探討抗原性醣類SSEA-4及Globo H之結合蛋白

Investigation of antigenic glycans SSEA-4 and Globo H binding proteins

洪鼎鈞(Ting-Chun Hung)

指導教授 ： 翁啟惠

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00725

摘要

異常的醣化是癌症的一個特徵。現今癌症研究的焦點在於醣基化所造成的進一步惡化或者抑制。然而研究大多僅止於此，主要是因為醣類在於癌症的功能性很難劃清界定。為了評估癌症的異常化醣類之功能性，其中一個必要的步驟就是確定找出其專一性的醣類結合蛋白，而這類的蛋白通常可能和調整細胞間交互作用，訊息傳遞，以及免疫反應有密切關係。然而，探尋專一性的醣類結合蛋白到目前為止是醣科學的其中一項主要挑戰，主因在於醣類結合蛋白以及醣類本身的親和力弱，而且也缺乏有力的探尋工具。
醣脂質是一種由碳水化合物部分共價性結合到脂質的一種醣複合物，而它們廣泛存在於真核細胞膜上。癌症最明顯的特點就是其異常化的醣脂質，尤其是那些唾液酸化以及岩藻醣化的醣脂質。許多證據顯示這類的醣脂質參與大多數的細胞吸附、增生、受體活化、細胞間辨識、分化、以及癌變，而這些行為都和癌症的擴散有相關聯。SSEA-4和Globo H分別是唾液酸化以及岩藻醣化的醣脂質，而它們長期被當作胚胎幹細胞以及癌細胞的標記。近來研究指出，SSEA-4涉及細胞癌變，像是癌細胞的侵入以及轉移。此外，Globo H也被發現大量表現於癌細胞上，而且相對於Globo H陰性的病人，有Globo H陽性反應的病人通常有較短壽命。然而由於SSEA-4以及Globo H的專一性結合蛋白仍屬未知，所以此二者醣脂質的明確功能性仍未被闡明。目前我的研究著重在SSEA-4以及Globo H專一性結合蛋白的探尋。而我所使用的策略以及工具是使用醣鍵結之磁珠之親和性捕捉、醣晶片、以及蛋白質晶片，將化學合成之SSEA-4和Globo H在不同的來源找尋專一性結合蛋白。結合這些方法，FK-506 binding protein 4 (FKBP4)以及artemin (ART)分別被發現到可辨識SSEA-4以及Globo H。而且，我們也發現到抑制MCF-7乳癌細胞內之FKBP4可以減少其表面SSEA-4表現量，這個現象可能代表著FKBP4可以藉由當作SSEA-4於細胞質及細胞膜間傳送之載體而給予調控SSEA-4於細胞膜表現之功能。

關鍵字

SSEA-4 ； Globo H ； 醣類結合蛋白 ； 磁珠 ； 醣晶片 ； 人類蛋白質晶片 ； FK-506 binding protein (FKBP4) ； artemin (ART)

並列摘要

Aberrant glycosylation is a characteristic feature of cancers. Glycosylation promoting or inhibiting cancerous progression is of crucial importance in current cancer research. Nevertheless, there are only limited studies in this field mainly because the functional roles of glycans in cancer are difficult to delineate. To evaluate the functional role of aberrant glycans in cancers, one essential step is to identify specific glycan-binding proteins (GBPs), which may mediate cell-cell interactions, signaling, and immune responses. However, owing to the low affinity of GBPs toward glycans and the lack of efficient tools available for investigation, this study therefore remains one of the major challenges in the field.
Glycolipids are a type of glycoconjugates with their carbohydrate moieties covalently bound to lipids and are widely found in the cell membrane of eukaryotic cells. Aberrant glycolipids, especially the sialylated and fucosyl ones, are the most characteristic patterns of a given cancer. Much evidence has shown their involvement in most cellular adhesiveness, proliferation, receptor activation, cellular recognition, differentiation and oncogenesis, and all of which are related to metastasis of cancer. SSEA-4 (stage-specific embryonic antigen-4, a sialyl-glycolipid) and Globo H (a fucosyl glycolipid), have long been used as cell surface markers for pluripotent human embryonic stem cells and breast cancer cells respectively. Recent studies show that SSEA-4 is implicated in the malignant process of cancers, such as invasion and metastasis of cancer cells. Furthermore, Globo H is highly expressed in cancer cells and patients with Globo H-positive tumors show a shorter survival in comparison to patients with Globo H-negative tumors. However, due to the lack of well-defined SSEA-4 and Globo H-binding proteins, the definite functional roles of these two glycolipids have not been demonstrated. In my current research, I focused on the investigation of SSEA-4 and Globo H-binding proteins. Taking advantage of the well-established glycan-conjugated bead affinity-capture, glycan microarrays, and protein microarrays, chemical synthesized SSEA-4 and Globo H were modified for investigation of GBPs in different sources. Two proteins FK-506 binding protein 4 (FKBP4) and artemin (ART), were found to specifically recognize SSEA-4 and Globo H analogues respectively. Furthermore, we also found that inhibition of cytoplasmic FKBP4 could reduce the expression of surface SSEA-4 in MCF-7 breast cancer cells, which suggested that FKBP4 might exert a regulatory function for transporting SSEA-4 from cytoplasm to cell membrane.

並列關鍵字

SSEA-4 ； Globo H ； glycan-binding protein (GBP) ； magnetic beads ； glycan microarray ； human protein microarray ； FK-506 binding protein 4 (FKBP4) ； artemin (ART)

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