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At least 19 recordsLinked to original sources
PubMed1999

Characterization of a hyaluronan receptor on rat sinusoidal liver endothelial cells and its functional relationship to scavenger receptors.

Hyaluronan is a widely distributed extracellular component of connective tissue with several mechanical and cell biological functions. The serum level of hyaluronan is elevated in rheumatic and liver diseases and in certain malignancies. The major route of hyaluronan clearance from the blood is via the liver, taken up predominantly by sinusoidal liver endothelial cells. We have purified a novel hyaluronan binding protein from liver that also has an affinity for the N-terminal propeptide of type I procollagen, a physiological scavenger receptor ligand. A polyclonal antibody raised against the protein was found to inhibit the binding and degradation of hyaluronan as well as two scavenger receptor ligands by cultured sinusoidal liver endothelial cells. Immunostaining of nonpermeabilized liver cells and liver sections showed that the antibody specifically stains the surface of sinusoidal liver endothelial cells. After pretreatment with monensin to block the recirculation of endocytic receptors, the immunostaining was specifically associated with early endosomes of these cells. Thus, this rat sinusoidal liver endothelial cell hyaluronan receptor shares functional properties with the scavenger receptor family, a group of proteins shown to play a key role in the uptake of atherogenic lipids and other waste products from the tissues.

Animals↗
PubMed2005

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

Animals↗
PubMed1993

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

Amino Acid Sequence↗
PubMed1994

The regulation of sperm motility by a novel hyaluronan receptor.

OBJECTIVE: To determine if a novel receptor for hyaluronan, termed RHAMM, is responsible for the previously observed increase in sperm locomotion in response to hyaluronan and to assess whether expression of the RHAMM protein is involved in sperm motility. DESIGN: The RHAMM protein was localized on human sperm by immunofluorescence of fixed cells, fluorescence-activated cell sorter (FACS) of cell surface phenotype, and Western transblot analysis of cell proteins. The effect of monospecific antibodies on sperm motility was examined using computer-assisted image analysis. Results of motility studies were assessed statistically with analysis of variance. SETTING: Samples were collected from donors from the University of Manitoba donor insemination program. SUBJECTS: Semen was collected twice from four participants and a total of 10,000 sperm per sample were evaluated. RESULTS: A hyaluronan receptor, RHAMM, was localized by immunofluorescence along the tail, the midpiece, and the head of sperm. Positive staining obtained with FACS analysis indicated that RHAMM occurred on the surface of sperm, whereas immunoblot analysis of sperm cell lysates revealed RHAMM proteins of MWE 58 and 64 kd, consistent with the size of RHAMM localized from fibroblasts. A polyclonal antibody specific to a peptide encoded in the fibroblast RHAMM complementary DNA significantly decreased the motility of sperm. Analysis of this inhibition is consistent with an effect of the antibody on flagellar function. CONCLUSIONS: The presence of RHAMM on sperm surfaces and the ability of monospecific antibodies to inhibit sperm motility suggest an important role for this novel glycoprotein in sperm motility.

Analysis of Variance↗
PubMed2003

A blocking antibody to the hyaluronan receptor for endocytosis (HARE) inhibits hyaluronan clearance by perfused liver.

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.

Animals↗
PubMed1999

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor.

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.

Animals↗
PubMed1996

Transforming growth factor-beta1 stimulates multiple protein interactions at a unique cis-element in the 3'-untranslated region of the hyaluronan receptor RHAMM mRNA.

The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.

Animals↗
PubMed1997

AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program.

Animals↗
PubMed2006

Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1.

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.

Animals↗
PubMed2006

Role of the hyaluronan receptor CD44 during porcine oocyte maturation.

Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.

Animals↗
PubMed1994

Hyaluronan receptors are expressed on human malignant mesothelioma cells but not on normal mesothelial cells.

