決定牛乳β-lactoglobulin-vitamin D complex之結晶結構並利用胺基酸定點突變及生物資訊探討其生理與物理生化功能

Abstract

β-Lactoglobulin (LG)為牛乳中主要乳清蛋白之一，calyx公認為維生素D的主要結合區，而第二個維生素D結合區之存在仍受到許多爭論。在本研究中，利用螢光光譜分析，得知維生素D與LG結合比例為2，表示LG可能具有兩個維生素D結合區。藉由降低pH值及加熱破壞calyx結合疏水性分子之能力，LG仍具有結合維生素D之能力，其結合比例為1，此結果證實LG除calyx以外具有第二個維生素D結合區。運用生物資訊程式(Insight II、Q-SiteFinder、GEMDOCK)發現有兩個區域可能為第二個維生素D結合區，且GEMDOCK 幫助我們在具有潛力的第二維生素D結合區上尋找額外電子雲密度。藉由改變製備共同結晶的pH值及起始維生素D/LG之比率來優化ligand佔有率及增加複合體中維生素D的電子雲密度。在pH 8 及起始維生素D/LG之比率為3的條件下，製備LG與維生素D複合體(complex)結晶，進行同步輻射光束繞射確切得知LG上第二個維生素D結合區所在區域。第二維生素D結合區位於LG分子之C端表面，此結合區具有約17.91 Å之長度，而維生素D分子約為12.51 Å，代表此結合區足以容納維生素D分子。第二維生素D結合區由α-helix 提供非極性胺基酸Phe136、Ala139、Leu140與β-strand I提供非極性胺基酸Ile147，經由疏水性loop (Ala142、Leu143、Pro144、Met145)連接形成一個疏水性結合區，促進維生素D結合。另一方面，利用定點突變進行系統化分析發現γ-trun loop上Leu143、Pro144、Met145為LG第二維生素D結合區上參與結合之重要胺基酸。更進一步支持證據為維生素D抑制專一性辨識γ-trun loop之單株抗體的免疫反應。眾所皆知，LG具有中心calyx及第二維生素D結合區，然而生理上LG能攜帶維生素D通過腸胃道，增加維生素D吸收之能力至今尚未證實。利用小鼠作為動物模式，起初證實LG為牛乳中攜帶維生素D及增加維生素D吸收之主要蛋白質。深入探討加熱對於LG增加維生素D吸收之影響，發現LG具有兩個維生素D結合區，餵食老鼠添加維生素D之LG，其血液中維生素D濃度於維生素D/LG比率2時達到飽和，而heated LG只具有第二維生素結合區，餵食老鼠添加維生素D之heated LG，其血液中維生素D濃度於維生素D/LG比率1時就已達到飽和。因LG第二維生素結合區不受加熱破壞，其具有攜帶維生素D及增加維生素D吸收之優勢。本研究總結發現於牛乳中添加維生素D將能有效增加維生素D為人體吸收。

β-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D3 binding, although the existence of another site beyond the calyx is controversial. In this study, using fluorescence spectral analyses, we showed the binding ratio for vitamin D3 to LG to be 2:1 and a ratio of 1:1 when the calyx was “disrupted” by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. The bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) identified the two potential regions for this secondary vitamin D binding site. It was concluded that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D3/LG in the cocrystal preparation. Finally, we identified the secondary site (defined as the exosite) for vitamin D binding using a crystal prepared at pH 8 with a vitamin D3/LG ratio of 3:1. The exosite, however, is near the surface at the C-terminus (residues 136-149) containing part of an α-helix and a β-strand I with 17.91 Å in length, while the span of vitamin D3 is about 12.51 Å. A remarkable feature of the exosite is that it combines amphipathic α-helix providing nonpolar residues (Phe136, Ala139, and Leu140) and β-strand I providing a nonpolar (Ile147), which are linked by a hydrophobic loop (Ala142, Leu143, Pro144, and Met145). Thus, the binding pocket of the exosite furnishes strong hydrophobic force to stabilize vitamin D3 binding. On the other hand, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the γ-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D3 to block the binding of a specific mAb in the γ-turn loop. LG contains a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Using the mouse (n=95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D3 revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2:1 (vitamin D3/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1:1. We have demonstrated for the first time that the exosite of LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.