Laboratory of Cytophysiology, State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
Laboratory of Cytophysiology, State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
J Biol Chem. 2018 May 25;293(21):8151-8160. doi: 10.1074/jbc.RA118.002113. Epub 2018 Apr 9.
Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface-localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.
烟酰胺腺嘌呤二核苷酸磷酸（NAADP）是一种钙动员的第二信使，调节广泛的生物活性。然而，其生物发生的机制仍存在争议。CD38 是唯一已知能够催化 NADP 和烟酸合成 NAADP 的酶。CD38 介导的催化需要酸性 pH 值，表明 NAADP 可能在酸性内溶酶体中产生，但这一假设尚未得到验证。在这项研究中，我们使用人类细胞系，专门将 CD38 导向内溶酶体系统，并评估细胞内的 NAADP 产生。首先，我们发现针对 CD38 C 端结构域上各种表位的纳米抗体可以与细胞表面定位的 CD38 结合，并诱导其内化。我们还发现 CD38 的内化是通过网格蛋白依赖的途径进行的，将 CD38 递送至内溶酶体，并提高细胞内的 NAADP 水平。我们还创建了一种用于溶酶体特异性表达的 CD38 变体，它不仅能耐受溶酶体中的降解环境，而且在提高细胞内 NAADP 水平方面比 WT CD38 更活跃。向表达 CD38 的细胞补充烟酸会显著增加细胞内的 NAADP 水平。这些结果表明，人细胞中的内溶酶体 CD38 可以产生 NAADP。它们进一步表明，CD38 向溶酶体的区室化可能允许通过底物进入来调节其活性，而不是通过酶激活，从而为调节细胞内 NAADP 产生提供了一种可靠的机制。
