title: Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.
abstract: Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers.
authors.affiliation: Centre for Biomedical Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, U.K.
authors.forename: Arnold Junior
authors.initials: AJ
authors.lastname: Tatsinkam
authors.affiliation: Centre for Biomedical Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, U.K.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mulloy
authors.affiliation: Centre for Biomedical Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 0EX, U.K.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Rider
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D055415:Bone Morphogenetic Protein 4 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016207:Cytokines; D005544:Forecasting; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D055415:Bone Morphogenetic Protein 4; D016207:Cytokines; D006493:Heparin; D036341:Intercellular Signaling Peptides and Proteins; C525809:BMP4 protein, human; C113178:Grem1 protein, mouse
keywords: CAN family; bone morphogenetic protein; bone morphogenetic protein antagonist; gremlin; heparan sulfate; heparin