title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.
abstract: Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Jinju
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Qin
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Benqiong
authors.initials: B
authors.lastname: Xiang
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Wei
mesh_terms: D019703:Calcineurin / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism
chemical_list: C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D019703:Calcineurin