利用化學酵素合成兩價以及四價唾液酸化樹枝狀分子:與流行性感冒病毒H5N1上之HA親和力分析
Abstract
蛋白質與多醣的作用力 (Protein-glycan interaction) 在生化反應過程中扮演著很重要的角色，其參與的範圍包括: 細胞黏著 (cell adhesion)、血管新生(Angiogenesis) 以及免疫反應 (Imunity)。
近年來，流行性感冒病毒不僅在世界各地流行，且出現跨越物種的新型病毒而造成死亡。病毒在進入宿主的階段， 病毒上的HA 會辨識宿主的唾液酸 (SA)，為解開病毒傳染禽類與人類間重要的關鍵因素，HA 與唾液酸間作用力的研究也就變得相當的重要。但是 HA 與 單一的唾液酸共軛物 (例如: 3’SL 以及6,SL ) 之間的作用力很弱 (只有 mM等級)，使得研究較為不易，為改善此缺點，我們設計了利用樹狀分子 (dendrimer) 末端帶有多價鍵結的特性，將唾液酸共軛物修飾在樹枝狀分子末端，以提高醣類與蛋白質間的親和力 (binding affinity)。
在本篇論文中，我們建立了一套有效率的化學酵素合成系統，藉由固相胜肽合成法 (SPPS) 合成具有兩價以及四價的乳糖化樹枝狀高分子，再利用 α2,6-唾液酸轉移酶 (α2,6-sialotransferase) 將 SA 修飾在乳糖的六號碳位置上，其催化產率大於 90%，最後將合成好的唾液酸化乳糖樹枝狀化合物與兩種類型的流行性感冒病毒蛋白 (H5N1原生株以及突變株 L133S) 做親合力 (KD) 測試。其中, G1-PEG-26SL (16) 與原生株的HA以及突變株L133S HA之親和力，分別比原本α2,6單醣提高至4000倍與605倍。
Protein-carbohydrate interaction plays an important role in cell adhesion、angiogenesis and immunity. In particular, the transmission of human and avian influenza viruses relies on the engagement of the viral envelope protein hemagglutinin (HA) to the sialic acid (SA)-terminated carbohydrate side chain on the host cell. The host cell tropism was determined by the α-glycosidic linkage between SA and penultimate sugar; namely the avian influenza viruses preferentially bind a-2,3-liked sialyloligosacchrides while the human influenza viruses bind to a-2,6-liked ones. In order to monitor the SA/oligosaccharide-HA interaction, we have synthesized poly (L-lysine) dendrimer scaffold conjugated to 2 and 4 α-2,6 sialylactose units using 2,6-sialotransferase in a peptide synthesizer. These α-2,6 sialylactose clusters-HA interaction are measured by surface plasmon resonance spectroscopy. Both the wild type and L133S mutant HAs were tested for α-2,6 sialylactose binding. It was found that the binding avidity of G1-PEG-26SL was 4000 and 605 fold more effective than the monomeric α2-6 sialylactose for the wild type and L133S HA, respectively.
Protein-carbohydrate interaction plays an important role in cell adhesion、angiogenesis and immunity. In particular, the transmission of human and avian influenza viruses relies on the engagement of the viral envelope protein hemagglutinin (HA) to the sialic acid (SA)-terminated carbohydrate side chain on the host cell. The host cell tropism was determined by the α-glycosidic linkage between SA and penultimate sugar; namely the avian influenza viruses preferentially bind a-2,3-liked sialyloligosacchrides while the human influenza viruses bind to a-2,6-liked ones. In order to monitor the SA/oligosaccharide-HA interaction, we have synthesized poly (L-lysine) dendrimer scaffold conjugated to 2 and 4 α-2,6 sialylactose units using 2,6-sialotransferase in a peptide synthesizer. These α-2,6 sialylactose clusters-HA interaction are measured by surface plasmon resonance spectroscopy. Both the wild type and L133S mutant HAs were tested for α-2,6 sialylactose binding. It was found that the binding avidity of G1-PEG-26SL was 4000 and 605 fold more effective than the monomeric α2-6 sialylactose for the wild type and L133S HA, respectively.
Description
Keywords
樹枝狀分子, 唾液酸, 流行性感冒病毒, 化學酵素催化, 固相胜肽合成, dendrimer, α2, 6-sialotransferase, SPPS, influenza, sialic acid