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LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.

Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG - J. Cell Biol. (1999)

Bottom Line: Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA.However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels.Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Molecular Immunology Group, Nuffield Department of Medicine, John Radcliff Hospital, Headington, Oxford OX3 9DU, United Kingdom.

ABSTRACT
The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

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LYVE-1 binds both immobilized and soluble HA. LYVE-1, expressed as a soluble IgFc fusion protein in transiently transfected human  COS fibroblasts, was compared with  CD44 for binding to HA and other  glycosaminoglycans. A shows LYVE-1  and CD44H Fc fusion proteins isolated from the supernatants of [35S]  methionine/cysteine-labeled transfectants and electrophoresed on a 7.5%  polyacrylamide SDS-PAGE gel. Samples were the protein A–Sepharose  adsorbed proteins from control untransfected cells (lane 1), CD44H  Fc transfected cells (lane 2), and  LYVE-1 transfected cells (lane 3).  The LYVE-1 fusion protein comprises residues 1–232 of the extracellular domain fused to the hinge (H),  CH2, and CH3 domains of human  IgG1. Details of the CD44H Fc protein, which includes residues 1–200 of  the extracellular domain, have been  published previously (1). For ligand  binding assays, LYVE-1 Fc was compared with CD44H Fc and the negative control fusion proteins CD33 Fc  and ICAM-2 Fc for adhesion to immobilized and soluble HA in 96-well  microtiter plates (see Materials and  Methods). B shows binding of the fusion proteins to immobilized HA, in  the absence of competing glycosaminoglycans; C shows binding (LYVE-1  Fc only) in the presence of free chondroitin-4-SO4, chondroitin-6-SO4, or  heparin; and D shows binding to soluble biotinylated HA. Detection of  bound fusion protein and biotinylated HA was carried out using peroxidase-conjugated anti–human IgFc  antibody and peroxidase-conjugated  streptavidin, respectively. Values are  the mean ± SEM of at least three replicates.
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Figure 4: LYVE-1 binds both immobilized and soluble HA. LYVE-1, expressed as a soluble IgFc fusion protein in transiently transfected human COS fibroblasts, was compared with CD44 for binding to HA and other glycosaminoglycans. A shows LYVE-1 and CD44H Fc fusion proteins isolated from the supernatants of [35S] methionine/cysteine-labeled transfectants and electrophoresed on a 7.5% polyacrylamide SDS-PAGE gel. Samples were the protein A–Sepharose adsorbed proteins from control untransfected cells (lane 1), CD44H Fc transfected cells (lane 2), and LYVE-1 transfected cells (lane 3). The LYVE-1 fusion protein comprises residues 1–232 of the extracellular domain fused to the hinge (H), CH2, and CH3 domains of human IgG1. Details of the CD44H Fc protein, which includes residues 1–200 of the extracellular domain, have been published previously (1). For ligand binding assays, LYVE-1 Fc was compared with CD44H Fc and the negative control fusion proteins CD33 Fc and ICAM-2 Fc for adhesion to immobilized and soluble HA in 96-well microtiter plates (see Materials and Methods). B shows binding of the fusion proteins to immobilized HA, in the absence of competing glycosaminoglycans; C shows binding (LYVE-1 Fc only) in the presence of free chondroitin-4-SO4, chondroitin-6-SO4, or heparin; and D shows binding to soluble biotinylated HA. Detection of bound fusion protein and biotinylated HA was carried out using peroxidase-conjugated anti–human IgFc antibody and peroxidase-conjugated streptavidin, respectively. Values are the mean ± SEM of at least three replicates.

Mentions: To confirm the presence of a functional HA-binding domain in the LYVE-1 receptor, we expressed the extracellular domain with its own NH2-terminal leader (residues 1–232) as a soluble fusion protein with the hinge, CH2, and CH3 domains of human IgG1 (total length 440 residues). The purified protein migrated as a single 80-kD band on SDS-PAGE, similar to a CD44H Fc fusion protein (total length 434 residues, Fig. 4 A). The discrepancy with the 46,900-D molecular mass predicted from the mature 440 residue primary sequence again is largely due to N- and O-glycosylation of the LYVE-1 core polypeptide (see above).


LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.

Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG - J. Cell Biol. (1999)

LYVE-1 binds both immobilized and soluble HA. LYVE-1, expressed as a soluble IgFc fusion protein in transiently transfected human  COS fibroblasts, was compared with  CD44 for binding to HA and other  glycosaminoglycans. A shows LYVE-1  and CD44H Fc fusion proteins isolated from the supernatants of [35S]  methionine/cysteine-labeled transfectants and electrophoresed on a 7.5%  polyacrylamide SDS-PAGE gel. Samples were the protein A–Sepharose  adsorbed proteins from control untransfected cells (lane 1), CD44H  Fc transfected cells (lane 2), and  LYVE-1 transfected cells (lane 3).  The LYVE-1 fusion protein comprises residues 1–232 of the extracellular domain fused to the hinge (H),  CH2, and CH3 domains of human  IgG1. Details of the CD44H Fc protein, which includes residues 1–200 of  the extracellular domain, have been  published previously (1). For ligand  binding assays, LYVE-1 Fc was compared with CD44H Fc and the negative control fusion proteins CD33 Fc  and ICAM-2 Fc for adhesion to immobilized and soluble HA in 96-well  microtiter plates (see Materials and  Methods). B shows binding of the fusion proteins to immobilized HA, in  the absence of competing glycosaminoglycans; C shows binding (LYVE-1  Fc only) in the presence of free chondroitin-4-SO4, chondroitin-6-SO4, or  heparin; and D shows binding to soluble biotinylated HA. Detection of  bound fusion protein and biotinylated HA was carried out using peroxidase-conjugated anti–human IgFc  antibody and peroxidase-conjugated  streptavidin, respectively. Values are  the mean ± SEM of at least three replicates.
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Figure 4: LYVE-1 binds both immobilized and soluble HA. LYVE-1, expressed as a soluble IgFc fusion protein in transiently transfected human COS fibroblasts, was compared with CD44 for binding to HA and other glycosaminoglycans. A shows LYVE-1 and CD44H Fc fusion proteins isolated from the supernatants of [35S] methionine/cysteine-labeled transfectants and electrophoresed on a 7.5% polyacrylamide SDS-PAGE gel. Samples were the protein A–Sepharose adsorbed proteins from control untransfected cells (lane 1), CD44H Fc transfected cells (lane 2), and LYVE-1 transfected cells (lane 3). The LYVE-1 fusion protein comprises residues 1–232 of the extracellular domain fused to the hinge (H), CH2, and CH3 domains of human IgG1. Details of the CD44H Fc protein, which includes residues 1–200 of the extracellular domain, have been published previously (1). For ligand binding assays, LYVE-1 Fc was compared with CD44H Fc and the negative control fusion proteins CD33 Fc and ICAM-2 Fc for adhesion to immobilized and soluble HA in 96-well microtiter plates (see Materials and Methods). B shows binding of the fusion proteins to immobilized HA, in the absence of competing glycosaminoglycans; C shows binding (LYVE-1 Fc only) in the presence of free chondroitin-4-SO4, chondroitin-6-SO4, or heparin; and D shows binding to soluble biotinylated HA. Detection of bound fusion protein and biotinylated HA was carried out using peroxidase-conjugated anti–human IgFc antibody and peroxidase-conjugated streptavidin, respectively. Values are the mean ± SEM of at least three replicates.
Mentions: To confirm the presence of a functional HA-binding domain in the LYVE-1 receptor, we expressed the extracellular domain with its own NH2-terminal leader (residues 1–232) as a soluble fusion protein with the hinge, CH2, and CH3 domains of human IgG1 (total length 440 residues). The purified protein migrated as a single 80-kD band on SDS-PAGE, similar to a CD44H Fc fusion protein (total length 434 residues, Fig. 4 A). The discrepancy with the 46,900-D molecular mass predicted from the mature 440 residue primary sequence again is largely due to N- and O-glycosylation of the LYVE-1 core polypeptide (see above).

Bottom Line: Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA.However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels.Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Molecular Immunology Group, Nuffield Department of Medicine, John Radcliff Hospital, Headington, Oxford OX3 9DU, United Kingdom.

ABSTRACT
The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Show MeSH
Related in: MedlinePlus