Guangdong Laboratory for Lingnan Modern Agriculture, Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100083, China.
Int J Mol Sci. 2022 Jul 10;23(14):7617. doi: 10.3390/ijms23147617.
The protozoan pathogen infects intestinal epithelial cells and causes diarrhea in humans and young animals. Among the more than 20 genes encoding insulinase-like metalloproteinases (INS), two are paralogs with high sequence identity. In this study, one of them, INS-16 encoded by the cgd3_4270 gene, was expressed and characterized in a comparative study of its sibling, INS-15 encoded by the cgd3_4260 gene. A full-length INS-16 protein and its active domain I were expressed in , and antibodies against the domain I and an INS-16-specific peptide were produced in rabbits. In the analysis of the crude extract of oocysts, a ~60 kDa fragment of INS-16 rather than the full protein was recognized by polyclonal antibodies against the specific peptide, indicating that INS-16 undergoes proteolytic cleavage before maturation. The expression of the gene peaked at the invasion phase of in vitro culture, with the documented expression of the protein in both sporozoites and merozoites. Localization studies with antibodies showed significant differences in the distribution of the native INS-15 and INS-16 proteins in sporozoites and merozoites. INS-16 was identified as a dense granule protein in sporozoites and macrogamonts but was mostly expressed at the apical end of merozoites. We screened 48 candidate INS-16 inhibitors from the molecular docking of INS-16. Among them, two inhibited the growth of in vitro (EC = 1.058 µM and 2.089 µM). The results of this study suggest that INS-16 may have important roles in the development of and could be a valid target for the development of effective treatments.
原生动物病原体 感染肠道上皮细胞，导致人类和幼小动物腹泻。在编码胰岛素样金属蛋白酶（INS）的 20 多个基因中，有两个是具有高度序列同一性的基因。在这项研究中，其中一个基因 cgd3_4270 编码的 INS-16，与它的基因 cgd3_4260 编码的 INS-15 进行了比较表达和特性研究。全长 INS-16 蛋白及其活性结构域 I 在 中表达，并在兔中产生了针对该结构域 I 和 INS-16 特异性肽的抗体。在对卵囊粗提物的分析中，针对该特异性肽的多克隆抗体识别了约 60 kDa 的 INS-16 片段，而不是全长蛋白，表明 INS-16 在成熟前经历了蛋白水解切割。在体外培养的入侵阶段， 基因的表达达到峰值，该蛋白在孢子和裂殖子中均有表达。用抗体进行的定位研究表明，天然 INS-15 和 INS-16 蛋白在孢子和裂殖子中的分布存在显著差异。INS-16 被鉴定为孢子中的致密颗粒蛋白和大配子体，但主要在裂殖子的顶端表达。我们从 INS-16 的分子对接中筛选了 48 种候选 INS-16 抑制剂。其中两种在体外抑制 的生长（EC = 1.058 µM 和 2.089 µM）。本研究结果表明，INS-16 可能在 的发育中具有重要作用，并且可能成为开发有效治疗方法的有效靶标。
