乳球蛋白中N端胺基酸與磷脂質結合的相關性

Abstract

β-乳球蛋白(LG)是一個已被廣泛探討的蛋白質，在牛乳中是一個重要的成分，約佔牛乳中所有蛋白質的15%。LG具有162個胺基酸，分子量大約為18.4kDa。先前研究藉由分析LG結晶結構發現，一個主要由β-結構所組成的LG可以具有兩個維生素D的結合位置:一個位於中心calyx，另一個則位於結構表面的疏水性袋口中。而表面的疏水性袋口是由第130到149的胺基酸序列及存有一個3-turn的雙極性α-helix所構成。

LG除了有與其配體結合的能力以外，還能跟磷脂質DMPG結合，然而 其結合區仍然是模糊不清的。一開始我們利用了建構在包含了第19個色氨酸的N端片段的LG 重組蛋白，來了解是否在這區域會有與脂質結合的位置。所以首先藉由螢光光譜儀探測其蛋白質中第19個色氨酸，發現了N端LG的重組蛋白與DMPG的相互作用跟自然界中的LG有相似結果，即它們都可以隨著不同劑量的DMPG而有相對應的變化。接下來這個位於LG中殘基1-112(一個大部分由β-結構所組成的重組蛋白)在圓二色光譜儀分析中，與DMPG相互作用的時候可以大幅增加helical content，此結果證明LG的N端胺基酸與DMPG的交互作用有關係。

因此我們假設LG的殘基1-112可能有形成α-helix的區域。利用一個預測二級結構的電腦程式 (PBIL LYON-GERLAND, HNN secondary structureprediction method) 找出存在有潛力發展成α-helix的區域主要是在殘基12-29的位置上。值得注意的是，此段序列的胺基酸位置具有一邊是疏水性胺基酸(Ile, Leu, Val, Trp, Ile)，另一邊則是親水性胺基酸(Thr, Lys, Ser, Asp, Met, Gln, Tyr, Ser)的特性，而使形成一個雙極性的α-helix。

下一階段我們在LG中殘基1-62建構了更小片段的重組蛋白，用來進一步探討與DMPG相互作用的關係。分別重複上述實驗發現此片段的重組蛋白也可以隨著與DMPG交互作用的結果發出自體螢光並且也有增加α-helical content的現象。實驗結果更確實了有潛力發展出α-helix的胺基酸位置。

由於在體內LG是經由與生物膜交互作用後直接進入到小腸來達到吸收 的效果，所以在LG中N端的胺基酸造成其特有α-helix結構的變化可能可以增進穿過生物膜達到輸送維他命D到體內的效果。

Lactoglobulin (LG), one of the most investigated proteins, is a major bovine milk component comprising about 15% of the total proteins. It consists of 162 amino-acids with a molecular weight of 18.4 kDa. By crystallographic study, we have shown that LG (predominantly a β-configuration) comprises two vitamin D binding sites: one is located at the central calyx, the other is at the surface hydrophobic pocket between residue 130 and 141 with a three-turns of α-helix.

In addition to the ligand binding properties, it has been shown that LG also binds to phospholipid dimyristoylphophatidylglycerol (DMPG), but the binding domain(s) located at the LG remains elusive and undetermined. The present study was initially to use a recombinant N-terminus of LG (residues 1-112) to determine whether it possessed a lipid-binding site. First, we showed that this N-terminus containing Trp 19, similar to native or recombinant LG, could interact with DMPG as a dose-dependent manner using a fluorescent anisotropic analysis. Second, we showed residues 1-112 (a major domain with predominantly a β-configuration) significantly increasing the helical content upon the interacting with DMPG using a circular dichroic (CD) analysis, it further demonstrated the interaction between the N-terminus of LG and DMPG.

We hypothesized that there might be a helical-forming region in residues 1-112. Using a computer secondary-structure-prediction program (PBIL LYON-GERLAND, HNN secondary structure prediction method), we identified residues 12-29 being a potential helical-forming region. Of remarkable interest, it orients as an amphipathic helix between hydrophobic and hydrophilic residues with Ile, Leu, Val, Trp, Ile clustered over one side and with Thr, Lys, Ser, Asp, Met, Gln, Tyr, Ser over the other without an exception.

Next, we prepared a recombinant fragment corresponding to residues 1-62, the minimum amino-acid residues we were able to express in E. coli thus far, and to test its interaction with DMPG. Similar to the studies mentioned above, residues 1-62 was able to interact with DMPG by increasing the intrinsic fluorescence and α -helical content using fluorescent and CD spectral analyses, respectively. The data proved a potential helical forming region (residues 12-29) being located in residues 1-62.

Because LG uptake is directly pass through the small intestine via a membrane interaction in vivo, the conformational change plus the unique helical structure at the N-terminus of lactoglobulin residues upon the phospholipid binding may significantly enhance its role in transporting vitamin D across the cell membrane.