Galectin-4 interacts with the drug transporter human concentrative nucleoside transporter 3 to regulate its function.
The intracellular N-terminal domain of the nucleoside and drug transporter human concentrative nucleoside transporter (hCNT)3 was used as bait in a glutathione S-transferase pull-down approach, to identify hCNT3 protein partners, using human colon homogenates as a prey source. Galectin (Gal)-4 was identified as a potential hCNT3 partner in the colon. The biochemical validation of the Gal-4-hCNT3 interaction was verified by targeted pull-down assays and coimmunoprecipitation experiments in HT-29 cells, which endogenously express hCNT3 and Gal-4. Furthermore, Gal-4 was shown to colocalize with hCNT3 in HT-29 cells. The biologic significance of this interaction was obtained from experiments in which Gal-4 was knocked down, showing that this protein is a regulator of hCNT3 trafficking and retention at the cell membrane, reducing its plasma membrane location by 70%. Conversely, the addition of Gal-4 increased hCNT3 location at the plasma membrane by 77%, thereby demonstrating that this lectin modulates hCNT3 function in colonic cells. The integrity of this partnership may be clinically relevant, because hCNT3 may be responsible for the translocation of thiopurines, such as 6-mercaptopurine, a front-line treatment in inflammatory bowel disease. The expression of Gal-4 and hCNT3 proteins is not impaired in inflamed colon from patients with Crohn's disease, thereby anticipating the integrity of this system for drug targeting.
*Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain; Oncology Program, National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER EHD), Instituto de Salud Carlos III, Madrid, Spain; and Department of Gastroenterology, Digestive System Research Unit, Institut de Recerca Vall d'Hebron, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, CIBER EHD, Barcelona, Spain pfernandezcalotti@gmail.com mpastor@ub.edu.
Paula
P
Fernández-Calotti
*Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain; Oncology Program, National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER EHD), Instituto de Salud Carlos III, Madrid, Spain; and Department of Gastroenterology, Digestive System Research Unit, Institut de Recerca Vall d'Hebron, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, CIBER EHD, Barcelona, Spain.
Olga
O
Casulleras
*Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain; Oncology Program, National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER EHD), Instituto de Salud Carlos III, Madrid, Spain; and Department of Gastroenterology, Digestive System Research Unit, Institut de Recerca Vall d'Hebron, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, CIBER EHD, Barcelona, Spain.
María
M
Antolin
*Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain; Oncology Program, National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER EHD), Instituto de Salud Carlos III, Madrid, Spain; and Department of Gastroenterology, Digestive System Research Unit, Institut de Recerca Vall d'Hebron, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, CIBER EHD, Barcelona, Spain.
Francisco
F
Guarner
*Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain; Oncology Program, National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER EHD), Instituto de Salud Carlos III, Madrid, Spain; and Department of Gastroenterology, Digestive System Research Unit, Institut de Recerca Vall d'Hebron, University Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, CIBER EHD, Barcelona, Spain pfernandezcalotti@gmail.com mpastor@ub.edu.
Marçal
M
Pastor-Anglada
D003106:Colon / Q000378:metabolism* / Q000473:pathology; D003424:Crohn Disease / Q000378:metabolism* / Q000473:pathology; D037541:Galectin 4 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D055785:Gene Knockdown Techniques; D019073:HT29 Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015122:Mercaptopurine / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference
D037541:Galectin 4; D026901:Membrane Transport Proteins; C065171:cif nucleoside transporter; D015122:Mercaptopurine
CNT; Crohn’s disease; IBD; membrane transporter; protein interaction