第二型人類岩藻醣水解酶和半乳糖凝集素-3針對 幽門螺旋桿菌之協同殺菌作用

Abstract

在全世界的人口中約有一半的人遭受幽門螺旋桿菌感染，大多數的感染者並沒有明顯的症狀，即使產生症狀，會有不同程度的病徵，例如胃炎、胃潰瘍和胃癌。這可能是因為細菌和胃表皮細胞本身的變異性；但也有可能是因細菌和宿主細胞不同的交互作用。根據先前文獻指出，受幽門螺旋桿菌感染時，胃部表皮細胞會分泌第三型半乳糖凝集素 ( galectin-3) 到胃表皮黏膜層，當 galectin-3結合到細菌上之脂多醣 ( Lipopolysaccharides, LPS) 時，會使細菌產生聚集和具有殺菌效果。然而幽門螺旋桿菌 LPS 所含外端的 O-antigen結構上，已知會有不同程度的岩藻醣化修飾 (fucosylation)，本論文目的在探討 LPS上岩藻醣化的程度是否會影響到 galectin-3 的結合力和功能，進一步瞭解 galectin-3抑制細菌生長的詳細機制。首先合成數個具有岩藻醣修飾之路易士抗原分子 (LeY-LeX、LeX-LeX 和 α 1,2-fucosyl type 2-tetraose)， 測試它們與 galectin-3的結合能力，這些分子和細菌 LPS 上 O-antigen 結構相似。研究結果顯示出路易士抗原分子具有岩藻醣修飾，會明顯降低 galectin-3的結合力。另外，過去的文獻指出細菌感染時，宿主胃部表皮細胞會釋放出第二型岩藻醣水解酶 (FUCA2)，本研究使用LC-MS/MS質譜分析，發現該酵素會對具有岩藻醣修飾的寡糖產生水解作用，而且該酵素對於不同鍵結岩藻醣的專一性依序為 α 1-2 > α 1-3> α 1-4；醣體上若具有多重 α 1-3 鍵結，會偏好非還原末端的 α 1-3 鍵結。相對地，只存在溶酶體的人類第一型岩藻醣水解酶 (FUCA1)，卻只能移除 α 1-2 鍵結之岩藻醣；因此 FUCA2能將 LPS 上大部分岩藻醣去除，暴露出醣體骨幹結構 poly- or oligo-LacNAc，使得水解產物與 galectin-3的結合力相較於未修飾的 LPS 提升了十倍，而殺菌效果增加一倍；這個結果說明了在 FUCA2和Galectin-3的協同作用下能增強殺菌效果。

Helicobacter pylori has persistently colonized over 50% of the global human population. Although most of infected remain asymptomatic, minor percentage of people develop gastritis, gastric ulcers, and gastric carcinomas. The reason is due to the highly stringent host immunity against pathogens and existence of dynamic host-pathogen interplay. More recent studies have shown that galectin-3 is up-regulated and secreted out by the gastric epithelial cells into the surface mucus layer as a response to H. pylori infection. This lectin can directly bind to the lipopolysaccharides (LPS), leading to bacterial aggregation and subsequent bactericidal effects. However, fucose decorated O-antigen of H. pylori LPS is not a preferred epitope for galectin-3 binding. Thus, it is of significant scientific interest to decipher how fucosylated LPS influences binding affinity of galectin-3 and its effect on galectin-3 mediated bactericidal effects. In order to evaluate the interaction between galectin-3 and H. pylori LPS, we synthesized various fucosylated Lewis antigens (including LeY-LeX, LeX-LeX and α 1,2-fucosyl type 2-tetraose) which are similar to O-antigen, representing the differential levels of fucosylation. The results clearly demonstrated the modulatory role of fucose, as fucose residues reduced the binding affinity of galectin-3. Our lab previously reported that human FUCA2 is secreted upon H. pylori infection. Interestingly, our current data shows that FUCA2 could remove fucose on LPS with the order of cleavage preference as α 1-2 > α 1-3> α 1-4. Since H. pylori LPS has many α 1-3 fucose residues, the preference of cleavage is from non-reducing end of O-antigen. On the other hand, FUCA1, a human lysosomal fucosidase could only remove α 1-2 linkage. Hence, the FUCA2 treatment was able to remove fucose residues on its LPS to expose the poly- or oligo-LacNAc structure that consistently, appeared to be a favored epitope for galectin-3 binding (10-fold higher binding ability, compared with the untreated LPS). Consequently, the galectin-3-mediated bactericidal effect is increased in FUCA2-treated H. pylori. Taken together, these results clearly depict that galectin-3 and human FUCA2 act synergistically to enhance bactericidal effects.