title: Structural determinants of heparin-transforming growth factor-β1 interactions and their effects on signaling.
abstract: Transforming growth factor-β1 (TGF-β1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-β1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-β1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-β1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-β1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-β1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-β1 in hMSCs.
authors: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore 117456 Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648.
authors: Jonathan
authors: J
authors: Lee
authors: Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore 138673.
authors: Sheena
authors: S
authors: Wee
authors: Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore 138673.
authors: Jayantha
authors: J
authors: Gunaratne
authors: Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648.
authors: R J E
authors: RJ
authors: Chua
authors: Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648.
authors: Raymond A A
authors: RA
authors: Smith
authors: Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648.
authors: Ling
authors: L
authors: Ling
authors: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
authors: David G
authors: DG
authors: Fernig
authors: Department of Biological Sciences, National University of Singapore, Singapore, Singapore 117543.
authors: Kunchithapadam
authors: K
authors: Swaminathan
authors: Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648 Lee Kong Chian School of Medicine, Nanyang Technological University-Imperial College, Singapore, Singapore 639798 simon.cool@imb.a-star.edu.sg victor.nurcombe@imb.a-star.edu.sg.
authors: Victor
authors: V
authors: Nurcombe
authors: Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore 138648 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 119228 simon.cool@imb.a-star.edu.sg victor.nurcombe@imb.a-star.edu.sg.
authors: Simon M
authors: SM
authors: Cool
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: C497782:SMAD2 protein, human; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D006493:Heparin
keywords: heparan sulfate; heparin; proteoglycans; transforming growth factor-β1