Abstract
The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.
Highlights
Hughes Medical Institute, University of California, San Francisco, CA 94143. 2 To whom correspondence should be addressed
Like CD44, lymphatic vessel endothelial receptor-1 (Lyve-1) is an integral membrane glycoprotein and a member of the HA-binding Link protein superfamily [13]. Each representative of this group contains a single copy of the consensus Link module ( known as the proteoglycan tandem repeat [16, 17]), a conserved domain resembling the C-type lectin fold composed of two antiparallel  sheets made from a total of six  strands (1– 6) and two ␣ helices stabilized by a pair of conserved disulfide bridges [18, 19]
Unlike CD44, in which HA binding is dominated by mixed hydrophobic and hydrogen bonding character, the Lyve-1 binding patch appears to be dominated by charged residues that support mainly electrostatic interactions
Summary
Lyve-1, lymphatic vessel endothelial receptor-1; BRET, bioluminescent resonance energy transfer; BRETeff, BRET efficiency; HA, hyaluronan; HABD, hyaluronan-binding domain; Fl-HA, fluoresceinconjugated hyaluronan; TEV, tobacco etch virus; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting; Luc, luciferase; GFP, green fluorescent protein; bHA, biotinylated hyaluronan. Unlike CD44, which can be activated by inflammatory agents such as tumor necrosis factor-␣ and lipopolysaccharide in cells expressing the appropriate sialidase activity [25,26,27], the factors required for unmasking Lyve-1 remain elusive. These various observations suggest that Lyve-1, similar to CD44, may have quite distinct properties, possibly explaining why two such related receptors might have evolved such markedly segregated patterns of expression. These characteristics indicate unexpected differences in HA binding properties between Lyve-1 and CD44 that are likely to have important consequences for biological function
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