Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes.
Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes.
复制标题
DOI：
10.3389/fphys.2018.00421
复制
发表时间：
2018
影响因子：
4
通讯作者：
Tissot JD
中科院分区：
文献类型：
--
作者：
Prudent M;Delobel J;Hübner A;Benay C;Lion N;Tissot JD
红细胞浓缩物（EC）的储存诱导损伤，这些损伤显著影响红细胞（RBC）的代谢、蛋白质活性、变形性以及氧的释放。带3是在RBC的离体老化期间受影响的蛋白质之一。这种膜蛋白是阴离子转运蛋白，是细胞骨架和其他膜蛋白的锚位点，也是糖酵解酶的结合位点，并携带血型抗原。在本研究中，从平均储存7天和42天的RBC（n = 3）以及从微泡（n = 3）中分离带3复合物。用含脱氧胆酸盐的缓冲液提取膜蛋白后，将带3复合物在用两种抗带3抗体包被的磁珠上共免疫沉淀。在SDS-PAGE上分离总膜蛋白提取物和洗脱液（含有条带3复合物），并通过自下而上蛋白质组学进行分析。结果表明，长时间保存和短时间保存的内皮细胞的带3复合物中分别有3种蛋白存在或缺失，而膜蛋白含量保持不变。储存诱导的RBC老化的这些潜在标志物是腺苷酸琥珀酸裂解酶（ADSL）、α-内收蛋白和flotillin-2，并使用蛋白质印迹法进行了进一步分析。在总膜蛋白和带3复合物中，ADSL丰度在储存期间趋于增加，而α-内收蛋白主要倾向于停留在膜提取物上。有趣的是，flotillin-2等效地存在于总膜蛋白中，而它在储存期间与条带3复合物明显共免疫沉淀（1.6倍变化，p = 0.0024）。此外，与微泡（MV）相比，红细胞中的flotillin-2富集（几乎三倍）（p < 0.001），并且MV中发现的量与条带3复合物相关。已知红细胞中存在不同类型的带3复合物，需要进一步研究以更好地了解这种蛋白质在红细胞储存期间参与微泡形成。
The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.
登录
查看更多内容
影响因子：
6.5
作者：
Burger, Patrick;Kostova, Elena;van Bruggen, Robin
通讯作者：
van Bruggen, Robin
影响因子：
2.9
作者：
Bosman, Giel J. C. G. M.;Lasonder, Edwin;De Grip, Willem J.
通讯作者：
De Grip, Willem J.
影响因子：
2.7
作者：
Bardyn, M.;Maye, S.;Prudent, M.
通讯作者：
Prudent, M.
影响因子：
3.7
作者：
Bardyn, Manon;Rappaz, Benjamin;Prudent, Michel
通讯作者：
Prudent, Michel
影响因子：
2.9
作者：
Ciana, A;Balduini, C;Minetti, G
通讯作者：
Minetti, G