禽類醣質體特定結構之系統性質譜分析

Abstract

中文摘要 根據西方墨點法(western blotting)和凝集素法(lectin blotting)的研究顯示：大部分在分類學上被歸類為Neoaves的現代禽類都能表現Galα1-4Gal這個末端醣抗原，而那些被歸類為Ratitae或是Galloanserae的禽類，則沒有辦法表現此特定醣結構。然而這些帶有Galα1-4Gal末端醣抗原結構的完整醣質結構，目前有被定出完整序列的僅有那些源自鴿子的蛋白及其血清IgG的醣質。本論文計畫主要在研發並應用各式串聯式質譜儀，針對現有超過一千多種禽類蛋白的珍貴庫藏樣品，開始系統性的分析禽類蛋白醣質體，以期更精準的鑑定Galα1-4Gal在禽類各網目的分佈，並藉以偵測是否有其它新穎醣抗原結構的存在，探討醣生物學與禽分類學上的相關聯性。此外，本計畫特著重於建立末端醣抗原修飾如Gal-Gal與NeuAc-Gal等的鍵結位置的質譜分析法，尤其是有鑒於後者與禽流感病毒的感染性有密切關係。現階段已完成近23隻大多在分類學來自不同「目」的禽類蛋白醣質體的分析，其流程是先以酵素或化學的方式將來自於禽類的蛋白或IgG上的醣切下，予以泛甲基化，經由質譜分析初步得到各自的醣質體表現圖譜，再以串聯式質譜儀高、低能量撞擊斷裂模式定序特定醣質結構，或再搭配離子井式多重斷裂模式分析(MSn)，鑑定末端結構如Galα1-4Gal，NeuAc，及硫酸化(sulfation)的鍵結位置。在一些特定例子中，進一步藉由離子井的快速掃瞄效率與敏感度，在MS2的層級取得醣質體圖譜，藉由特定斷片離子篩出帶有特定醣抗原結構如Galα1-4Gal的醣類分子。另外亦嘗試經由膠體電泳分離禽類的醣蛋白，再以酵素就各別呈現的醣蛋白取得其修飾的醣質，進一步探討此禽類的醣質分佈是否具有蛋白專一性的表現，亦可藉此再視需求鑑定出該蛋白身份。 綜合上述的實驗流程與方法，目前在禽類醣質體學上已得到的具體成果包含：鑑定多種複雜的 hybrid type和complex type的N-linked醣的廣泛存在、確認Galα1-4Gal的表現與禽類的網目分類有關聯、發現Gal-Gal的末端醣抗原除了可存在於N-linked醣外，亦存在於少數禽類的O-linked醣；並額外發現了一些新型的末端醣抗原結構，如Galβ1-4Gal，diLacNAc，LacdiNAc與硫酸化修飾。簡言之，禽類蛋白中N-linked醣的歧異度主要是源自不完全的α-/β-galactosylation、有無bisecting GlcNAc、唾液酸化與硫酸化。硫酸化修飾存在於大部分的禽類蛋白上的N-linked醣，其中硫酸化的位置可以是在LacNAc末端的Gal，或是Gal-Gal-GlcNAc中的GlcNAc的第六號碳上。在唾液酸的鍵結方面，發現大部分源自於海鷗蛋白上N-linked醣之唾液酸是屬於α2,3鍵結的，而源自於海鷗、turkey、guineafowl和peafowl IgG上N-linked醣之唾液酸則大多是屬於α2,6鍵結。最後，從醣質體分析延伸，搭配膠體電泳的使用，得以快速鑑定特定醣質結構之分佈於個別蛋白上並鑑定其身份。結果顯示有些禽類的個別蛋白會帶有顯著不同的醣化修飾，某些種類卻不然，因此對其蛋白生合成醣化的調控機制與結果仍有待進一步的探討。

Abstract Phylogenetic expression of Galα1-4Gal glycotope among the avians has recently been systematically investigated by lectin and western blotting. In short, it was shown that only the egg white glycoproteins of avian species belonging to Neoaves but not those of Ratitae and Galloanserae would express this glycotope. However, the precise structures of its glycan carriers have thus far been established only for the pigeon egg white and IgG. Making use of a precious collection of egg white samples from over 1,000 species in hand, a systematic avian egg white glycomic analysis by mass spectrometry was initiated, aiming to map more extensively the phylogenic expression of Galα1-4Gal, along with any other novel epitopes, as well as to define the linkages of terminal sialylation in relation to influenza virus infection. To this end, egg white samples from over 20 avian species representing major taxonomical divisions were processed to release both N- and O-glycans and profiled by MALDI-MS after permethylation. Tentative assignment of the avian glycomic constituents was supported by MS/MS sequencing based on complementary low and high energy collision induced dissociation, as well as multistage activation MSn to define the linkage of sulfation, terminal galactosylation and sialylation. Additional glycomic mapping at the MS2 level by the total ion mapping functionality on a linear ion trap was developed and applied to define the entire sub-glycome on which the Gal-Gal glycotope is carried. Moreover, gel based glycomic analysis of individual protein bands enables a quick assessment of the distribution of individual glycans among distinct egg white glycoproteins. Collectively, applications of the developed experimental glycomic workflow successfully confirmed that Galα1-4Gal is phylogenetically distributed among the avian egg whites. A wide diversity of hybrid and complex type N-glycan structures were structurally defined, along with the identification of terminal Galβ1-4Gal, diLacNAc, LacdiNAc and sulfation. In short, the avian egg white N-glycomic heterogeneity was shown to be mostly due to incomplete α-/β-galactosylation, with or without bisecting GlcNAc, sialylation, and sulfation. In addition, Gal-Gal capping was found to occur not only on the egg white N-glycans but also on O-glycans, albeit much more restrictedly. Sulfation on the avian egg white N-glycans was widely detected and shown to be 6-linked to the terminal Gal residue of a LacNAc antenna, or the GlcNAc residue of the Gal-Gal-GlcNAc sequence. NeuAc-sialylation on gull egg white glycoproteins was shown to be predominantly in α2,3 linkage, whereas those on gull, turkey, guineafowl, and peafowl IgG samples prevailed in α2,6 form. Finally, gel-based analysis of a selected few egg white samples demonstrated that a significant difference in glycosylation profiles may exist among individual glycoprotein carriers for at least one avian species, but can be rather similar for others.