State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu 210093, China.
J Biol Chem. 2019 Oct 18;294(42):15293-15303. doi: 10.1074/jbc.RA119.010010. Epub 2019 Aug 21.
The CD38 molecule (CD38) catalyzes biogenesis of the calcium-mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, and type III CD38, with its catalytic domain facing the cytosol, has low abundance but is efficient in cyclizing cytosolic NAD to produce cADPR. The role of cell surface type II CD38 in cellular cADPR production is unknown. Here we modulated type II CD38 expression and assessed the effects of this modulation on cADPR levels. We developed a photoactivatable cross-linking probe based on a CD38 nanobody, and, combining it with MS analysis, we discovered that cell surface CD38 interacts with CD71. CD71 knockdown increased CD38 levels, and CD38 knockout reciprocally increased CD71, and both could be cocapped and coimmunoprecipitated. We constructed a chimera comprising the N-terminal segment of CD71 and a CD38 nanobody to mimic CD71's ligand property. Overexpression of this chimera induced a dramatically large decrease in CD38 via lysosomes. Remarkably, cellular cADPR levels did not decrease correspondingly. Bafilomycin-mediated blockade of lysosomal degradation greatly elevated active type II CD38 by trapping it in the lysosomes but also did not increase cADPR levels. Retention of type II CD38 in the endoplasmic reticulum (ER) by expressing an ER construct that prevented its transport to the cell surface likewise did not change cADPR levels. These results provide first and direct evidence that cADPR biogenesis occurs in the cytosol and is catalyzed mainly by type III CD38 and that type II CD38, compartmentalized in the ER or lysosomes or on the cell surface, contributes only minimally to cADPR biogenesis.
CD38 分子（CD38）催化钙动员信使环 ADP-核糖（cADPR）的生物合成。CD38 具有双重膜取向，其催化结构域面向细胞质的 III 型 CD38 丰度低，但在将细胞质 NAD 环化为产生 cADPR 方面效率很高。细胞表面 II 型 CD38 在细胞内 cADPR 产生中的作用尚不清楚。在这里，我们调节了 II 型 CD38 的表达，并评估了这种调节对 cADPR 水平的影响。我们开发了一种基于 CD38 纳米抗体的光活化交联探针，并结合 MS 分析，发现细胞表面 CD38 与 CD71 相互作用。CD71 敲低增加了 CD38 水平，CD38 敲除则相反地增加了 CD71，并且两者都可以共帽和共免疫沉淀。我们构建了一种嵌合体，包含 CD71 的 N 端片段和 CD38 纳米抗体，以模拟 CD71 的配体特性。该嵌合体的过表达通过溶酶体显著降低了 CD38 的水平。值得注意的是，细胞内 cADPR 水平并没有相应降低。通过用巴弗洛霉素阻断溶酶体降解，通过将其困在溶酶体中，大大增加了活性 II 型 CD38 的含量，但也没有增加 cADPR 水平。通过表达阻止其转运到细胞表面的内质网（ER）构建体，将 II 型 CD38 保留在内质网中，也没有改变 cADPR 水平。这些结果提供了直接的证据，证明 cADPR 的生物合成发生在细胞质中，主要由 III 型 CD38 催化，而定位于内质网或溶酶体或细胞表面的 II 型 CD38 对 cADPR 的生物合成贡献很小。
