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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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Specificity of a  LYVE-1 receptor polyclonal  serum. The specificity of an  affinity-purified rabbit polyclonal antiserum generated  against soluble LYVE-1 receptor and its capacity to  block HA binding were assessed in microtiter plate  binding assays. In the top  panel, wells coated with either LYVE-1 Fc (filled circles), the CD44H ectodomain fragment CD44158his  (triangles), or the control fusion protein ICAM-2 Fc  (squares) were incubated  with appropriately diluted  LYVE-1 specific polyclonal  antiserum, and binding was  detected with peroxidase-conjugated anti–human Ig  (see Materials and Methods).  As a control, a second set of LYVE-1–coated wells was reacted  with appropriately diluted preimmune serum (open circles). In  the bottom panel, wells coated with LYVE-1 Fc were incubated  with soluble biotinylated HA (5 μg/ml), in the presence of increasing concentrations of either LYVE-1 antiserum or control  preimmune serum followed by detection of bound HA as described in Materials and Methods. Data in each case are the  mean ± SEM of triplicate determinations.
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Figure 7: Specificity of a LYVE-1 receptor polyclonal serum. The specificity of an affinity-purified rabbit polyclonal antiserum generated against soluble LYVE-1 receptor and its capacity to block HA binding were assessed in microtiter plate binding assays. In the top panel, wells coated with either LYVE-1 Fc (filled circles), the CD44H ectodomain fragment CD44158his (triangles), or the control fusion protein ICAM-2 Fc (squares) were incubated with appropriately diluted LYVE-1 specific polyclonal antiserum, and binding was detected with peroxidase-conjugated anti–human Ig (see Materials and Methods). As a control, a second set of LYVE-1–coated wells was reacted with appropriately diluted preimmune serum (open circles). In the bottom panel, wells coated with LYVE-1 Fc were incubated with soluble biotinylated HA (5 μg/ml), in the presence of increasing concentrations of either LYVE-1 antiserum or control preimmune serum followed by detection of bound HA as described in Materials and Methods. Data in each case are the mean ± SEM of triplicate determinations.

Mentions: To explore the expression pattern of the LYVE-1 protein in more detail, we generated a polyclonal serum by immunizing rabbits with purified LYVE-1 Fc fusion protein (see above). The serum was then preabsorbed on human IgG Sepharose to remove contaminating Fc-reactive antibodies, followed by affinity chromatography on a LYVE-1–Sepharose column. The specificity of the purified antiserum was established using immobilized LYVE-1 Fc fusion protein in an ELISA assay. As shown in the top panel of Fig. 7, the LYVE-1 antiserum was highly specific for LYVE-1 Fc at dilutions below 1:100 and reacted only weakly with the control ICAM-2 fusion protein, which in common with LYVE-1 Fc, contains the hinge, CH2, and CH3 domains of human IgG1. Importantly, the LYVE-1 antiserum was unreactive with a CD44H ectodomain construct CD44158his which contains a functional Link module (residues 1–158) expressed as a histidine-tagged bacterial fusion protein (3), with CD44H Fc and with CD44-transfected COS 1 cells (not shown). Finally, the LYVE-1 antiserum (1:100 dilution) had the capacity to completely block binding of soluble bHA (Fig. 7, bottom). These data indicate the polyclonal serum is specific for LYVE-1 and does not exhibit reactivity with CD44, its closest homologue.


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)

Specificity of a  LYVE-1 receptor polyclonal  serum. The specificity of an  affinity-purified rabbit polyclonal antiserum generated  against soluble LYVE-1 receptor and its capacity to  block HA binding were assessed in microtiter plate  binding assays. In the top  panel, wells coated with either LYVE-1 Fc (filled circles), the CD44H ectodomain fragment CD44158his  (triangles), or the control fusion protein ICAM-2 Fc  (squares) were incubated  with appropriately diluted  LYVE-1 specific polyclonal  antiserum, and binding was  detected with peroxidase-conjugated anti–human Ig  (see Materials and Methods).  As a control, a second set of LYVE-1–coated wells was reacted  with appropriately diluted preimmune serum (open circles). In  the bottom panel, wells coated with LYVE-1 Fc were incubated  with soluble biotinylated HA (5 μg/ml), in the presence of increasing concentrations of either LYVE-1 antiserum or control  preimmune serum followed by detection of bound HA as described in Materials and Methods. Data in each case are the  mean ± SEM of triplicate determinations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132933&req=5

Figure 7: Specificity of a LYVE-1 receptor polyclonal serum. The specificity of an affinity-purified rabbit polyclonal antiserum generated against soluble LYVE-1 receptor and its capacity to block HA binding were assessed in microtiter plate binding assays. In the top panel, wells coated with either LYVE-1 Fc (filled circles), the CD44H ectodomain fragment CD44158his (triangles), or the control fusion protein ICAM-2 Fc (squares) were incubated with appropriately diluted LYVE-1 specific polyclonal antiserum, and binding was detected with peroxidase-conjugated anti–human Ig (see Materials and Methods). As a control, a second set of LYVE-1–coated wells was reacted with appropriately diluted preimmune serum (open circles). In the bottom panel, wells coated with LYVE-1 Fc were incubated with soluble biotinylated HA (5 μg/ml), in the presence of increasing concentrations of either LYVE-1 antiserum or control preimmune serum followed by detection of bound HA as described in Materials and Methods. Data in each case are the mean ± SEM of triplicate determinations.
Mentions: To explore the expression pattern of the LYVE-1 protein in more detail, we generated a polyclonal serum by immunizing rabbits with purified LYVE-1 Fc fusion protein (see above). The serum was then preabsorbed on human IgG Sepharose to remove contaminating Fc-reactive antibodies, followed by affinity chromatography on a LYVE-1–Sepharose column. The specificity of the purified antiserum was established using immobilized LYVE-1 Fc fusion protein in an ELISA assay. As shown in the top panel of Fig. 7, the LYVE-1 antiserum was highly specific for LYVE-1 Fc at dilutions below 1:100 and reacted only weakly with the control ICAM-2 fusion protein, which in common with LYVE-1 Fc, contains the hinge, CH2, and CH3 domains of human IgG1. Importantly, the LYVE-1 antiserum was unreactive with a CD44H ectodomain construct CD44158his which contains a functional Link module (residues 1–158) expressed as a histidine-tagged bacterial fusion protein (3), with CD44H Fc and with CD44-transfected COS 1 cells (not shown). Finally, the LYVE-1 antiserum (1:100 dilution) had the capacity to completely block binding of soluble bHA (Fig. 7, bottom). These data indicate the polyclonal serum is specific for LYVE-1 and does not exhibit reactivity with CD44, its closest homologue.

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