Lys-gingipain (Kgp) is a major cysteine proteinase produced by the oral anaerobic bacterium Porphyromonas gingivalis, and has been implicated as a major pathogen in the development and progression of advanced adult periodontitis. This enzyme is believed to act as a major virulence factor of the disease, yet there exist no convenient and sensitive substrates for analyzing its biological activity. For a better understanding of the importance of this enzyme in the organism, there is an urgent need for specific substrates. Here we designed and synthesized two peptide 4-methyl-coumaryl-7-amides (MCA), carbobenzoxy (Z)-His-Glu-Lys-MCA, and Z-Glu-Lys-MCA, and tested their possible use as sensitive substrates for Kgp with limited specificity. Both substrates exhibited greater k(cat)/K(m) values than the best known Kgp substrates described so far. Both substrates were resistant to Arg-gingipain, another pathogenic cysteine proteinase from P. gingivalis, as well as trypsin and cathepsins B, L, and H. The levels of Kgp in various microorganisms and human cells were determined with Z-His-Glu-Lys-MCA. Little or no Kgp-like activity was detected in either other microorganisms or human cells tested. These results indicate that the present substrates are a valuable and fast tool for routine assays and for mechanistic studies on Kgp.

译文

Lys-gingipain (Kgp) 是由口腔厌氧菌牙龈卟啉单胞菌产生的主要半胱氨酸蛋白酶,已被认为是晚期成人牙周炎的发生和发展的主要病原体。该酶被认为是该疾病的主要毒力因子,但没有方便而敏感的底物来分析其生物活性。为了更好地了解这种酶在生物体中的重要性,迫切需要特定的底物。在这里,我们设计并合成了两种肽4-甲基-香豆基-7-酰胺 (MCA),即碳苯氧基 (Z)-His-Glu-Lys-MCA和Z-Glu-Lys-MCA,并测试了它们作为敏感底物的可能用途Kgp的特异性有限。与迄今为止描述的最著名的Kgp衬底相比,两种衬底均显示出更大的k(cat)/K(m) 值。两种底物均对牙龈卟啉单胞菌的另一种致病性半胱氨酸蛋白酶,以及胰蛋白酶和组织蛋白酶B,L和H具有抗性。用Z-His-Glu-Lys-MCA测定各种微生物和人类细胞中Kgp的水平。在其他测试的微生物或人类细胞中几乎没有或没有检测到Kgp样活性。这些结果表明,本发明的底物是常规测定和Kgp机理研究的有价值且快速的工具。

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