哺乳动物原钙粘蛋白18生物学功能初探
摘要
钙粘蛋白是一类钙离子依赖的粘附分子,为单次跨膜的糖蛋白,对细胞的粘附具有重要作用,广泛参与到细胞的识别、通讯、运动、分化等过程,而钙粘蛋白表达的改变可以引起肿瘤的发生、迁移和侵袭等过程。原钙粘蛋白18(Protocadherin18, PCDH18)是一个62原钙粘蛋白,斑马鱼中发现有两个PCDH18同源蛋白,Pcdhl8a和Pcdhl8k,实验表明,尽管缺少β-连环蛋白(β-Catenin)结合位点,斑马鱼Pcdhl8a能够介导细胞粘附,在胚胎发育中的细胞迁移过程中发挥非常重要的作用。但是,我们对哺乳动物PCDH18蛋白的基本生物学功能仍缺乏了解,包括：(1)哺乳动物PCDH18蛋白的组织表达情况是怎样的,(2)哺乳动物PCDH18蛋白能否介导细胞粘附,(3) PCDH18缺少β-连环蛋白结合位点,它是如何与β-连环蛋白、从而与细胞骨架相联系的。
β-Catenin首先是作为一种具有粘附功能的分子得到重视,它与E-钙粘蛋白(E-cadherin)相互作用形成复合物,从而介导同型细胞间的粘附功能。我们发现支架蛋白MAGI-1(Membrane-associated guanylate kinase inverted-1, MAGI-1)能够与PCDH18相互作用,可能提供了PCDH18与β-连环蛋白之间的联系,从而在PCDH18介导的细胞粘附过程中发挥重要作用。MAGI-1作为一个支架蛋白,含有多个介导蛋白相互作用的结构域,包括1个GK结构域,2个WW结构域,6个PDZ结构域,在多个组织中发挥了把膜蛋白和细胞骨架联系在一起的桥梁作用。我们发现MAGI-1通过其PDZ3结构域与PCDH18相互作用,而有研究表明MAGI-1能通过其PDZ5结构域结合β-连环蛋白,因此,我们推测MAGI-1提供了PCDH18与p-连环蛋白之间的联系,从而在PCDH18介导的细胞粘附过程中扮演了重要角色。
我们利用多种分子和细胞生物学手段对小鼠PCDH18蛋白的功能进行了初步探讨。RT-PCR检测PCDH18mRNA在小鼠各组织中的表达情况,显示PCDH18mRNA在小鼠各组织中广泛表达；荧光显微镜检测PCDH18-GFP的亚细胞定位表明PCDH18在细胞与细胞连接处表达量较高。免疫共沉淀实验和GST-pull down技术检测PCDH18、 MAGI-1、β-连环蛋白形成蛋白复合体的情况表明PCDH18的胞内段能与MAGI-1的PDZ3结构域相互作用,MAGI-1和p-连环蛋白能在体外结合,但是PCDH18, MAGI-1和β-连环蛋白三种蛋白形成复合物的情况由于时间限制和条件摸索原因需要再进一步的检测。我们还尝试利用L细胞研究PCDH18介导细胞粘附的功能,目前转染体外培养的L细胞后检测不到PCDH18的表达,因此还需进一步优化实验条件。
本研究不仅能帮助我们初步认识PCDH18的表达模式和生物学功能,也为进一步深入了解PCDH18介导细胞粘附的机制打下了良好的基础。
Cadherin is a kind of calcium ion-dependent adhesion molecule. It is single-stranded transmembrane glycoprotein and plays an important role in cell adhesion. Meanwhile, it gets involved in identification, communication, movement and differentiation of cell widely. The change of its expression can cause the occurrence, migration and invasion of tumor. Protocadherin18(PCDH18) is a δ2protocadherin. Two PCDH18homologous proteins are found in zebrafish, namely, Pcdhl8a and Pcdhl8k. Though there is no β-catenin binding site, the experiment shows that Pcdhl8a in zebrafish can mediate cell adhesion and play an important role in the process of cell migration during embryonic development. However, we still lack knowledge of the basic biological function of PCDH18protein of mammals, including:(1) what is the tissue expression of PCDH18protein of mammals,(2) whether PCDH18protein of mammals can mediate cell adhesion,(3) since PCDH18lacks β-catenin binding site, how can it connect with β-catenin, and then connect with cytoskeleton.
β-Catenin receives attention as a kind of molecule with adhesion function at first. It interacts with E-cadherin and forms complex and thus mediates the adhesion function among homoplastic cells. We found that scaffolding protein MAGI1(Membrane-associated guanylate kinase inverted-1) can interact with PCDH18and this may offer the connection between PCDH18and β-catenin, and thereby play an important part in cell adhesion process mediated by PCDH18. MAGI-1, as a scaffolding protein, contains multiple structural domains formed through interaction among mediated proteins, including one GK domain, two WW domains and six PDZ domains. They function as a bridge to connect membrane protein with cytoskeleton in multiple tissues. We find that MAGI-1interacts with PCDH18through its PDZ3domain Meanwhile, there is study showing that MAGI-1can combine with β-Catenin through its PDZ5domain; therefore, we guess that MAGI-1offers the connection between PCDH18and β-catenin and thus plays an important role in cell adhesion process mediated by PCDH18.
