protein into the SmaI site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 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 of Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.