基于抗体M-19的胞内CD38拓扑学研究

CD38 is a multifunctional protein ubiquitously present in cells. It is also the dominant enzyme for metabolizing cyclic ADP-ribose (cADPR), an intracellular Ca2+ mobilizing messenger. CD38 is believed to be a Type II transmembrane protein, with its catalytic domain on the outside. How CD38 can then synthesize and hydrolyze intracellular cADPR has been controversial. To resolve the issue, we proposed a "Type III CD38" hypothesis, in which we proposed that cells express a certain amount of CD38 in the Type III orientation together with the majority of Type II CD38, and published the first supporting evidence. In the current proposal, we will test this hypothesis in another direction by using antibody M-19, which can recognize intracellular CD38 with the reduced C-terminal domain but not the extracellular form with the oxdized C-terminus. The preliminary data shows that the orientation of CD38 is inheritable and we isolated two clones with exclusively intra- or extra-cellular CD38 expression. We will study the topology of the intracellular CD38 and try to investigate the molecular mechanism by using the techneques such as comparative transcriptomic and proteomic analysis. We will use M-19 and another antibody, Ab90, which recognizes Type II CD38, to study the expression profiles of both Type II and Type III CD38 in different mouse tissues. Meanwhile, we will measure the cellular contant of nicotinamide adenine dinucleotide (NAD) and cADPR in the Type III/Type II CD38-expressing cells and tissues to reveal any possible correlation of orientation and activity and provide clues for the further functional studies of the CD38/cADPR signaling pathway.

膜蛋白CD38是一个多功能酶，它是合成和水解第二信使cADPR的主要蛋白。一般认为CD38是II型膜蛋白，其催化结构域位于细胞外，所以CD38代谢胞内cADPR的机制一直存在争议。为了诠释该拓扑学悖论，我们提出了"III型CD38"假说并发表了首个证据。本项目拟从另一角度论证该假说，即利用可识别还原性CD38 C端结构域的抗体论证胞内III型CD38的存在和发生机制。我们发现抗体M-19仅识别胞内CD38（还原态），而不识别胞外CD38（氧化态）。将用M-19和II型CD38抗体Ab90，对小鼠不同组织进行III型和II型CD38的表达谱分析，测定胞内NAD和cADPR含量来揭示这两型CD38表达量之间的关系。我们筛选到仅表达胞内CD38的细胞株，将以之为模型验证胞内CD38的拓扑学方向是否为III型，用比较转录组/蛋白组学等方法鉴定出调节CD38拓扑学方向的关键基因，同时阐述其发生机制。

膜蛋白CD38是一个多功能酶，它是合成和水解第二信使cADPR的主要蛋白。一般认为CD38是II型膜蛋白，其催化结构域位于细胞外，所以CD38代谢胞内cADPR的机制一直存在争议。为了诠释该拓扑学悖论，我们提出了"III型CD38"假说并发表了首个证据。本项目拟从另一角度论证该假说，即利用可识别还原性CD38 C端结构域的抗体论证胞内III型CD38的存在和发生机制。我们首先利用识别非糖基化CD38的抗体，研究了决定CD38膜拓扑学方向的N端氨基酸；然后开发CD38不同膜透性的拮抗剂，试图区分II型和III型的CD38；再次，开发了CD38的纳米抗体，用于靶向II型和III型的CD38。一开始立题的抗体M-19，我们认为它仅识别胞内CD38（还原态），而不识别胞外CD38（氧化态），但后续的研究发现，造成该差异识别的不是CD38的氧化还原状态，而是多聚化状态。对于CD38膜拓扑学的研究还在进行中，这个项目的成果为后续深入的机制阐述提供了研究工具。