N-domain of human adhesion/growth-regulatory galectin-9: Preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution |
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| Authors: | Dolores Solís María Jesus Maté Michaela Lohr João P Ribeiro Lara López-Merino Sabine André Eliza Buzamet F Javier Cañada Herbert Kaltner Martin Lensch Federico M Ruiz Gunter Haroske Uwe Wollina Matthias Kloor Jürgen Kopitz José L Sáiz Margarita Menéndez Jesús Jiménez-Barbero Antonio Romero Hans-Joachim Gabius |
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| Institution: | 1. Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain;2. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Bunyola, Mallorca, Illes Baleares, Spain;3. Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain;4. Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, Veterinärstr. 13, 80539 München, Germany;5. Krankenhaus Dresden-Friedrichstadt, Abteilungen für Dermatologie/Allergologie und Pathologie, Friedrichstr. 41, 01067 Dresden, Germany;6. Abteilung für Angewandte Tumorbiologie, Institut für Pathologie, Klinikum der Ruprecht-Karls-Universität, Im Neuenheimer Feld 220, 69120 Heidelberg, Germany;1. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, BioMol-Lab, Campus do Pici S/N, 60440-970 Fortaleza, Ceará, Brazil;2. Departamento de Ciências Biológicas, Universidade Regional do Cariri, Campos Sales, Ceará, Brazil;3. Departamento de Biologia Molecular, Universidade Federal da Paraíba, Campus I, Cidade Universitária, 58059-900 João Pessoa, Paraíba, Brazil;4. Departamento de Engenha de Pesca, Universidade Federal do Ceará, BioMol-Group, Campus do Pici S/N, Fortaleza, Ceará, Brazil;5. Departamento de Física, Universidade Federal do Ceará, Campus do Pici S/N, 60455-760 Fortaleza, Ceará, Brazil;6. Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, 90035-003 Porto Alegre, Rio Grande do Sul, Brazil;1. Division of Gastroenterology/Hepatology, University of Colorado, Aurora, Colorado;4. Division of Pulmonary Medicine, University of Colorado, Aurora, Colorado;3. Institute of Cellular Medicine, Newcastle University Medical School, Newcastle upon Tyne, United Kingdom;1. Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinärstr. 13, 80539 München, Germany;2. Department of Chemistry, University of Debrecen, PO Box 20, 4010 Debrecen, Hungary;3. Richter Gedeon Rt., Gyömröi út 19-21, 1103 Budapest, Hungary;4. NMR and Structural Analysis Unit, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium;1. Abteilung für Angewandte Tumorbiologie, Zentrum Pathologie, Klinikum der Ruprecht-Karls-Universität, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany;2. Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, Veterinärstraße 13, 80539 München, Germany;3. Funktionelle Proteomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany;1. Aquatic Environment and Health Management Division, ICAR- Central Institute of Fisheries Education, Mumbai 61, India;2. Fish Genetics and Biotechnology Division, ICAR- Central Institute of Fisheries Education, Mumbai 61, India |
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| Abstract: | Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the β-sandwich fold by an antiparallel β-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without α2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays. |
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