title: Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.
abstract: The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.
authors.affiliation: From the Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
authors.forename: Suneale
authors.initials: S
authors.lastname: Banerji
authors.affiliation: From the Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Lawrance
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
authors.forename: Clive
authors.initials: C
authors.lastname: Metcalfe
authors.affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Biology, Medicine, and Health, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: David C
authors.initials: DC
authors.lastname: Briggs
authors.affiliation: Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan, and.
authors.forename: Akira
authors.initials: A
authors.lastname: Yamauchi
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
authors.forename: Omer
authors.initials: O
authors.lastname: Dushek
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
authors.forename: P Anton
authors.initials: PA
authors.lastname: van der Merwe
authors.affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Biology, Medicine, and Health, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Anthony J
authors.identifier: http://orcid.org/0000-0002-1415-3134
authors.initials: AJ
authors.lastname: Day
authors.affiliation: From the Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom, david.jackson@imm.ox.ac.uk.
authors.forename: David G
authors.identifier: http://orcid.org/0000-0002-4133-9364
authors.initials: DG
authors.lastname: Jackson
mesh_terms: D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004729:Endothelium, Lymphatic / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D055503:Protein Multimerization / Q000502:physiology*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; C119248:LYVE1 protein, human; D033921:Vesicular Transport Proteins; D006820:Hyaluronic Acid; D003545:Cysteine
keywords: LYVE-1; avidity; dimer; disulfide; endothelium; hyaluronan; labile; lymphatic; receptor; redox regulation