Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
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 |
_version_ | 1782469643519131648 |
---|---|
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. |
format | Online Article Text |
id | pubmed-5122770 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT banerjisuneale homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT lawrancewilliam homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT metcalfeclive homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT briggsdavidc homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT yamauchiakira homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT dushekomer homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT vandermerwepanton homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT dayanthonyj homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium AT jacksondavidg homodimerizationofthelymphvesselendothelialreceptorlyve1througharedoxlabiledisulfideiscriticalforhyaluronanbindinginlymphaticendothelium |