利用結締組織細胞株L929探討小白鼠子宮分泌24p3蛋白質的內吞作用機制

Abstract

24p3蛋白為分子量25.8 kD的醣蛋白，屬於疏水性分子結合蛋白家族中的一員。24p3蛋白會在雌鼠動情前期之子宮組織表現並分泌至子宮腔內。小鼠副睪是雄鼠唯一發現24p3蛋白表現的生殖系統組織。此蛋白可結合於精蟲的頭端，增強其活動性，但抑制由牛血清蛋白引發的精蟲獲能作用並減低小鼠受孕能力。在血球細胞上被認為與環境壓力和細胞凋亡有關，但在生物體確實的功能至今尚未明瞭。 疏水性分子結合蛋白家族可能是藉由細胞表面受體調控的內吞作用攜帶疏水性分子進入細胞中促成進一步的訊號傳遞，因此推測24p3蛋白會與細胞表面蛋白結合，引發細胞生理作用的改變。實驗室之前的研究中發現24p3蛋白可進入小鼠結締組織細胞株L929，有引發細胞凋亡的可能，因此利用L929細胞株探討24p3蛋白進入細胞的路徑及尋找細胞上未知的24p3蛋白結合蛋白，以期待未來24p3蛋白對傳遞分子訊息的研究有所幫助。本實驗證實了24p3蛋白確實會與L929細胞表面進行專一性結合。實驗中使用影響受體調控內吞作用的藥物則可影響24p3蛋白在細胞內的傳遞，確實了解24p3蛋白進入細胞之路徑。由24p3蛋白與溶酶體膜上的Lamp-2蛋白在L929細胞內的結合位置一致的觀察結果，以及內吞至細胞內biotin標記24p3蛋白的減少，顯示24p3蛋白可能內吞至細胞後進入分解的階段。萃取細胞膜蛋白進行二維電泳後，轉漬到PVDF膜，進行24p3蛋白結合實驗結果顯示有一較強的結合蛋白，並利用24p3蛋白建立的親和性管柱分析，分離出幾個可能和24p3蛋白結合的膜蛋白，希望未來能藉由質譜儀定序的結果找出L929細胞膜上與24p3蛋白結合的蛋白質，提供24p3蛋白在細胞功能研究的基礎。

24p3 protein is a 25.8 kD glycoprotein which belongs to the lipocalin superfamily. It is expressed and secreted from uterine endometrial epithelia during the proestrous phase of female mice, but is constitutively expressed in epididymis. The protein could associate with sperm head and then enhance the sperm motility, but was suggested as a stressor of sperm capacitation and fertility. It was also considered as an apoptotic factor or inflammatory factor in blood cells. The real function is unclear. Our previous studies showed that 24p3 protein could internalize into the cell and had effects on mouse connective tissue cell line, L929. Lipocalin has been proposed internalizing into the cells via receptor-mediated endocytosis and triggering further signal transduction. The unclearity of the 24p3 protein interacting molecule in cell membrane encouraged us to find out the interacting molecule(s) from the cell. Using L929 cell line as a model, we attempted to demonstrate the unknown interacting protein and transition pathway. The competition assay and endocytosis inhibition studies indicated that 24p3 protein could bind to the cell surface specifically and transport into the cell via receptor-mediated endocytosis pathway. Observation the locations of FITC-LAMP-2 Ab(lysosome- associated membrane protein-2 antibody) and the Alexa555-24p3 protein under fluorescence microscope revealed that the 24p3 protein should be localized in lysosome. The degradation of internalized 24p3 protein in L929 cells coincided with the colocalization of 24p3 and lysosome. Western blotting analysis of L929 cell membrane protein extract with 24p3 protein showed stress-70 protein might be the interacting protein of 24p3 protein in the PVDF membrane. We constructed a 24p3 protein-coupled affinity column to separate several 24p3 protein associated-membrane protein candidates after flow in the L929 cell membrane protein extract. Furthermore, we will identify the interacting protein of 24p3 protein from L929 cell line by the mass spectrum analysis and elucidate the 24p3 protein signal transduction in the cells.