Podocalyxin/Gp135 藉雙重訊息正確運送至細胞頂域

A bipartite signal regulates the faithful delivery of apical domain marker podocalyxin/Gp135

10.6342/NTU.2008.00410

余俊頴

極性 ； Gp135 ； 頂部區域 ； 蛋白質運送 ； 乳醣凝集素 ； EBP50 ； GP135 ； apical domain ； sorting ； polarity ； Galectin ； EBP50

臺灣大學分子醫學研究所學位論文

2008年

博士

周祖述

英文

PC/Gp135近來發現參予了細胞頂部區域的形成，此功能的執行需要Gp135被正確的運送至頂部區域。我們發現Gp135藉由雙訊息的雙重保障，確保了正確的運送。此雙重訊息由Gp135蛋白上兩區域所組成，一者為細胞外富涵Ｏ型醣化修飾的區域，另一為細胞內可與PDZ結構域辨認結合的序列。我們藉由接合喪失此序列的Gp135與此序列所辨認反應的PDZ結構域蛋白(EBP50)，來確認序列的功能。根據我們的觀察，EBP50在高基式體即與Gp135接合，加速Gp135的多單位體形成，一同組成運送至頂部區域的複合體。缺少序列，無法與EBP50反應的Gp135，或是剃除細胞內的EBP50，皆會造成Gp135延遲離開脂筏，無法順利組成多單位體，並且錯誤運送至MDCK細胞的側邊區域。這些錯誤運送的Gp135會藉由PKC酵素所影響的機制，快速的被細胞內噬。另一方面，我們發現了乳糖凝集素-8可與細胞外富涵O型醣修飾的區域結合，促成Gp135的多單位體形成，與中和Ｏ型醣上由唾液酸修飾所造成的負電荷，避免負電排斥力造成運送液泡的分解。與剔除EBP50的反應類似，將細胞內的乳糖凝集素-8剃除，亦能使Gp135延遲離開脂筏，並錯誤的運送至MDCK細胞的側邊區域。因此，我們提出了一種模型，說明了高負電價的膜蛋白，可藉由細胞內的接合蛋白(EBP50)與細胞外的中和接合蛋白(乳糖凝集素-8)，形成運送至頂部區域的複合體，有效且正確的運送Gp135至頂部區域。此外，我們發現細胞可藉由不同的乳糖凝集素，將具有相對應醣修飾的蛋白聚集成相異的 運送液泡。藉此機制，細胞可利用醣化來控制蛋白質的運送。

Podocalyxin (PC)/Gp135 was recently demonstrated to participate in the formation of a pre-apical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation rich region and the intracellular PDZ domain binding motif. The function of this PDZ binding motif could be substituted with a fusion construct of Gp135 with Ezrin binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at Golgi apparatus, and facilitates oligomerization and sorting of Gp135 into clustering complex. Defective connection between Gp135 and EBP50 or EBP50 knock-down results in a delayed exit from the detergent resistant microdomain (DRM), failure of oligomerization, and basolateral missorting of Gp135. The basolaterally missorted EBP50 binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. Moreover, we found that Galectin-8 could cluster Gp135 and neutralize the negative charges of Gp135 through interacting with extracellular O-glycosylation rich region. Knock-down of Galectin also delay the exist from DRM and causes the basolateral missorting of Gp135.According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aids of its cytoplasmic partner EBP50 and extracellular partner Galectin-8. We also found that Galectins could segregate glycoproteins into different sorting cargos. We propose that cells could direct the sorting and timing of glycoprotein by controlling the glycosylation and Galectin expression.