The sedoheptulose kinase carbohydrate kinase-like protein (CARKL) is critical for immune cell activation, reactive oxygen species (ROS) production, and cell polarization by restricting flux through the pentose phosphate pathway (PPP). To date, little is known about CARKL in regulating immune responses in marine invertebrates. In this study, we first cloned and characterized the CARKL gene from Apostichopus japonicus (designated as AjCARKL). Time-course analysis revealed that Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly downregulated AjCARKL mRNA expression. Furthermore, AjCARKL overexpression in cultured coelomocytes not only significantly inhibited the mRNA expression level of the rate-limiting enzyme glucose-6-phosphate dehydrogenase of the PPP but sharply decreased coelomocyte proliferation, ROS production, and phagocytic rate. Additionally, AjCARKL overexpression in mouse peritoneal macrophages (RAW264.7 cells) significantly attenuated the intracellular ROS production and sensitized the M2 phenotype macrophage polarization. These results revealed that AjCARKL serves as a rheostat for cellular metabolism and is required for proper immune response by negatively regulating PPP in pathogen-challenged A. japonicus.

中文翻译：

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Sedoheptulose 激酶在病原体攻击的海参 Apostichopus japonicus 中架起了磷酸戊糖途径和免疫反应。

景天庚酮糖激酶碳水化合物激酶样蛋白 (CARKL) 通过限制通过戊糖磷酸途径 (PPP) 的通量，对免疫细胞激活、活性氧 (ROS) 产生和细胞极化至关重要。迄今为止，关于 CARKL 在调节海洋无脊椎动物的免疫反应方面知之甚少。在这项研究中，我们首先克隆并表征了刺参（Apostichopus japonicus）（命名为 AjCARKL）的 CARKL 基因。时程分析显示，体内灿烂弧菌挑战和体外脂多糖刺激显着下调 AjCARKL mRNA 表达。此外，培养的体腔细胞中 AjCARKL 的过表达不仅显着抑制了 PPP 限速酶葡萄糖-6-磷酸脱氢酶的 mRNA 表达水平，而且显着降低了体腔细胞的增殖，ROS 的产生和吞噬率。此外，小鼠腹腔巨噬细胞（RAW264.7 细胞）中的 AjCARKL 过表达显着减弱了细胞内 ROS 的产生并使 M2 表型巨噬细胞极化变得敏感。这些结果表明，AjCARKL 作为细胞代谢的变阻器，是通过负调节病原体攻击的日本 A. japonicus 中的 PPP 来进行适当免疫反应所必需的。