We have a research on the function of PCDH18protein of murine through making use of various molecules and methods of cell biology. Detection of the expression of PCDH18mRNA in different tissues of murine through RT-PCR shows that PCDH18mRNA is widely expressed in the different tissues of murine and; detection of subcellular localization of PCDH18through fluorescence microscope shows that PCDH18is relatively more in junctions of cells. Detection of protein complex formation of PCDH18, MAGI-1and β-Catenin through co-immunoprecipitation assay and GST-pull down technology shows that intracellular domain of PCDH18can interact with PDZ3structural domain of MAGI-1, MAGI-1can interact with β-Catenin in vitro. PCDH18, MAG11and β-Catenin can combine in vitro, but it needs to further detect the formation of complex through interaction of PCDH18, MAGI-1and β-Catenin because of time limit and condition exploration.
This research not only can help us know expression pattern and biological functi-on of PCDH18preliminarily, but also lays good foundation for further understanding mechanism of PCDH18mediated cell adhesion.
β-Catenin首先是作为一种具有粘附功能的分子得到重视,它与E-钙粘蛋白(E-cadherin)相互作用形成复合物,从而介导同型细胞间的粘附功能。我们发现支架蛋白MAGI-1(Membrane-associated guanylate kinase inverted-1, MAGI-1)能够与PCDH18相互作用,可能提供了PCDH18与β-连环蛋白之间的联系,从而在PCDH18介导的细胞粘附过程中发挥重要作用。MAGI-1作为一个支架蛋白,含有多个介导蛋白相互作用的结构域,包括1个GK结构域,2个WW结构域,6个PDZ结构域,在多个组织中发挥了把膜蛋白和细胞骨架联系在一起的桥梁作用。我们发现MAGI-1通过其PDZ3结构域与PCDH18相互作用,而有研究表明MAGI-1能通过其PDZ5结构域结合β-连环蛋白,因此,我们推测MAGI-1提供了PCDH18与p-连环蛋白之间的联系,从而在PCDH18介导的细胞粘附过程中扮演了重要角色。
我们利用多种分子和细胞生物学手段对小鼠PCDH18蛋白的功能进行了初步探讨。RT-PCR检测PCDH18mRNA在小鼠各组织中的表达情况,显示PCDH18mRNA在小鼠各组织中广泛表达；荧光显微镜检测PCDH18-GFP的亚细胞定位表明PCDH18在细胞与细胞连接处表达量较高。免疫共沉淀实验和GST-pull down技术检测PCDH18、 MAGI-1、β-连环蛋白形成蛋白复合体的情况表明PCDH18的胞内段能与MAGI-1的PDZ3结构域相互作用,MAGI-1和p-连环蛋白能在体外结合,但是PCDH18, MAGI-1和β-连环蛋白三种蛋白形成复合物的情况由于时间限制和条件摸索原因需要再进一步的检测。我们还尝试利用L细胞研究PCDH18介导细胞粘附的功能,目前转染体外培养的L细胞后检测不到PCDH18的表达,因此还需进一步优化实验条件。
本研究不仅能帮助我们初步认识PCDH18的表达模式和生物学功能,也为进一步深入了解PCDH18介导细胞粘附的机制打下了良好的基础。
Cadherin is a kind of calcium ion-dependent adhesion molecule. It is single-stranded transmembrane glycoprotein and plays an important role in cell adhesion. Meanwhile, it gets involved in identification, communication, movement and differentiation of cell widely. The change of its expression can cause the occurrence, migration and invasion of tumor. Protocadherin18(PCDH18) is a δ2protocadherin. Two PCDH18homologous proteins are found in zebrafish, namely, Pcdhl8a and Pcdhl8k. Though there is no β-catenin binding site, the experiment shows that Pcdhl8a in zebrafish can mediate cell adhesion and play an important role in the process of cell migration during embryonic development. However, we still lack knowledge of the basic biological function of PCDH18protein of mammals, including:(1) what is the tissue expression of PCDH18protein of mammals,(2) whether PCDH18protein of mammals can mediate cell adhesion,(3) since PCDH18lacks β-catenin binding site, how can it connect with β-catenin, and then connect with cytoskeleton.
β-Catenin receives attention as a kind of molecule with adhesion function at first. It interacts with E-cadherin and forms complex and thus mediates the adhesion function among homoplastic cells. We found that scaffolding protein MAGI1(Membrane-associated guanylate kinase inverted-1) can interact with PCDH18and this may offer the connection between PCDH18and β-catenin, and thereby play an important part in cell adhesion process mediated by PCDH18. MAGI-1, as a scaffolding protein, contains multiple structural domains formed through interaction among mediated proteins, including one GK domain, two WW domains and six PDZ domains. They function as a bridge to connect membrane protein with cytoskeleton in multiple tissues. We find that MAGI-1interacts with PCDH18through its PDZ3domain Meanwhile, there is study showing that MAGI-1can combine with β-Catenin through its PDZ5domain; therefore, we guess that MAGI-1offers the connection between PCDH18and β-catenin and thus plays an important role in cell adhesion process mediated by PCDH18.
