摘要
NK细胞在机体的免疫监视中发挥着重要作用。它可以识别并杀伤肿瘤细胞和病毒感染的细胞。NK细胞对靶细胞的识别和杀伤依赖于它表面表达的各种激活型和抑制型的受体。这两类NK细胞受体与靶细胞上配体结合后促进或者抑制NK细胞的杀伤作用。除了NKG2D、NKp30、NKp44和NKp46等受体以外,NK细胞表面还表达一类特殊受体,可以识别Nectin/Necl家族蛋白。Nectin/Necl家族共有9个成员,分别是Nectin-1,2,3,4和Neel-1,2,3,4,5这个家族蛋白的主要功能是介导细胞之间的粘附。其中Nectin-2、Necl-2和Necl-5还可以作为配体被NK细胞受体识别。DNAM-1和TIGIT分别是激活型受体/抑制型受体,它们识别共同的配体Nectin-2和Necl-5。CRTAM是一种激活型的受体,它识别的配体是Necl-2。CD96的配体也是Necl-5,但其作为激活型还是抑制型的受体仍存争议。本研究旨在阐明CRTAM、CD96和DNAM-1与配体的相互作用机制。
CRTAM与配体Necl-2的相互作用可以促进NK细胞和T细胞对肿瘤细胞的杀伤。除此之外,二者相互作用可以促进CD8+T细胞分泌Y干扰素,促进CD8+T细胞在淋巴结里的停留和活化。它们还在自身免疫病中发挥作用,在小鼠的自身免疫性脱毛症模型中,活化的CD8+T通过CRTAM杀伤表达Necl-2的毛囊角化细胞。但CRTAM与Necl-2相互作用的分子机制并不清楚。此外,CRTAM和Necl-2本身还分别参与了细胞粘附作用,这种细胞粘附作用与它们介导的免疫识别有什么关系也亟待研究解决。
本研究中,在体外成功制备了CRTAM/Necl-2复合体蛋白,并解析了复合物的晶体结构。在CRTAM/Necl-2复合体的结构中,我们在两个蛋白的接触面上找到了一对“双锁钥”区域负责他们之间的相互结合；我们进一步在双“锁钥”区域引入突变,通过表面等离子共振实验和四聚体染色实验验证了区域上关键氨基酸对于复合物形成的重要作用。同时,我们通过体外生化实验证实CRTAM和Necl-2本身都可以形成同源二聚体,有意思的是,它们形成同源二聚体的结合面与它们形成异源二聚体的结合面相同。这就揭示了同源二聚化和异源二聚化的竞争关系。我们通过体外的细胞接合实验进一步验证了上面的竞争模式。由于CRTAM和Necl-2的同源二聚化介导了细胞粘附,而它们互相二聚化介导了免疫识别,这也体现了细胞粘附和免疫识别相互竞争的关系。通过我们的结构学研究发现的这种免疫分子结合的竞争关系在生理上可能具有重要意义,例如Necl-2同源二聚化介导的肿瘤细胞的粘附和聚集会促进它们向血管内皮细胞的侵入和转移,而CRTAM与Necl-2的识别一方面会促进NK细胞对肿瘤细胞的杀伤,另一方面会抑制肿瘤细胞自身的粘附、聚集和向组织的转移。
DNAM-1作为NK细胞另一个重要的表面受体在NK细胞对肿瘤细胞的杀伤中发挥重要作用。如DNAM-1与配体Nectin-2和Necl-5的相互作用促进了NK细胞对结肠癌细胞、大肠癌细胞、宫颈癌细胞和黑色素瘤细胞等多种肿瘤细胞的杀伤。除此之外,DNAM-1与配体的作用还可以调节T细胞的发育与分化,参与NK细胞和T细胞与抗原呈递细胞之间的互作等。DNAM-1在很多自身免疫病中发挥作用,如DNAM-1与配体参与了移植物抗宿主病、系统性红斑狼疮症和II型顽固性乳糜泻中等疾病造成的免疫损伤。然而DNAM-1与配体Nectin-2和Necl-5是如何相互作用的?DNAM-1、TIGIT和CD96共用相同的配体Necl-5,它们与配体的作用机制是否相同?这就是我们想要研究的问题。我们在体外表达了DNAM-1和CD96蛋白,及其配体Necl-5和Nectin-2,并成功制备DNAM-1和两个配体的复合体蛋白。我们进一步解析了人和小鼠DNAM-1以及人CD96的结构。通过与前人解析的TIGIT/Necl-5的结构比较,我们在DNAM-1、CD96以及对应配体上找到了负责结合的可能氨基酸。从而在分子水平揭示了NK细胞上这类受体结合配体的共有分子机制。
综上所述,我们的研究阐明了NK细胞上CRTAM、CD96和DNAM-1等受体与其配体的相互作用分子基础,为NK细胞免疫识别的机制以及可能得后续的基于结构的药物设计打下了基础。
NK cells play important roles in immune surveillance. They could detect and kill tumor cells or virus infected cells directly through lymphocyte cytotoxicity. The recognition and killing of target cells depend on activating and inhibiting receptors expressed on NK cells, which activates or inhibits the cytotoxity respectively. Besides common receptors such as NKG2D、NKp30、NKp44and NKp46, NK cells also express a specific kind of receptors recognizing proteins which belongs to Nectin/Necls family members whose major role are mediating cell adhesion. There are9members in this family, Nectin1,2,3,4and Necl-1,2,3,4,5.Nectin-2, Necl-2and Necl-5are also ligands for a spefic kind of NK cell receptors, which includes CRTAM, DNAM-1,CD96and TIGIT. Though DNAM-1and TIGTI are stimulating and inhibitory receptors respectively, they share the same ligands nectin-2and necl-5. CRTAM is an activating receptor which recognize Necl-2. CD96recognize Necl-5. Whether CD96is an activating or inhibitory receptor is still controversial.Our research aims to delineate the mechanism of how CRTAM,CD96and DNAM-1recognize their ligand.
