Volynsky Pavel, Efremov Roman, Mikhalev Ilya, Dobrochaeva Kira, Tuzikov Alexander, Korchagina Elena, Obukhova Polina, Rapoport Evgenia, Bovin Nicolai
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation; Higher School of Economics, Myasnitskaya ul. 20, Moscow, 101000, Russian Federation.
Mol Immunol. 2017 Oct;90:87-97. doi: 10.1016/j.molimm.2017.06.247. Epub 2017 Jul 11.
Human blood contains a big variety of natural antibodies, circulating throughout life at constant concentration. Previously, we have found natural antibodies capable of binding to trisaccharide Galα1-4Galβ1-4Glc (P) practically in all humans. Intriguingly, the same trisaccharide is a key fragment of glycosphingolipid globotriaosylceramide (Gb3Cer) - normal component of erythrocyte and endothelial cell membrane, i.e. the antibodies and their cognate antigen coexist without any immunological reaction.
To explain the inertness of human anti-P antibodies towards own cells.
We used a combination of immunochemical and molecular dynamics (MD) experiments. Antibodies were isolated using affinity media with P trisaccharide, their epitope specificity was characterized using ELISA (enzyme-linked immunosorbent assay) with a set of synthetic glycans related to P synthetic glycans and FACS (Fluorescence-Activated Cell Sorting) analysis of cells with inserted natural Gb3Cer and its synthetic analogue. Conformations and clustering of glycolipids immersed into a lipid bilayer were studied using MD simulations.
Isolated specific antibodies were completely unable to bind natural Gb3Cer both inserted into cells and in artificial membrane, whereas strong interaction took place with synthetic analogue differing by the presence of a spacer between trisaccharide and lipid part. MD simulations revealed: i) although membrane-bound glycans do not form stable long-living aggregates, their transient packing is more compact in natural Gb3 as compared with the synthetic analog, ii) similar conformation of P glycan in composition of the glycolipids, iii) no effect on the mentioned above results when cholesterol was inserted into membrane, and iv) better accessibility of the synthetic version for interaction with proteins.
Both immunochemical and molecular dynamics data argue that the reason of the "tolerance" of natural anti-P antibodies towards cell-bound Gb3Cer is the spatial inaccessibility of P glycotope for interaction. We can conclude that the antibodies are not related to the blood group P system.
人类血液中含有多种天然抗体，其在整个生命过程中以恒定浓度循环。此前，我们发现几乎在所有人类中都存在能够结合三糖Galα1-4Galβ1-4Glc（P）的天然抗体。有趣的是，相同的三糖是糖鞘脂球三糖神经酰胺（Gb3Cer）的关键片段，Gb3Cer是红细胞和内皮细胞膜的正常成分，即抗体及其同源抗原共存且无任何免疫反应。
解释人类抗P抗体对自身细胞的惰性。
我们采用了免疫化学和分子动力学（MD）实验相结合的方法。使用带有P三糖的亲和介质分离抗体，通过酶联免疫吸附测定（ELISA），利用一组与P合成聚糖相关的合成聚糖来表征其表位特异性，并对插入天然Gb3Cer及其合成类似物的细胞进行荧光激活细胞分选（FACS）分析。使用MD模拟研究浸入脂质双层中的糖脂的构象和聚集情况。
分离出的特异性抗体完全无法与插入细胞中的天然Gb3Cer以及人工膜中的天然Gb3Cer结合，而与三糖和脂质部分之间存在间隔基团的合成类似物发生了强烈相互作用。MD模拟显示：i）尽管膜结合聚糖不会形成稳定的长寿聚集体，但与合成类似物相比，其在天然Gb3中的瞬时堆积更为紧密；ii）糖脂组成中P聚糖的构象相似；iii）将胆固醇插入膜中对上述结果没有影响；iv）合成版本与蛋白质相互作用的可及性更好。
免疫化学和分子动力学数据均表明，天然抗P抗体对细胞结合的Gb3Cer“耐受”的原因是P糖位在空间上无法进行相互作用。我们可以得出结论，这些抗体与血型P系统无关。
