Hoekstra D, Düzgüneş N
Biochemistry. 1986 Mar 25;25(6):1321-30. doi: 10.1021/bi00354a020.
The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide (Gb3) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. The extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA1) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer much less than LacCer less than Gb3, while a reversed order was obtained for Ca2+- and spermine-induced aggregation and fusion, indicating an enhanced steric interference on close approach of bilayers with increasing head group size. The lectin-mediated agglutination rates for LacCer- and Gb3-containing vesicles increased by an order of magnitude when Ca2+ was also included in the medium, at a concentration that did not induce aggregation per se. Charge neutralization could not account for this observation as the polyvalent cation spermine did not display this synergistic effect with RCA1. Addition of Ca2+ to preagglutinated vesicles substantially reduced the threshold cation concentration for fusion (micromolar vs. millimolar). Quantitatively, this concentration decreased with decreasing carbohydrate head group size, indicating that the head group protrusion determined the interbilayer distance within the vesicle aggregate. The distinct behavior of Ca2+ vs. spermine on RCA1-induced agglutination on the one hand and fusion on the other indicated that Ca2+ regulates the steric orientation of the carbohydrate head group, which appears to be related to its ability to dehydrate the bilayer. As a result, lectin agglutinability becomes enhanced while fusion will be interrupted as the interbilayer distance increases, the threshold head group size being three carbohydrate residues (Gb3). Finally, GalCer-containing vesicles were not agglutinated by RCA1 at ambient temperature, irrespective of the presence of Ca2+. Above 25 degrees C, RCA1 facilitated Ca2+-induced fusion of the vesicles, which was abolished by the haptenic sugar lactose. Since Gb3- and LacCer-containing vesicles displayed a similar behavior, a temperature-induced alteration in the supporting lipid matrix is suggested, which apparently affects lectin/glycolipid interaction.
将糖脂半乳糖脑苷脂（GalCer）、乳糖神经酰胺（LacCer）和三己糖神经酰胺（Gb3）插入由磷脂酰乙醇胺和磷脂酸组成的磷脂囊泡中。通过测定在有或没有凝集素的情况下囊泡对I型蓖麻凝集素（RCA1）诱导的凝集以及Ca²⁺和精胺诱导的聚集和融合的敏感性，来研究它们的碳水化合物头部基团伸出囊泡表面的程度以及它们对膜接近的干扰。初始凝集速率按GalCer远小于LacCer小于Gb3的顺序增加，而对于Ca²⁺和精胺诱导的聚集和融合则得到相反的顺序，这表明随着头部基团尺寸的增加，双层膜紧密接近时的空间位阻干扰增强。当培养基中也包含Ca²⁺时，含LacCer和Gb3的囊泡的凝集素介导的凝集速率增加了一个数量级，该浓度本身不会诱导聚集。电荷中和不能解释这一观察结果，因为多价阳离子精胺与RCA1没有显示出这种协同效应。向预凝集的囊泡中添加Ca²⁺会大幅降低融合的阈值阳离子浓度（微摩尔与毫摩尔）。定量地说，该浓度随着碳水化合物头部基团尺寸的减小而降低，这表明头部基团的伸出决定了囊泡聚集体内双层膜之间的距离。一方面，Ca²⁺与精胺在RCA1诱导的凝集上的不同行为，另一方面在融合上的不同行为表明，Ca²⁺调节碳水化合物头部基团的空间取向，这似乎与其使双层膜脱水的能力有关。结果，凝集素的凝集能力增强，而随着双层膜间距离增加融合将被中断，阈值头部基团尺寸为三个碳水化合物残基（Gb3）。最后，无论是否存在Ca²⁺，含GalCer的囊泡在环境温度下都不会被RCA1凝集。在25℃以上，RCA1促进Ca²⁺诱导的囊泡融合，而这种融合被半抗原糖乳糖消除。由于含Gb3和LacCer的囊泡表现出类似的行为，提示支持脂质基质存在温度诱导的变化，这显然会影响凝集素/糖脂相互作用。
