蛋白質的醣化修飾對鱟血漿外源凝集素在結構變異性、熱穩定性及配體專一性的影響

Effects of protein glycosylation on structural variation, thermal stability and ligand specificities of two Tachypleus plasma lectins

謝慶霖(Ching-Lin Hsieh)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10397

摘要

源自三棘鱟（Tachypleus tridentatus）血清中的鱟血清外源凝集素-1及-2可分別辨認革蘭氏陽性菌細胞壁上的肽聚醣(peptidoglycan)及革蘭氏陰性菌的脂多醣(lipopolysaccharide) (1, 2)。這兩種蛋白在天賦免疫系統上擔任病原相關分子型態辨識受體(pattern recognition receptor)的角色以執行第一線的防禦任務。先前的研究中，我們使用酵母菌表現系統Pichia pastoris表達有醣化修飾的鱟血清外源凝集素-1及-2 (yTPL-1及yTPL-2)，兩種重組蛋白皆有活性但表達量卻偏低(3)。因此，在本研究中，我們嘗試利用原核表達系統E. coli大量表現此兩種蛋白質(EcTPL-1及EcTPL-2)，並將這些缺乏醣化修飾的蛋白質與先前具有醣化的蛋白質做比較，看看對他們的配體專一性、熱穩定性及結構變異性有無影響。EcTPL-1失去原本具有與肽聚醣結合的能力，對N-acetyl單醣的親和力也大大下降；而EcTPL-2可以專一地與L-Rha及綠膿桿菌的脂多醣結合，但原先可與大腸桿菌含有O抗原的脂多醣結合之能力卻已消失。在二級結構上，EcTPL-1及yTPL-1皆主要由β–sheet構成，但EcTPL-1有較多的random coil；EcTPL-2及yTPL-2也主要由β–sheet構成，而yTPL-2反而含有較多的random coil。從分析式超高速離心機(analytic ultracentrifugation)的結果發現，EcTPL-1會形成二聚體，而yTPL-1則為單聚體及二聚體的混合物。雖然在非還原的SDS-PAGE下EcTPL-2及yTPL-2皆泳動為二聚體，他們的四級結構卻有所不同，二聚體EcTPL-2間會利用非共價作用力進一步形成八聚體。利用掃描式熱差分儀(differential scanning calorimetry)所得的熱變性曲線可發現，不具醣化的血清外源凝集素比起醣化的血清外源凝集素有較高的Tm值，可見在這個例子中，醣化修飾並沒有增進蛋白質的熱穩定性。
我們也利用圓二色光譜(circular dichroism)及分析式超高速離心機來觀察配體所引起的結構改變及蛋白質原聚體間的聚合狀態。加入MurNAc的EcTPL-1會由二聚體變成四聚體，然而MurNAc卻會造成yTPL-1的單聚體化。若加入yTPL-1本身更強效的配體muramyl-dipeptide，也會引起yTPL-1的單聚體化，並使其二級結構變得更不規則。處理綠膿桿菌脂多醣的EcTPL-2仍維持在八聚體，而原本的β–sheet結構則變得更明顯；相對之下，處理大腸桿菌脂多醣的yTPL-2則有顯著的聚合作用及些微的二級結構改變。最後，我們利用恆溫滴定熱卡計(isothermal titration calorimetry)及動態光散射儀(dynamic light scattering)來探討脂多醣-鱟血清外源凝集素-2複合體的物理性質及其結合時熱力學的變化。結果發現，鱟血清外源凝集素-2可以破壞脂多醣微團(micelle)間的疏水性作用力，使微團分解成較小的寡聚體，而此能量的消耗可由熵(entropy)所驅動鱟血清外源凝集素-2與脂多醣的結合所代償，聚合化的蛋白更能藉由多價效應(multivalency)增強與脂多醣的親和力而達到穩定的效果。總括而論，本研究證實鱟血清外源凝集素的醣化修飾確實影響其配體專一性及結構變異性，但不一定會改變蛋白質的熱穩定性。

關鍵字

鱟血漿外源凝集素

並列摘要

Two lectins, Tachypleus plasma lectin-1 (TPL-1) and Tachypleus plasma lectin-2 (TPL-2), derived from hemolymph of Tachypleus tridentatus (horseshoe crab) recognize peptidoglycan (PGN) in Gram-positive bacteria and lipopolysaccharide (LPS) in Gram-negative bacteria, respectively (1, 2). They serve as pattern recognition receptors (PRRs) and exert important roles in innate immunity of host. Previously, we utilized yeast Pichia pastoris to express glycosylated TPL-1 and TPL-2 (yTPL-1 and yTPL-2) with PGN-binding and LPS-trapping activity, but expression yields of both proteins were low (3). In this study, I tried to overexpress non-glycosylated TPL-1 and TPL2 (EcTPL-1 and EcTPL-2) using prokaryotic system and compared their ligand specificities with glycosylated equivalents. Non-glycosylated EcTPL-1 lost the PGN-binding activity, and its affinity to N-acetylmonosaccharide was weaker compared to that of yTPL-1. EcTPL-2 specifically interacted with L-rhamnose (L-Rha) and LPS from P. aeruginosa, but other O-antigen-containing LPS from E. coli, effective ligands for yTPL-2, could not bind to EcTPL-2. Both EcTPL-1 and yTPL-1 were dominated by β–sheet structure, whereas EcTPL-1 contained more random coil. The secondary structure of EcTPL-2 was also predominant in a β–sheet conformation differing from more unordered yTPL-2. As evidenced by analytic ultracentrifugation (AUC), EcTPL-1 formed a dimer and yTPL-1 were mixtures of monomer and dimer. Both EcTPL-2 and yTPL-2 migrated as a dimer under non-reducing SDS-PAGE analysis, and dimeric EcTPL-2 further associated to become an octamer. In the thermal unfolding profiles of differential scanning calorimetry (DSC), both non-glycosylated TPLs have Tm values a little higher than glycosylated counterparts, suggesting that in the present case glycosylation modification of protein could not contribute to its thermal stability.
Ligand-induced conformational change and variation of association status of proteins were further examined by circular dichroism (CD), AUC. The addition of N-acetylmuramic acid (MurNAc), a common ligand for both EcTPL-1 and yTPL-1, rendered EcTPL-1 a tetramer and induced monomerization of yTPL-1. Moreover, monomerization of yTPL-1 upon addition of muramyl-dipeptide correlated to more random coil structure. P. aeruginosa LPS-treated EcTPL-2 remained as an octamer and gained more compact β–sheet conformation. By contrast, E. coli LPS-bound yTPL-2 exhibited significant oligomerization and minor conformational change. Finally, we utilized isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) to investigate the physical property of LPS-TPL-2 complex and corresponding thermodynamic scenario. TPL-2 possessed disaggregating effect to disrupt the hydrophobic force among LPS micelle, and the expense of energy might be compensated by entropy-driven protein-ligand interaction and further stabilized by the multivalency of oligomeric TPL-2. Overall, these studies demonstrate that glycosylation modification of TPLs does influence its ligand specificities and causes structural variations even though it does not change thermostability.

並列關鍵字

Tachypleus plasma lectins

參考文獻

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