大腸癌細胞醣脂質之癌相關抗原結構質譜分析與生合成機制探討

在癌症生物學的研究當中，常發現由不正常醣化結構所構成的癌相關抗原，即癌細胞常會表現出一般正常細胞中不會表現的特殊醣質結構。一般而言，以 Galb1-4GlcNAc，亦即 LacNAc，所聚合組成的第二型醣鍊為人體內較常表現的醣結構，通常可再線性延伸並形成分支狀。相對而言，構成第一型醣鍊的 Galb1-3GlcNAc 則通常不會再延伸。90年代初期，在大腸直腸癌細胞株 Colo205 細胞膜上第一次發現具有延伸性的第一型醣鍊位於醣脂質上，搭配其不同程度的岩藻醣化 (fucosylation) 形成二聚體的Leb/a-Lea 特殊抗原。本論文的研究目標開始於應用一些已經確定結構的醣質標準品，以及表現在 Colo205 醣脂質上的第一型醣鍊及其衍生物為樣品，去探討並建立一綜合高撞擊能量與低撞擊能量串聯式質譜去分析、定序及區分第一型以及第二型醣鍊的方法。接下來，利用此互輔性的質譜分析法，搭配酵素、高效能液相層析分離以及化學反應，鑑定出 Colo205 醣脂質上由三個 Lewis 單位聚合組成的醣鍊，含延伸性和分支狀的純第一型醣鍊，及第一型與第二型不同排列複合組成的醣鍊。由於第一型醣鍊的延伸性醣脂質為一罕見結構並與癌化細胞相關，遂從已知的人類醣轉移酵素中，藉由體外酵素合成的方式來分析何種酵素可能具有分支第一型醣鍊的能力。實驗結果顯示已知的三種分支合成酵素 b6-N-acetyl-glucosaminyltransferases (IGnTs) 皆含有此活性，其中以 IGnT2 的活性最高，並為 Colo205 細胞內表現量較為顯著的酵素。配合質譜分析鑑定其合成結構，可進而確定其分支點位置。此外，藉由分析此酵素合成產物，得以再度應證已建立的串聯式質譜分析方法的確能有效分析、辨識第一型以及第二型醣鍊。搭配醣轉移酵素的應用，亦可有效合成的各式第一型以及第二型醣鍊結構，形成多種抗原組合以探討抗體對不同結構的辨識能力，進而定出其相對應之抗原結構。總言之，本論文的主要研究成果在於建立並有效應用高敏感度串聯式質譜分析，搭配高效能液相層析以及化學和酵素的反應，成功的鑑定表現於大腸癌細胞醣脂質上罕見的第一型延伸性醣鍊，並探討其相關生合成酵素，遂再以生化應用之角度辨別最小抗原決定結構。

關鍵字

延伸性和分支狀第一型醣鍊；不正常醣化結構

並列摘要

Tumor associated antigens arising from aberrant glycosylation is a well recognized phenomenon in cancer biology. Among the more notable structural features is the rare occurrence of extended type 1 chain on the lactosylceramides of the human colonic adenocarcinoma cell line, Colo205, concomitant with multiple fucosylation to give multimeric Lewisb/a terminal epitopes. While a dimeric Leb/a-Lea determinant has been well characterized previously, a trimeric stretch of type 1 units, -3Galb1-3GlcNAcb1-, and its fucosylated variants have not been unequivocally demonstrated. In a broader context of developing facile mass spectrometry (MS) methodologies for the sequencing of linear and branched poly-N-acetyl-lactosamine (polyLacNAc), this thesis demonstrates that the type 1 unit can be not only linearly extended to three or more tandem repeats, but also branched at C6 of 3-linked Gal in a manner similar to its type 2 polyLacNAc counterparts. A combination of chemical and enzymatic approaches, HPLC purification, and both low and high energy CID tandem mass spectrometry (MS/MS) analyses showed that the glycan chains carried on the lactosylceramides of Colo205 comprise a complex mixture of type 1 and 2 hybrids as well as those exclusively of extended type 1 chain. Complementary fragmentation characteristics were established to allow discrimination of linkage specific branching versus linear extension. Identification of these novel structures further prompted an investigation into the candidate glycosyltransferases that may be responsible for mediating their aberrant synthesis. Based on in vitro enzymatic synthesis systems, linear extended type 1 and 2 chains and their hybrids were first synthesized which serve as authentic standards to validate MS/MS fragmentation pattern established; as well as substrates for further enzymatic (a-fucosylation and to assay for the specific activities of various I branching enzyme, (b6-N-acetyl-glucosaminyltransferases (IGnT or(b6GnT). The former allows a reconstitution of authentic oligomeric Lewis X and A antigens to be used in conjunction with other analytical methods to define the specificity of a monoclonal antibody which apparently recognizes only the GSLs from Colo205 of a size larger than those carrying a dimeric Lewis A epitopes. Variably fucosylated hybrid type 1 and 2 chains with at least an internal Lewis X epitope were established as being the most likely carrier of the epitope recognized. The latter led to a successful demonstration that extended type 1 and the hybrid chains can be branched in a manner similar to type 2 polyLacNAc chains by all three available human IGnTs albeit at a lower reactivity. IGnT2 is the only transcript expressed at any significant level in Colo205 and the most active in branching an extended type 1 chain with the branched Gal position critically established by further MS/MS analysis of the synthesized products. In short, this work has unambiguously identified the presence of linear and branched extended type 1 chains on the GSLs of a colonic adenocarcinoma cell line based on advanced MS/MS analysis. With the following up in vitro enzymatic synthesis studies coupled with further MS/MS analysis of the products made, the implicated glycosyltransferases were shown to be capable of making the novel tumor-associated structures and the derived synthetic glycan epitopes were further used successfully to define the specificity of a monoclonal antibody raised against the tumor-associated antigens on GSLs, as well as to validate the MS/MS methodologies developed.

並列關鍵字

Tumor associated antigens ； human colonic adenocarcinoma ； branched / extended type 1 chain

參考文獻

Berg, E. L., Robinson, M. K., Warnock, R. A. and Butcher, E. C. (1991) The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor. J Cell Biol 114, 343-349

Bierhuizen, M. F., Mattei, M. G. and Fukuda, M. (1993) Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1, 6-N-acetylglucosaminyltransferase gene family. Genes Dev 7, 468-478

Bochner, B. S., Alvarez, R. A., Mehta, P., Bovin, N. V., Blixt, O., White, J. R. and Schnaar, R. L. (2005) Glycan array screening reveals a candidate ligand for Siglec-8. J Biol Chem 280, 4307-4312

Dell, A. (1987) F.A.B.-mass spectrometry of carbohydrates. Adv Carbohydr Chem Biochem 45, 19-72

Dell, A., Morris, H. R., Egge, H., von Nicolai, H. and Strecker, G. (1983) Fast-atom-bombardment mass-spectrometry for carbohydrate-structure determination. Carbohydrate Research 115, 41-52

國際替代計量

大腸癌細胞醣脂質之癌相關抗原結構質譜分析與生合成機制探討

全文下載

主題瀏覽