Hyaluronan-binding sites were demonstrated on the cell surface of three malignant mesothelioma cell lines derived from human tumors using either [3H]hyaluronan or fluorescein-tagged hyaluronan. No hyaluronan-binding activity was observed on normal human mesothelial cells. The absence of hyaluronan receptors on normal human mesothelial cells was not due to a down-regulation by endogenously synthesized hyaluronan, since no binding sites appeared when the cells were cultured under conditions known to suppress hyaluronan synthesis (in starvation medium containing either hydrocortisone or n-butyrate) or to degrade endogenously synthesized hyaluronan (in the presence of Streptomyces or testicular hyaluronidase). The binding of [3H]hyaluronan on mesothelioma cells could be partially inhibited by prior incubation of the cells with trypsin, indicating that the hyaluronan-binding site is a protein. The binding sites on human malignant mesothelioma cells were shown to be saturable with about 54,000 hyaluronan molecules (M(r) 1.4 x 10(6)) bound per cell with a Kd of 0.3 x 10(-9) M. The binding was specific for hyaluronan inasmuch as a number of other macromolecules gave negligible inhibition of the binding. High molecular weight preparations of hyaluronan inhibited the binding more effectively than low molecular weight preparations; hyaluronan oligosaccharides down to a length of six monosaccharide units showed competing activity. The hyaluronan receptor appeared to be related to CD44 (a cell surface glycoprotein previously suggested to function as a hyaluronan receptor) since Hermes-1 monoclonal antibodies which inhibit the binding of hyaluronan to CD44 blocked a major part of the binding of hyaluronan to the mesothelioma cells. However, there was no strict correlation between the hyaluronan-binding activity on the mesothelioma cell lines tested and the levels of CD44 molecules on their cell surface, suggesting that only a subfraction of the CD44 molecules bound hyaluronan or that other hyaluronan-binding proteins also exist on those cells. The presence of hyaluronan receptors on mesothelioma cells, but not on their normal counterparts, may be of importance for the migration of the transformed cells in hyaluronan-enriched matrices and for their ability to form metastases.

Antibodies, Monoclonal↗
PubMed1992

The hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan.

The hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, the binding of lymphocytes to high endothelial cells during extravasation, and conferring metastatic potential to carcinoma cells. Here, we demonstrate that the receptor also participates in the uptake and degradation of hyaluronan by both transformed fibroblasts (SV-3T3 cells) and alveolar macrophages. These cells were incubated with isotopically labeled hyaluronan for various periods of time, and the extent of degradation was determined by either molecular-sieve chromatography or centrifugation through Centricon 30 microconcentrators. The macrophages degraded the hyaluronan at a faster rate than the SV-3T3 cells, which may reflect the fact that they contained a greater number of receptors. More importantly, in both cell types, the degradation of hyaluronan was specifically blocked by antibodies directed against the receptor. However, the receptor by itself did not have the ability to degrade hyaluronan, since preparations of SV-3T3 membranes containing the receptor did not break down hyaluronan. Subsequent experiments revealed that macrophages can internalize fluorescein-tagged hyaluronan, and this process was blocked by antibodies against the receptor. Furthermore, the subsequent degradation of hyaluronan was inhibited by agents that block the acidification of lysosomes (chloroquine and NH4Cl). Thus, the most likely explanation for these results is that the receptor mediates the uptake of hyaluronan into the cell where it can be degraded by acid hydrolases in lysosomes. The ability of cells expressing the receptor to degrade hyaluronan may be important during tissue morphogenesis and cell migration.

Ammonium Chloride↗
PubMed1992

Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility.

A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.

3T3 Cells↗
PubMed1991

Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix.

In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serum-containing or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed.

Animals↗
PubMed1998

The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells.

The receptor for hyaluronan mediated motility (RHAMM) has been reported to mediate migration, transformation, and metastatic spread of murine fibroblasts. Here we describe the expression of two human RHAMM isoforms, which are generated by alternative splicing of the primary gene transcript, by a series of human breast carcinoma cell lines. A polyclonal antibody, raised against a bacterially expressed RHAMM fusion protein, detected an 85-90 kDa protein by western blot analysis. No correlation between the level of RHAMM mRNA and protein expression with known metastatic/malignant potential of the tumour cell lines was observed. Interestingly, we found that the antibody did not stain the cell surface but the cytoplasm of breast cancer cells. The intracellular localisation of RHAMM was confirmed by subcellular fractionation studies. RHAMM proteins were capable of binding to hyaluronan, but not to heparin or chondroitin sulphate, in an vitro binding assay. We also provide evidence that a potential hyaluronan-binding motif in the N terminus of the protein is not involved in the interaction of RHAMM with hyaluronan. Our findings lead us to conclude that RHAMM does not function as a conventional motility receptor for HA in human breast cancer cells and we suggest the term RHAMM be substituted by 'intracellular hyaluronic acid binding protein' (IHABP).

