Supplementary Material for: Down regulation of Endo-beta-N-acetylglucosaminidase in Caenorhabditis elegans improves stress adaptivity

Introduction: Endo-beta-N-acetylglucosaminidase (ENGASE) is one of the key enzymes involved in the structural and functional regulations of glycoproteins. Although its enzymatic activities and applications have been well studied in vitro, its biological function in vivo yet remains to be illustrated. In this study, the biological function of ENGASE in Caenorhabditis elegans (C. elegans) was explored in detail.
Methods: An Engase gene knockout in C. elegans (CeEng-1 or CeEngase) was constructed and subjected to a panel of phenotypical and glycomics analysis. In addition, in vitro and in vivo ENGASE inhibition assays were performed.
Results: Engase knockout worm’s adaptivity to environmental stresses (heat and osmotic) was significantly improved, and its longevity was also increased mildly. A clustered change in basement membrane proteins (e.g., LAM-1, LAM-2, and EPI-1) was illustrated by Nglycopeptide analysis, suggesting ENGASE is involved in a basement membrane-based stress regulation. Then, the heat stress phenotype was further supported by in vivo CeEngase knockdown assay and in vitro and in vivo small compound inhibitory assay of CeENGASE, indicating ENGASE is a potential drug target for stress management.
Conclusion: Engase is actively involved in a basement membrane-mediated stress adaptation, and could serve as a potential target for healthcare products.

β-N-乙酰葡萄糖胺内切酶（Endo-beta-N-acetylglucosaminidase，ENGASE）是糖蛋白结构与功能调控的关键酶之一。尽管其酶活性及体外应用已得到充分研究，但其体内生物学功能仍有待阐明。本研究详细探究了ENGASE在秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）中的生物学功能。
方法：构建了秀丽隐杆线虫Engase基因敲除株（CeEng-1或CeEngase），并对其进行了一系列表型分析和糖组学分析。此外，还开展了ENGASE的体外及体内抑制实验。
结果：Engase基因敲除线虫对环境胁迫（热胁迫和渗透胁迫）的适应性显著提升，寿命也略有延长。通过N-糖肽分析发现基底膜蛋白（如LAM-1、LAM-2及EPI-1）存在聚类变化，提示ENGASE参与基于基底膜的胁迫调控。随后，体内CeEngase敲低实验及CeENGASE的体外、体内小分子化合物抑制实验进一步验证了热胁迫表型，表明ENGASE是潜在的胁迫管理药物靶点。
结论：Engase积极参与基底膜介导的胁迫适应过程，且可作为保健品的潜在靶点。