Smolarek Dorota, Hattab Claude, Buczkowska Anna, Kaczmarek Radoslaw, Jarząb Anna, Cochet Sylvie, de Brevern Alexandre G, Lukasiewicz Jolanta, Jachymek Wojciech, Niedziela Tomasz, Grodecka Magdalena, Wasniowska Kazimiera, Colin Aronovicz Yves, Bertrand Olivier, Czerwinski Marcin
Laboratory of Glycoconjugate Immunochemistry Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland; INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King's College London, Guy's Hospital, London, United Kingdom.
INSERM, UMR_S1134, F-75015 Paris, France; Institut National de la Transfusion Sanguine, F-75015 Paris, France; Universite Paris-Diderot, Sorbonne Paris Cité, F-15013, Paris, France; Laboratory of Excellence GR-Ex, Paris, France.
PLoS One. 2015 Feb 23;10(2):e0116472. doi: 10.1371/journal.pone.0116472. eCollection 2015.
Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.
趋化因子的达菲抗原受体（DARC）作为一种血型抗原、趋化因子受体以及间日疟原虫裂殖子唯一已知的受体，在人类健康中发挥着多种作用。它是鼠抗Fy6单克隆抗体2C3的靶点，该抗体与第一个细胞外结构域（ECD1）结合，但所识别表位的确切性质一直是相互矛盾报道的主题。在此，我们通过一系列复杂实验，包括在大肠杆菌和K562细胞中表达Fy6表位内具有氨基酸替换的DARC、酶联免疫吸附测定（ELISA）、表面等离子体共振（SPR）和流式细胞术，解决了先前发表报告之间的差异，并表明2C3抗体识别的基本表位是22FEDVW26，其中22F和26W是最重要的残基。此外，我们证明30Y在结合中起辅助作用，特别是当该残基被硫酸化时。使用2C3衍生的Fab和合成肽进行的STD核磁共振（NMR）研究证实了这些结果中的大部分，并且与分子建模一起表明25V不参与与抗体的直接相互作用，但决定表位主链的折叠。
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