利用單株抗體探討β-乳球蛋白之結構與功能

Abstract

□-乳球蛋白在牛乳中是一種熱不穩定蛋白。因此□-乳球蛋白易因加熱而導致結構之改變，在先前研究中我們製作出可辨識受熱變性後□-乳球蛋白之專一性單株抗體。於本研究中將利用此抗體深入探討□-乳球蛋白之結構與功能。在研究中發現當生乳加熱溫度達攝氏70到80度時，此抗體與□-乳球蛋白之結合能力會大幅地增加，證明□-乳球蛋白於加熱至攝氏70到80度時結構會明顯改變。為了找出這抗體於□-乳球蛋白所辨識之抗原決定位(epitope)，我們利用綜合策略之免疫分析法包含: 酵素及化學法分解蛋白質(胰蛋白酵素及CNBr)，化學修飾法(acetylation及carboxymethylation)，胜肽矩陣 (peptide array)與水溶性胜肽。 其結果準確的分析出抗原決定位於□-乳球蛋白之D股上(其胺基酸為66-76之位置)。其次於旋光雙級光譜(Circular dichroic spectral analysis)分析中，發現carboxymethylation之□-乳球蛋白不僅完全喪失二級結構，且與此單株抗體結合力遠超出經加熱後變性之□-乳球蛋白10倍以上，此證明□-乳球蛋白結構形成disorder時會被此抗體辨識。於抗體決定位做單點突變，結果發現Lys-69和glu-74是抗原決定位中與抗體結合之重要胺基酸。此外，我們更證明出結構變性後之□-乳球蛋白不能與疏水性分子(retinol和palmitic acid)結合，而此抗原決定位扮演重要的角色，當此抗原決定位結構改變被單株抗體所辨識，則□-乳球蛋白不能與疏水性分子結合。而當pH 9~10時□-乳球蛋白上之抗體決定位的Lys呈電中性，會造成其免疫反應能力與疏水性小分子結合能力下降。綜合上述結果，□-乳球蛋白上D股為熱變性之區域及參與疏水性分子結合之功能。

β-lactoglobulin (β-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured β-LG. In the present study, we show a dramatic increase in β-LG immunoreactivity when heating raw milk between 70 °C and 80 °C. To map out the specific epitope of β-LG recognized by this mAb, we used a combined strategies including tryptic digestion and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in-situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of β-LG. Circular dichroic spectral analysis shows that carboxymethylation on β-LG not only resulted in a substantial loss of β-configuration, but also exerted a 10 x increase in immunoreactivity as compared to heated β-LG. The result suggests that a further disordered structure occurred in β-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated β-LG and its binding to retinol or palmitic acid. Interestingly, at pH 9-10, which neutralizes the Lys groups of β-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of β-LG participated in the thermal denaturation between the temperatures of 70 °C and 80 °C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.