分枝桿菌噬菌體的O-醣基化之酵素特性描述嘗試

Abstract

分枝桿菌噬菌體是一群多樣化的噬菌體，他們能夠選擇性地感染分枝桿菌，包括結核分枝桿菌。最近已發表的研究揭示了分枝桿菌噬菌體生物學一個獨特的特性—部分噬菌體頭部或尾管具有O-醣基化。特殊的是這些噬菌體表的醣基化是由噬菌體自體編碼的醣基轉移酶所轉移的，與一般對病毒的認知不同，這些噬菌體並不會通過利用宿主的機制進行修飾，也因此導致不同分枝桿菌噬菌體之間具有不同的表面糖類之修飾。這些醣鏈通常很長、結構複雜且在代謝扮演著重要的角色。這些長醣鏈可以作為噬菌體的醣護盾，使病毒顆粒不易受抗體結合的影響。在小鼠接種後的初始階段，IgM和IgG抗體對未醣基化的噬菌體顆粒的親和力比對醣基化的病毒顆粒更強。雖然已經有論文發表了分枝桿菌噬菌體表面糖類的存在以及負責對病毒結構蛋白進行醣基化的噬菌體編碼醣基轉移酶，但仍有幾個方面尚未清楚，包括這些酵素的功能、基質特異性以及這些噬菌體表面醣類的化學組成。在這篇論文中，我試圖證明分枝桿菌噬菌體中醣基轉移酶的功能。首先，我通過使用各式生物訊息及結構工具與軟體鑑定出多個醣基轉移酶候選基因，再將所鑑定的醣基轉移酶在預測出的三維結構模型中找出保守的醣基轉移酶家族結構胺基酸序列，這些序列代表了潛在的醣基轉移酶功能位點。進一步的結構分析揭曉了這些醣基轉移酶具有類Rossman折疊，這是GT-A或GT-B家族的醣基轉移酶家族所擁有的特徵。其中的一組醣基轉移酶來自於分枝桿菌噬菌體Corndog（gp36、gp37和gp38），這組基因被挑選出來對其進行了克隆並且成功表達出這三個酵素。再使用合成的核苷酸醣和胜肽作為受體對Corndog的醣基轉移酶進行功能測試。儘管在不同的受體設計和反應條件下進行了多次嘗試，但Corndog醣基轉移酶未能將醣基轉移到受體上。這項結果表明幾種可能性：這些酵素的供體和受體特異性可能比最初預期的更複雜，或者用於測試的體外條件未能模擬酵素活性所需的環境。供體與受體的專一配對在本研究中是一個重大的挑戰，也因此成為了這些未知功能的醣基轉移酶功能測試的瓶頸。

Mycobacteriophages are a diverse group of bacteriophages that infect mycobacteria, including Mycobacterium tuberculosis. Recent investigations have revealed a fascinating aspect of mycobacteriophage biology, the O-glycosylation of capsid and/or tail tube subunits. This viral surface glycosylation is orchestrated by phage-encoded glycosyltransferases, bypassing the need to exploit the host's biosynthetic machinery, resulting in a distinct surface glycans across different mycobacteriophages. These glycans, often large and intricately structured, are proposed to play a pivotal role as a glycan shield, rendering viral particles less susceptible to antibody binding. Both IgM and IgG antibodies exhibit a stronger affinity for non-glycosylated phage particles than to glycosylated virions during the initial stages following mouse inoculation. While the presence of mycobacteriophage surface glycans and the phage-encoded glycosyltransferases responsible for glycosylating viral structural proteins have been discovered, several aspects remain fairly unknown. These include the precise function of these enzymes, their substrate specificity, and the chemical composition of the surface glycans. In this study, the primary focus is to demonstrate the function of these glycosyltransferases. Multiple glycosyltransferase candidates were identified by using of a number of online bioinformatic and structural tools and software. The identified glycosyltransferases exhibited conserved glycosyltransferase family structural motifs in high-confidence three-dimensional models showing potential functional sites. Further structural analysis suggests that these glycosyltransferases are likely to be soluble enzymes belonging to the GT-A or GT-B families with Rossman-like folds, which are characteristic of these glycosyltransferase families. A set of glycosyltransferases from these candidates belonging to the mycobacteriophage Corndog (gp36, gp37, and gp38) was successfully cloned with relatively high efficiency. The functional assays of the Corndog glycosyltransferases using synthesized nucleotide sugars and peptide as acceptors, although initially promising, did not result in detectable glycosylation. Despite multiple attempts with different acceptor designs and reaction conditions, the glycosyltransferases were unable to transfer sugar residues to the acceptors. This outcome suggests several possibilities: the donor and acceptor specificities of these enzymes may be more complex than initially anticipated, or the in vitro conditions used in the assays failed to simulate or fully replicate the native environment required for enzymatic activity. The identification of specific donor-acceptor pairs remains a significant challenge and a potential bottleneck in demonstrating the precise functionalities of these novel enzymes.