Distinctive properties of the hyaluronan-binding domain in the lymphatic endothelial receptor Lyve-1 and their implications for receptor function.
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.
Banerji
Suneale
S
Medical Research Council Human Immunology Unit, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
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Branwen R S
BR
James
John R
JR
Noble
Martin E M
ME
Jackson
David G
DG
D000595:Amino Acid Sequence; D004729:Endothelium, Lymphatic / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C497483:CD44 protein, human; D018960:Hyaluronan Receptors; C119248:LYVE1 protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D006820:Hyaluronic Acid