Jones D H, Lingwood C A, Barber K R, Grant C W
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Biochemistry. 1997 Jul 15;36(28):8539-47. doi: 10.1021/bi970183a.
Recognition of macromolecules by glycosphingolipids is closely correlated with the nature of the glycolipid carbohydrate; however, it is also thought to be secondarily modulated by the structure of the single fatty acid. In the present work, we sought insight into what physical effect a change in this fatty acid has on the extramembranous portion of globosides at liposomal surfaces mimicking systems for which modulated receptor function has been recorded in the past. Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of globotriaosylceramide (Gb3) were replaced with deuterium. In this location, the nonperturbing probe nuclei sampled cumulative conformational and orientational characteristics of the oligosaccharide chain at a sugar residue that is critical in specific binding of verotoxins. Deuterated Gb3 having 18:1 fatty acid was compared to the same species having 22:1 fatty acid, at 6.3 mol % in unsonicated bilayers of dipalmitoylphosphatidylcholine/cholesterol. Both produced narrow, apparently axially asymmetric 2H NMR spectra over a wide temperature range. Motional properties of the terminal sugar were measurably influenced by the fluidity of the host matrix; however, evidence was not found for conformational or orientational variation in this sugar brought about by the fatty acid alteration. In related experiments, acetate protons on the terminal N-acetyl galactosamine (GalNAc) residue of globotetraosylceramide (Gb4) were substituted with deuterium, and the natural fatty acid was replaced with 18:0 or 24:0 species deuterated at C2. Once again, species with short vs long fatty acid were examined for evidence of headgroup differences. Spectra of Gb4 were compared at 10 mol % in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and at 5 mol % in membranes containing 33 mol% cholesterol. Spectral splittings reflecting cumulative effects on conformation and order at the terminal deuterated sugar remained unchanged between species having 18:0 vs 24:0 fatty acid in POPC/cholesterol. In a pure POPC host matrix, there was clear evidence of a motional difference between the two--the longer chain Gb4 demonstrating spectral asymmetry--but the spectral width was unchanged. Transverse relaxation times, T2, were measured. Our findings appear to help correlate the conclusions of a number of workers dealing with the molecular basis of crypticity. We suggest that changes in glycolipid receptor function based on ceramide fatty acid variation have a major origin in the fatty acid's ability to determine the thermodynamics of interaction with the host matrix, as reflected in such parameters as glycolipid motional properties, local membrane curvature, and likely glycolipid time-dependent lateral associations. The result at low concentrations of glycolipid may often be only a subtly altered collective surface epitope, best detected by a specific recognition event.
糖鞘脂对大分子的识别与糖脂碳水化合物的性质密切相关；然而，人们也认为它会受到单一脂肪酸结构的二次调节。在本研究中，我们试图深入了解这种脂肪酸的变化对脂质体表面球苷脂膜外部分有何种物理影响，这些脂质体表面模拟了过去已记录到受体功能受到调节的系统。将球三糖神经酰胺（Gb3）末端半乳糖（Gal）残基上的环外羟甲基质子用氘取代。在此位置，无干扰的探针核在一个对维毒素特异性结合至关重要的糖残基处采样寡糖链的累积构象和取向特征。将含有18:1脂肪酸的氘代Gb3与含有22:1脂肪酸的同一物质进行比较，二者在二棕榈酰磷脂酰胆碱/胆固醇的未超声处理双层膜中占6.3 mol%。在很宽的温度范围内，二者均产生狭窄的、明显轴向不对称的2H NMR谱。末端糖的运动性质受到主体基质流动性的显著影响；然而，未发现脂肪酸改变导致该糖的构象或取向发生变化的证据。在相关实验中，将球四糖神经酰胺（Gb4）末端N - 乙酰半乳糖胺（GalNAc）残基上的乙酰基质子用氘取代，并将天然脂肪酸替换为在C2处氘代的18:0或24:0脂肪酸。同样，研究了具有短链和长链脂肪酸的物质，以寻找头基差异的证据。在1 - 棕榈酰 - 2 - 油酰磷脂酰胆碱的未超声处理流体双层膜中，Gb4的浓度为10 mol%，在含有33 mol%胆固醇的膜中，Gb4的浓度为5 mol%，对二者的光谱进行了比较。在POPC/胆固醇中，含有18:0与24:0脂肪酸的物质之间，反映对末端氘代糖构象和有序性累积影响的光谱分裂保持不变。在纯POPC主体基质中，有明确证据表明二者存在运动差异——长链Gb4表现出光谱不对称——但光谱宽度不变。测量了横向弛豫时间T2。我们的研究结果似乎有助于关联许多研究隐蔽性分子基础的工作者的结论。我们认为，基于神经酰胺脂肪酸变化的糖脂受体功能变化主要源于脂肪酸决定与主体基质相互作用热力学的能力变化，这体现在糖脂运动性质、局部膜曲率以及可能的糖脂时间依赖性横向缔合等参数中。低浓度糖脂时的结果可能往往只是一个微妙改变的集体表面表位，最好通过特异性识别事件来检测。
