人類細胞表面糖蛋白SCUBE2的單離，及其特性與訊號傳遞的相關性

Isolation and characterization of a secreted,cell-surface glycoprotein SCUBE2 from humans

蔡明孜(Ming-Tzu Tsai)

指導教授 ： 徐成金 教授

中山醫學大學/健康管理學院/營養學研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00056

摘要

SCUBE2 (Signal peptide-CUB-EGF-like domain containing protein 2) 是屬於SCUBE蛋白質家族成員之一，它的區段特性是在NH2端有9個epidermal growth factor (EGF)-like repeats，一個spacer region，三個cysteine-rich motifs，及在COOH端有一個CUB domain。雖然以有許多遺傳學分析證實斑馬魚之SCUBE2基因參與Hedgehog (HH) 之訊號傳遞，但在哺乳類上,SCUBE2之完整全長cDNA及其生化功能還沒被研究。在本篇研究中，本實驗室分離出人類完整全長SCUBE2之cDNA，以及研究其參與HH訊號傳遞上扮演的角色。當本實驗室表達人類SCUBE2於HEK-293T細胞中，此蛋白是一個分泌性，而且會嵌在細胞膜上的糖基化蛋白。當本實驗室對SCUBE2做不同區段的切割之後分析，而得知spacer region或cysteine-rich repeats參與細胞膜上之表達有關。更進一步生化及功能性的分析，證實人類SCUBE2可以和 Sonic Hedgehog (SHH) 及SHH receptor PTCH1做蛋白交互作用，而且可以增強SHH之訊號傳遞。此SCUBE2增強SHH之活性是發生在細胞膜上富含膽固醇之脂筏 (lipid rafts)。本實驗室的結果顯示人類 SCUBE2是一個嶄新，而在膜上正向調控HH訊號傳遞之分子，因此人類 SCUBE2在HH相關之生物學及病理學上，比如器官發育及癌症進展，可能扮演重要角色。

關鍵字

EGF-like repeats ； CUB 區段 ； 細胞表面蛋白 ； 糖基化蛋白 ； 訊號傳遞 ； Sonic Hedgehog

並列摘要

SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2) belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by 9 epidermal growth factor (EGF)-like repeats, a spacer region, 3 cysteine-rich repeat motifs, and one CUB domain at the C terminus. Despite several genetic analyses suggested that the zebrafish orthologue of the mammalian gene SCUBE2 participates in Hedgehog (HH) signaling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signaling remains uninvestigated. In this study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signaling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted, surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with Sonic HH (SHH) and SHH receptor Patched-1 (PTCH1), and enhance the SHH signaling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel, positive component of the HH signal, acting upstream in ligand reception at the plasma membrane. Thus, human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumor progression.

並列關鍵字

EGF-like repeats ； CUB domain ； cell-surface protein, ； glycoprotein, ； signal transduction, ； Sonic Hedgehog

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