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Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium

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 lymph...

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Autores principales: Banerji, Suneale, Lawrance, William, Metcalfe, Clive, Briggs, David C., Yamauchi, Akira, Dushek, Omer, van der Merwe, P. Anton, Day, Anthony J., Jackson, David G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122770/
https://www.ncbi.nlm.nih.gov/pubmed/27733683
http://dx.doi.org/10.1074/jbc.M116.736926
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author Banerji, Suneale
Lawrance, William
Metcalfe, Clive
Briggs, David C.
Yamauchi, Akira
Dushek, Omer
van der Merwe, P. Anton
Day, Anthony J.
Jackson, David G.
author_facet Banerji, Suneale
Lawrance, William
Metcalfe, Clive
Briggs, David C.
Yamauchi, Akira
Dushek, Omer
van der Merwe, P. Anton
Day, Anthony J.
Jackson, David G.
author_sort Banerji, Suneale
collection PubMed
description 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.
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spelling pubmed-51227702016-11-28 Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium Banerji, Suneale Lawrance, William Metcalfe, Clive Briggs, David C. Yamauchi, Akira Dushek, Omer van der Merwe, P. Anton Day, Anthony J. Jackson, David G. J Biol Chem Glycobiology and Extracellular Matrices 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. American Society for Biochemistry and Molecular Biology 2016-11-25 2016-10-12 /pmc/articles/PMC5122770/ /pubmed/27733683 http://dx.doi.org/10.1074/jbc.M116.736926 Text en © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Glycobiology and Extracellular Matrices
Banerji, Suneale
Lawrance, William
Metcalfe, Clive
Briggs, David C.
Yamauchi, Akira
Dushek, Omer
van der Merwe, P. Anton
Day, Anthony J.
Jackson, David G.
Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title_full Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title_fullStr Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title_full_unstemmed Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title_short Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium
title_sort homodimerization of the lymph vessel endothelial receptor lyve-1 through a redox-labile disulfide is critical for hyaluronan binding in lymphatic endothelium
topic Glycobiology and Extracellular Matrices
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122770/
https://www.ncbi.nlm.nih.gov/pubmed/27733683
http://dx.doi.org/10.1074/jbc.M116.736926
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