Zhao Yong Juan, Zhu Wen Jie, Wang Xian Wang, Zhang Li-He, Lee Hon Cheung
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 2199 Lishui Road, Nanshan District, Shenzhen, China; Department of Physiology, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong.
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 2199 Lishui Road, Nanshan District, Shenzhen, China.
Biochim Biophys Acta. 2015 Sep;1853(9):2095-103. doi: 10.1016/j.bbamcr.2014.10.028. Epub 2014 Nov 4.
CD38 catalyzes the synthesis of two structurally distinct messengers for Ca²⁺-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca²⁺-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
CD38分别催化由胞质底物NAD和NADP合成两种结构不同的用于钙离子动员的信使分子，即环ADP核糖（cADPR）和烟酰胺腺嘌呤二核苷酸磷酸（NAADP）。CD38通常被认为是一种II型膜蛋白，其催化位点面向细胞外。我们最近发现，CD38实际上以两种相反的膜取向存在。膜拓扑结构的决定因素尚不清楚。在此，我们设计并制备了针对III型CD38的特异性抗体。我们发现，将CD38 N端尾巴中的带正电残基突变可将其取向转变为III型，催化结构域面向细胞质，并且它在产生细胞内cADPR方面完全有活性。将丝氨酸残基替换为功能上等同于磷酸化的天冬氨酸也有类似效果。突变的CD38在细胞内表达且未进行糖基化。通过改变高度保守的二硫键也可以调节膜拓扑结构。结果表明，N端片段的净电荷在决定CD38的膜拓扑结构中很重要，并且III型取向可能是CD38用于钙离子信号传导的一种功能形式。本文是名为“第13届欧洲钙研讨会”的特刊的一部分。
