Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.
Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.
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DOI：
10.1074/jbc.m116.736926
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发表时间：
2016-11-25
期刊：
影响因子：
--
通讯作者：
Jackson DG
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作者：
Banerji S;Lawrance W;Metcalfe C;Briggs DC;Yamauchi A;Dushek O;van der Merwe PA;Day AJ;Jackson DG
淋巴管内皮受体LYVE-1参与透明质酸（HA）的摄取和白细胞向引流淋巴结的运输。然而，LYVE-1仅对透明质酸具有弱亲和力，并且依赖于受体簇和更高级配体组织以在淋巴内皮中持久结合。LYVE-1在其他HA受体中未发现的一个不寻常的特征是形成二硫键连接的同源二聚体的潜力。然而，它们对功能的影响尚未研究。在这里，我们表明LYVE-1同源二聚体是淋巴管内皮细胞在体外和体内的主要构型，并且形成仅需要膜近端结构域内的未配对半胱氨酸残基Cys-201，产生比单体高15倍的HA结合亲和力和低67倍的解离速率。此外，我们发现非二聚LYVE-1突变体即使在淋巴管内皮细胞中以高密度表达或与抗体人工交联时也不能结合HA。与这些发现一致，小角X射线散射（SAXS）表明Cys-201链间二硫键形成铰链，该铰链将同二聚体维持在“开放剪刀”构象，可能允许两个HA结合结构域的排列用于与配体相互接合。最后，我们证明了Cys-201链间二硫键是高度不稳定的，并且用TCEP-HCl选择性还原破坏LYVE-1同源二聚体，消除HA结合。这些发现表明，结合不仅取决于聚集，还取决于LYVE-1同二聚体的生化特性。他们还将LYVE-1标记为第一个需要共价同源二聚化功能的连接蛋白超家族成员，并表明链间二硫键在体内起氧化还原开关的作用。
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.