牛乳中β-lactoglobulin與維生素D之結合能力

Abstract

β-Lactoglobulin (LG)是牛乳主要蛋白之一，含量約10％，分子量約18.5kDa，二級結構上由9 個β-sheet 及1 個α-helix 所組成。中心為疏水性結構(calyx)，可結合維生素A和D、棕梠酸(palmitic acid)等疏水性分子。LG 是牛乳中主要熱敏感蛋白，在加熱70-80°C之間其二級結構開始改變。過去五年研究中，我們定義出LG 變性之溫度及時間，可提供業者乳品加工之依據(Chen et al, JDS, 2005) (1)。並定位出LG 加熱變性之區域，使其喪失結合疏水性分子之功能(Song et al, JBC, 2004) (2)。此外我們發現只可辨識還原乳而不辨識生乳之單株抗體(Chen et al, JDS, 2004 and 2006) (3-4)。反之，也製作出只辨識nativeLG 之單株抗體，將其用於偵測加工後鮮乳內尚未變性之LG (Liu et al, JDS, 2007) (5)。並發現LG能刺激hybridoma cell增生，經加熱後即完全喪失上述功能。並鑑定LG 之receptor為membrane Ig M，此為一重大發現。利用同步輻射光束繞射LG與維生素D三度空間複合體(complex)結晶，確切得知LG上第二個維生素D結合區所在區域(由本實驗室首次發現) (Yang et al, Proteins, 2007) (6)。當人們缺乏維生素D時，產生許多疾病如骨質疏鬆症、自體免疫失調(多發性硬化症、腦脊髓炎)。然而LG 生理上功能至今尚未清楚，唯獨在in vivo研究，LG對於胃酸抗性極佳，其通過胃仍保持完整結構。於母親喝牛乳後可在母乳中偵測到完整LG實驗驗證。因此LG具有攜帶疏水性分子通過腸胃道，增加疏水性分子吸收之能力。本計畫將利用定點突變進行系統化分析LG第二維生素結合區上重要參與結合之胺基酸，進一步利用Docking程式模擬出維生素D結合模式，另一方面利用動物實驗研究LG增加維生素D吸收之相關機制，以利於未來將LG應用於維生素D及其類似物輸送與吸收。本計畫之主要目標如下： 1. 利用專一性單株抗體分析LG 熱敏感區域及其與生理功能之相關性 (第一年)。 2. 利用大腸桿菌表現LG重組蛋白及進行單一胺基酸取代之定點突變 (第一年及第二年)。 3. 測定LG及突變型重組蛋白與維生素D之結合能力，分析LG第二結合區(Secondary binding site)上重要參與結合之胺基酸 (第一年及第二年)。 4. 利用Docking computer simulation程式比較各種疏水性分子與LG結合情形 (第一年及第二年)。 5. 利用分子模擬計算各突變型LG之維生素D結合能力，以設計增加維生素D結合能力之LG重組蛋白 (第一年及第二年)。 6. 測定LG增加維生素D吸收之效能(動物實驗)，並分析LG增加維生素D吸收之相關機制 (第一年及第二年)。 過去五年(2002~2006)我們非常努力，由於篩選抗體、生理實驗及定點突變表現及純化LG重組蛋白所需之時間較長，我們提出兩年之計畫，誠懇希望獲得支持。

β-Lactoglobulin (LG) is a major milk whey protein, constituting about 10% of the total protein mass. The molecular mass of LG is 18.5 kDa, belonging to the lipocalin family. In secondary structure, it consists of nine β strands and one α-helix. The central hydrophobic pocket (calyx) possesses the property to bind vitamin D, vitamin A, and fatty acids. LG is quite sensitive to thermal denaturation; the secondary structure is altered upon heating with a transition temperature at 70-80°C. In the last five years, we have constructed a detailed thermal denaturation curve for LG with its time and temperature, which provided the dairy industry with a valuable reference (Chen et al, 2005, JDS) (1). We have also mapped out a specific amino acid sequence region that is responsible for the thermal change above 80°C. Such changes also result in the loss of its ligand binding (retinol and palmitic acid) (Song et al, 2004, JBC) (2). In addition, we have reported that a monoclonal antibody can only recognize the dry milk, but not the fresh raw milk (Chen et al, JDS, 2004 and 2006) (3-4). Reversely, we also developed a monoclonal antibody that only recognizes the native form of LG, so that the un-denatured LG content in the processed milk can be determined (Liu et al, JDS, 2007) (5). More recently, we have shown that LG dramatically stimulates the proliferation of hybridoma B cells, but thermal denaturation abolishes this ability of LG. It is an important observation that the LG receptor was identified as membrane Ig M using mass spectrum. For the first time, we have identified a second vitamin D binding site using synchrotron X-ray (Yang et al, Proteins, 2007) (6). Patients with vitamin D deficiency are associated with some diseases such as osteoporosis, and autoimmune disorders. In vivo studies have shown that LG can be directly absorbed into the circulation through the gastro-intestinal system because of its acid-resistant property. The immunoreactive LG is recovered following 2h ingestion of bovine milk. The LG is therefore an effective vehicle in binding the hydrophobic liagands. In this proposal, we plan to identify the essential residues of the secondary binding site involved in vitamin D binding using site-directed mutagenesis. Further, we plan to simulate the binding mode using computer docking method to investigate whether LG mutant can enhance vitamin D binding and absorption. Therefore, the specific aims in the next 2 years will be to: 1. Use monoclonal antibody as a probe to analyze the thermal sensitive region of LG and to relate it in functional role (Year 1). 2. Express LG recombinant protein and its mutant type by site-directed mutagenesis in E coli. (Years 1 and 2). 3. Estimate vitamin D binding ability of LG recombinant protein and its mutant, and to map essential residues involved in the secondary vitamin D binding site (Years 1 and 2). 4. Compare binding mode between ligands and LG using Docking program (Years 1 and 2). 5. Estimate vitamin D binding ability of LG mutants using molecular dynamics simulation and docking in order to design LG mutant with high vitamin D binding affinity (Years 1 and 2). 6. Determine whether LG can enhance vitamin D absorption using mice as an animal model and define the mechanism by which LG enhances absorption of vitamin D. (Years 1 and 2).