使用液相層析串聯式質譜尋找醣基轉移酶GALNT14的新穎受質之醣蛋白體學研究

Glycoproteome-wide Identifications of Novel GALNT14 Substrates Using Tandem Mass Spectrometry

林郁樺(Lin Yu-Hua)

指導教授 ： 陳頌方

國立臺灣師範大學/理學院/化學系/碩士(2017年)

https://doi.org/10.6345/NTNU202202866

摘要

多肽氨端乙酰基半乳糖轉移酶14 (GALNT14)的基因型近幾年來被發現與許多癌症腫瘤的發生和其對治療後的反應相關。這些癌症種類包括肝癌、膽管癌、大腸癌、食道癌、神經母細胞瘤和乳癌。特別是從台灣900多例病患中發現GALNT14的基因型可作為預測肝癌化療療效反應的標記。然而，除了在癌細胞外緣負責傳遞凋亡訊號的死亡受體 (DR) 5外，我們並不清楚GALNT14的其他受質。於是，本研究的目標為，從醣蛋白體學的層面，透過凝集素親和性管柱再搭配高效能液相層析串聯式質譜儀 (HPLC-MS/MS) 來尋找出可能為GALNT14受質的醣基化蛋白質。首先，將GALNT14嵌入Huh7、J7和Mahlavu這三株人類肝癌細胞株內，使其穩定表達GALNT14，另外還有三株一樣的人類肝癌細胞株，但是並無嵌入GALNT14，作為對照組。接下來通過自製的凝集素親和性管柱純化出特定的醣基化蛋白質，其中凝集素是選用花生凝集素(PNA)與碗豆凝集素 (VVA)。使用胰蛋白酶將純化出的醣基化蛋白水解成生肽後進入高效能液相層析質譜儀分析。在結果中，扣除掉對照組重複鑑定到的蛋白質，三個細胞株搭配兩種凝集素，一共鑑定出305種醣基化蛋白質。我們發現這些醣基化蛋白質與核醣體這條路徑極為相關，其中許多蛋白質也清楚地顯示出與肝癌的相關性。因此本篇研究成功的進一步了解GALNT14與癌症的關聯，對未來要了解詳細致癌機制開啟了一個新視野。

關鍵字

GALNT14 ； 肝癌 ； 醣基化蛋白質 ； 凝集素親和性層析 ； 質譜

並列摘要

The glycosyltransferase GALNT14 was recently shown to mediate the oncogenesis and treatment responses of multiple cancers, including hepatocellular carcinoma, cholangiocarcinoma, colon cancer, esophageal cancer, neuroblastoma and breast cancer. Particularly, the genotype of GALNT14 was tightly associated to the therapeutic response of hepatocellular carcinoma in an accumulation of more than 900 patients treated in Taiwan. However, the substrates of GALNT14 are not entirely known, except the death receptor (DR) 5 which mediates the extrinsic apoptosis signaling of cancer cells. We aim to investigate novel substrates of GALNT14 using a lectin enrich glycoproteome-wide screening method combined with high performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The GALNT14 genomic DNA was cloned to plasmids which were then transfected to the hepatocellular carcinoma cell line including Huh7, J7 and Mahlavu, for generating stable GALNT14 overexpression cell lines. Their corresponded control cell lines were also established with only the plasmid backbone without the GALNT14 DNA. Glycoproteins of the three cell lines were captured using self-made lectin affinity column, which was packed with Peanut Agglutin lectin (PNA) or Vicia Villosa Lectin (VVA). The captured proteins were following by trypsin digestion and high performance liquid chromatography – tandem mass spectrometric analyses. In summary, 305 glycoproteins were identified in GALNT14 overexpressed cells but not in the control cells. They are found to be involved in ribosome pathway and clearly shown to be associated with hepatocellular carcinoma. Therefore, this study successfully demonstrated the relation between GALNT14 and cancers, and could pave the way for a deeper understanding of carcinogenesis studies.

並列關鍵字

GALNT14 ； hepatocellular carcinoma ； glycoprotein ； lectin affinity chromatography ； mass spectrometry

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