Analysis of the effects of mucous protease inhibitor on periodontopathic bacteria and their proteases

牙周病多见于50至60岁。它的病原是一些选定的微生物，即所谓的牙周病微生物，但我们观察到牙龈卟啉单胞菌和其他牙周病细菌习惯在牙周健康的儿童和20多岁的年轻人的口腔上。因此，这些观察结果表明，口腔内的抗菌药物等宿主因子在疾病的发生中起着重要作用，这些因子与引起组织损伤和炎症反应的蛋白酶、脂多糖等细菌致病因子之间的平衡似乎很重要。在这项研究中，我们研究了口腔细菌产生的蛋白酶的加工。此外，重点研究了粘膜蛋白酶抑制剂（mucous protease inhibitor， MPI）作为宿主因子对口腔细菌蛋白酶的影响。重组MPI表达：构建his标记的MPI表达载体，在大肠杆菌中表达重组MPI。为了纯化rMPI，从细菌裂解液中制备含有rMPI的包涵体，溶解后用亲和层析纯化rMPI。MPI对细菌蛋白酶的影响：研究了口服表皮葡萄球菌谷氨酸特异性蛋白酶（GluSE）、V8蛋白酶和胰蛋白酶对MPI的抑制作用。结果表明，MPI对这些蛋白酶没有明显的体外抑制作用。通过2D-PAGE、MS和氨基酸测序研究了GluSE的加工过程，并鉴定了信号序列和前肽序列。前肽显著抑制GluSE的蛋白水解活性。研究了前肽与MPI的序列相似性。

The onset of periodontal disease is mostly observed at the age from 50s to 60s. Its etiological agents are some selected microorganisms, so-called periodontopathic microorganisms, but we observed that Porphyromonas gingivalis and other periodontopathic bacteria habit on the oral cavity of periodontally healthy children and young adults at the age of 20s. Therefore, these observations suggest that host factors such as antibacterial agents in the oral cavity play an important role in the onset of the diseases, and that a balance between these factors and bacterial pathogenic factors like proteases and lipopolysaccharide that cause tissue damages and inflammatory responses seems important. In this study, we investigated the processing of proteases produced by oral bacteria. In addition, mucous protease inhibitor (MPI) was focused as a host factor and its effects on oral bacterial proteases were studied.1. Recombinant expression of MPI : His-tagged MPI expression vectors was constructed and recombinant MPI was expressed in Escherichia coli. In order to purify, inclusion body containing rMPI was prepared from bacterial lysate, dissolved, and then rMPI was purified by using affinity chromatography.2. Effects of MPI on bacterial proteases : Inhibition activity of MPI was studied with glutamic acid specific protease (GluSE) from oral Staphylococcus epidermidis and with V8 protease and trypsin. It was demonstrated that MPI did not significantly inhibit these proteases in vitro. Processing process of GluSE was investigated by 2D-PAGE, MS, and amino acid sequencing, and as a result, signal and prepeptide sequences were identified. Prepeptide significantly inhibited the proteolytic activity of GluSE. Sequence similarity between the prepeptide and MPI was investigated.