The interaction of CRTAM and Necl-2stimulate NK cells and T cells to kill tumor cells. Besides, binding between CRTAM and Necl-2faciliates CD8+T cells to secrete interferon y as well as regulates their retention in the lymph nodes.The excessive interaction beween these two proteins often results in autoimmune diseases. In mouse autoimmune alopecia model, activating CD8+T cells express CRTAM and kills necl-2positive hair follicle keratinocytes.But mechanism of the interaction between these two proteins is not clear. In addition, CRTAM and Necl-2are also involved in cell adhesion. What is relationship between cell adhesion and immune recognition both mediated by these proteins is also unkown.
In our research, we expressed CRTAM/Necl-2complex in vitro, found a "double lock and key" region located at the interface of CRTAM and necl-2, which was responsible for binding of these two proteins.Key amino acids in this region are identied and futher validated by biochemical assays such as surface plasmon resonance and tetramer staining. The same interafaces both in CRTAM and necl-2were also identified,which mediated CRTAM and necl-2self-homodimerization as well as their cross heterodimerization. Since the former interaction was involved in cell adhesion whereas the latter one contributed to immune recognition, the competion between them might also reflect their competing roles in vivo. This competing mode might be significant in vivo, as necl-2expressed on tumor cells facilitated their adhesion, aggregation, binding to endothelial cells and transferring to tissues. Binding between necl-2and CRTAM resulted in weaker tumor adhesion and their killing by immune cells.
The major function of DNAM-1is to stimulate the cytotoxity of NK cells. For instance, the interaction between DNAM-1and its ligands nectin-2and necl-5stimulates NK cells to kill colon cancer cells, colorectal cancer cells, cervical cancer cells and melanoma. Besides, they also contribute to other immune process. DNAM-1also participtes in regulating T cell development and differenciation, the interaction between NK cells and antigen presenting cells, binding of leukocytes to endothelial cells. DNAM-1and its ligands are involved in the immnopathogy of graft-versus-host disease, Systemic Lupus Erythematosus and Refractory Celiac Disease Type Ⅱ. However, how DNAM-1interact with its ligands nectin-2and necl-5is unclear. Besides, DNAM-1, TIGIT and CD96share the same receptor necl-5, whether they use common mechanism is unkown. This are the questions we want to solve. We prepare DNAM-1, CD96, necl-5and nectin-2. We successfully get the complex of DNAM-1with its two ligand. By comparing the the anino acid sequences and the crystal structures of DNAM-1, CD96with the previously reported TIGIT/Necl-5. Conserve amino acids that might be responsible for complex formation.were identified in DNAM-1, CD96and TIGIT as well as their corresponding ligands. Our analysis might reveal a common receptor-ligand binding mechasm in this specific family of NK cell receptors.
In summary, our research delineate our NK cell receptor CRTAM, CD96and DNAM-1recognize their ligands, which provides foundation for research on NK cell recognition and drug design
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