Alternative Splicing↗
PubMed1993

Antibody-induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody.

CD44 can function as a receptor for hyaluronan (HA). However, many cell lines and normal hematopoietic cells that express CD44 do not constitutively bind HA. A monoclonal antibody (mAb) specific for CD44 (IRAWB 14) has been described previously which induces CD44-mediated binding of HA rapidly (seconds to minutes) in some cell lines and in normal murine T cells. Of 16 CD44-specific mAb tested in the present study, only 3 exhibited this activity. Monovalent Fab fragments were prepared from two IgG2a antibodies that induce HA binding (IRAWB 14 and IRAWB 26) and used to determine whether multivalent binding was required for induction of HA receptor function. Fab from both antibodies had a tendency to form multivalent aggregates. After addition of iodoacetamide to prevent further aggregation, multimeric and monovalent forms were separated by gel filtration. This made it possible to compare the inducing activity of monovalent and multivalent antibody fragments of identical composition in the absence of Fc determinants. Multimeric forms were very active at inducing binding of fluorescein-conjugated HA (Fl-HA). Monovalent Fab fragments of both antibodies had 20- to 50-fold lower binding activity than intact antibody or multimer. IRAWB 26 Fab monomers were completely inactive in the induction of HA-binding. The observed weak inducing activity of IRAWB 14 Fab monomer could be attributed to very low levels of contaminating multimer. Induction of HA binding could also be achieved by using anti-immunoglobulin to cross-link Fab monomers of IRAWB 26. Thus, multivalent binding was required for the activation of HA binding by CD44-specific antibody, suggesting that the distribution of CD44 molecules on the cell surface is important for HA receptor function. In kinetic studies, induction of HA receptor function occurred simultaneously with antibody binding at 0 degrees C (ice water bath). Furthermore, antibody could induce HA binding in paraformaldehyde-fixed cells, which were permeable to propidium iodide and trypan blue, suggesting that intracellular signaling mechanisms were not involved in induction of receptor function. We conclude, therefore, that these CD44-specific antibodies are inducing HA binding by directly influencing the distribution of CD44 on the cell surface. The possibility of a concurrent change in CD44 conformation is not ruled out. We discuss possible mechanisms by which CD44 might be activated to bind HA in vivo.

Animals↗
PubMed1999

Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor.

The glycosaminoglycan hyaluronan (HA) and HA-binding proteins (HABPs) serve important structural and regulatory functions during development and in maintaining adult tissue homeostasis. Here we have identified and partially characterized the sequence and expression pattern of three putative novel HABPs. DNA sequence analysis revealed that two of the novel HABPs, WF-HABP and BM-HABP, form a unique HA-binding subfamily, whereas the third protein, OE-HABP, is more closely related to the LINK subfamily of HABPs. Northern blotting experiments revealed that the expression of BM-HABP was highly restricted, with substantial expression detected only in human fetal liver. In contrast, WF-HABP and OE-HABP mRNAs were detected in a number of tissues, with particularly prominent expression in highly vascularized tissues such as the heart, placenta, and lung. Additional studies showed that OE-HABP was expressed by cultured human endothelial cells, smooth muscle cells, and differentiated monocytes. However, only endothelial cells expressed WF-HABP mRNA, and its expression was regulated by growth state, being most prominent in quiescent endothelial cells. We further characterized the expression of WF-HABP in vivo and found that its expression colocalized with CD31-positive cells and was prominently expressed in microvessels in the human aorta and in atherectomy samples. Our data suggest that WF-HABP is an endothelial cell-specific HA receptor and that it may serve a unique function in these cells. The WF-HABP gene was localized to chromosome 3p21.31 and the OE-HABP gene to 15q25.2-25.3.

Adult↗
PubMed2004

Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE).

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains approximately 25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous (125)I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was approximately 8.5 min. The average K(d) for the binding of HA to the 190-kDa hHARE at 4 degrees C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 degrees C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 degrees C, none of the glycosaminoglycans tested, except HA, competed for (125)I-HA binding by 190hHARE cells at 4 degrees C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit (125)I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.

Animals↗