West Gun, Nylund Matts, Peter Slotte J, Mattjus Peter
Department of Biochemistry and Pharmacy, Abo Akademi University, Tykistökatu 6A, FI-20520, Turku, Finland.
Biochim Biophys Acta. 2006 Nov;1758(11):1732-42. doi: 10.1016/j.bbamem.2006.06.020. Epub 2006 Jul 7.
In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.
在本研究中，我们研究了糖脂转移蛋白（GLTP）在不同pH值和氯化钠浓度下转移蒽乙烯基 - 半乳糖神经酰胺的能力，以及三种不同突变体在供体膜和受体模型膜之间转移荧光标记半乳糖神经酰胺的能力。我们通过定点诱变构建了单色氨酸突变体，其中野生型人GLTP的三个色氨酸（W）中的两个被苯丙氨酸（F）取代，分别命名为W85 GLTP（W96F和W142F）、W96 GLTP（W85F和W142F）以及W142 GLTP（W85F和W96F）。野生型GLTP和W96 GLTP均能够转移蒽乙烯基 - 半乳糖神经酰胺，但两个变体W85 GLTP和W142 GLTP未表现出任何糖脂转移活性，这表明96位的色氨酸对于转移活性至关重要。色氨酸荧光发射显示，含糖脂的囊泡与野生型GLTP和W96 GLTP相互作用时，最大发射波长发生蓝移，而蛋白变体W85 GLTP和W142 GLTP未记录到蓝移。W96 GLTP蛋白的色氨酸发射量子产率最高，而W85 GLTP、W142 GLTP和野生型GLTP的量子产率较低且几乎相似。不同色氨酸突变体与含半乳糖神经酰胺的囊泡结合后，其寿命和各向异性衰减也发生了变化。同样，野生型GLTP和W96 GLTP在含糖脂囊泡存在时表现出相似的行为。综上所述，我们的数据表明W96不仅参与蛋白质的活性，还参与蛋白质与含糖脂膜之间的相互作用。
