Maeda H, Miyamoto M, Kokeguchi S, Kono T, Nishimura F, Takashiba S, Murayama Y
Department of Periodontology and Endodontology, Okayama University Dental School, Okayama, Japan.
FEMS Immunol Med Microbiol. 2000 Jul;28(3):219-24. doi: 10.1111/j.1574-695X.2000.tb01480.x.
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.
牙龈卟啉单胞菌是人类牙周病的一种假定病原体，它拥有一种60 kDa的热休克蛋白（hsp60，GroEL）。由于与人类hsp60的序列相似性，GroEL同源物被认为是自身免疫反应中的关键分子。在本研究中，通过用截短的牙龈卟啉单胞菌GroEL（PgGroEL）进行Western免疫印迹以及多针合成肽方法，对牙龈卟啉单胞菌GroEL上的B细胞表位进行了分析。为检测牙周炎患者体内自身抗体的产生，用人牙龈成纤维细胞进行了Western免疫印迹。从克隆的牙龈卟啉单胞菌groEL基因构建缺失突变体，并从这些缺失突变体中制备了4个C末端截短的PgGroEL和1个N末端截短的PgGroEL。牙周炎患者的血清与本研究中使用的所有截短的PgGroEL发生反应。结果表明，B细胞表位遍布于PgGroEL。为确定B细胞表位的详细位置，合成了覆盖整个PgGroEL的84个十肽，并检测了血清IgG对这些肽的反应。使用合成肽进行的表位定位证实，B细胞表位遍布PgGroEL全长，并显示PgGroEL与人类hsp60之间高度保守的肽段能被血清抗体识别。用30例牙周炎患者和10例牙周健康受试者的血清检测了对人牙龈成纤维细胞的免疫反应性。在两名患者中检测到了针对人牙龈成纤维细胞中65 kDa抗原（与人类hsp60分子量相同）的IgG抗体。虽然在牙周炎患者中针对人类hsp60产生IgG可能是罕见情况，但表位定位结果表明PgGroEL有与人hsp60发生交叉反应的可能性。
