Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Central Hospital, 00014, Helsinki, Finland; Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences, The Department of Biochemistry and Biotechniques, University of Helsinki, 00014, Helsinki, Finland.
Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Central Hospital, 00014, Helsinki, Finland; Department of Dental Medicine, Karolinska Institutet, Huddinge, 14104, Sweden.
Microb Pathog. 2020 Dec;149:104547. doi: 10.1016/j.micpath.2020.104547. Epub 2020 Sep 30.
Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10-50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.
光滑念珠菌（Candida glabrata，C. glabrata）细胞壁蛋白在毒力以及宿主初始免疫识别和反应中发挥作用。我们从临床医院的光滑念珠菌 T-1638 血液分离株中分离并鉴定了光滑念珠菌细胞壁蛋白酶，评估了其酶活性及其降解明胶和加工 proMMP-8 的能力，并评估了盐处理、巯基乙醇和越橘发酵汁对这些蛋白酶的调控作用。Vaccinium vitis idaea L. 细胞壁蛋白酶通过酶从细胞壁中释放出来，并用阴离子 DEAE-琼脂糖离子交换层析和凝胶过滤将 β-1,3-结合蛋白酶分为 10-50 kDa 和 >50 kDa 部分。用 MDPF-zymography 监测和分析蛋白质，并用平行银染凝胶切出五条明胶水解带进行 LC-MS/MS 分析。这些蛋白酶缺乏信号序列，表明它们是兼职蛋白酶。使用这两种级分进行了人 proMMP-8 激活测定，并使用 MMP-8 特异性抗体进行了 Western 免疫印迹验证。用发酵越橘汁抑制了 proMMP-8 转化为较低分子活性酶的转化。结果表明，兼职蛋白酶可能在光滑念珠菌的毒力中发挥作用。