We have a research on the function of PCDH18protein of murine through making use of various molecules and methods of cell biology. Detection of the expression of PCDH18mRNA in different tissues of murine through RT-PCR shows that PCDH18mRNA is widely expressed in the different tissues of murine and; detection of subcellular localization of PCDH18through fluorescence microscope shows that PCDH18is relatively more in junctions of cells. Detection of protein complex formation of PCDH18, MAGI-1and β-Catenin through co-immunoprecipitation assay and GST-pull down technology shows that intracellular domain of PCDH18can interact with PDZ3structural domain of MAGI-1, MAGI-1can interact with β-Catenin in vitro. PCDH18, MAG11and β-Catenin can combine in vitro, but it needs to further detect the formation of complex through interaction of PCDH18, MAGI-1and β-Catenin because of time limit and condition exploration.
This research not only can help us know expression pattern and biological functi-on of PCDH18preliminarily, but also lays good foundation for further understanding mechanism of PCDH18mediated cell adhesion.
引文
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2. Gumbiner, B.M., Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol,2005.6(8):p.622-34.
3. Ulyanova, T., et al., VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin. Blood,2005.106(1):p.86-94.
4. Dimitroff, C.J., et al., Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells. Cancer Res,2005.65(13):p.5750-60.
5. Gibson, R.M., et al., Lectin and epidermal growth factor domains of P-selectin at physiologic density are the recognition unit for leukocyte binding. Blood, 1995.85(1):p.151-8.
6. Kansas, G.S., et al., A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion. J Cell Biol,1994.124(4):p. 609-18.
7. Takeuchi, T., et al., Upregulated expression and function of integrin adhesive receptors in systemic lupus erythematosus patients with vasculitis. J Clin Invest, 1993.92(6):p.3008-16.
8. Hynes, R.O., Integrins:bidirectional, allosteric signaling machines. Cell,2002. 110(6). p.673-87.
9. van der Flier, A. and A. Sonnenberg, Function and interactions of integrins. Cell Tissue Res,2001,305(3):p.285-98.
10 Kilger, G, et al., Differential regulation of alpha 4 integrin-dependent binding to domains 1 and 4 of vascular cell adhesion molecule-1. J Biol Chem,1995. 270(11):p.5979-84.
11. Takeichi, M, Cadherins:a molecular family important in selective cell-cell adhesion. Annu Rev Biochem,1990.59:p.237-52.
12. Gumbiner, B, B. Stevenson, and A Grimaldi, The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J Cell Biol,1988.107(4):p.1575-87.
13. Renaud-Young, M. and W.J. Gallin, In the first extracellular domain of E-cadherin, heterophilic interactions, but not the conserved His-Ala-Val motif, are required for adhesion. J Biol Chem,2002.277(42):p.39609-16.
14. Blaschuk, O.W., et al., Identification of a cadherin cell adhesion recognition sequence. Dev Biol,1990.139(1):p.227-9.
15. Takeichi, M., Morphogenetic roles of classic cadherins. Curr Opin Cell Biol, 1995.7(5):p.619-27.
16. Hulpiau, P. and F. van Roy, Molecular evolution of the cadherin superfamily. Int J Biochem Cell Biol,2009.41(2):p.349-69.
17 Takeichi, M. and K. Abe, Synaptic contact dynamics controlled by cadherin and catenins. Trends Cell Biol,2005.15(4):p.216-21.
18. Suzuki, S.C. and M. Takeichi, Cadherins in neuronal morphogenesis and function. Dev Growth Differ,2008.50 Suppl 1:p. S119-30.
19. Vanhalst, K., et al., delta-Protocadherins:a gene family expressed differentially in the mouse brain. Cell Mol Life Sci,2005 62(11):p.1247-59.
20. Redies, C, K. Vanhalst, and F. Roy, delta-Protocadherins:unique structures and functions. Cell Mol Life Sci,2005,62(23):p.2840-52
21 Yu, J.S., et al., PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer. Oncogene,2008.27(34) p 4657-65.
22 Li, Z., et al., Role of PCDH10 and its hypermethylation in human gastric cancer. Biochim Biophys Acta,2012.1823(2):p.298-305.
23 Bertrand, K.C., et al., PCDH10 is a candidate tumour suppressor gene in medulloblastoma Childs Nerv Syst,2011.27(8):p.1243-9.
24 Yu, B., et al., High-resolution melting analysis of PCDH10 methylation levels in gastric, colorectal and pancreatic cancers Neoplasma,2010.57(3):p. 247-52.
25. Wang, K.H., et al., Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings. Cancer Sci,2009.100(11):p.2175-80.
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