Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis.
Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.
Maeda
H
H
Department of Periodontology and Endodontology, Okayama University Dental School, Okayama, Japan.
Miyamoto
M
M
Kokeguchi
S
S
Kono
T
T
Nishimura
F
F
Takashiba
S
S
Murayama
Y
Y
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D015153:Blotting, Western; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000276:immunology; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000276:immunology*; D014169:Transformation, Bacterial
D000942:Antigens, Bacterial; D018834:Chaperonin 60; D007106:Immune Sera; D010455:Peptides