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Originally published In Press as doi:10.1074/jbc.M307201200 on August 20, 2003

J. Biol. Chem., Vol. 278, Issue 44, 42802-42811, October 31, 2003
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Characterization of the Recombinant Rat 175-kDa Hyaluronan Receptor for Endocytosis (HARE)*

Janet A. Weigel and Paul H. Weigel{ddagger}

From the Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

Received for publication, July 5, 2003 , and in revised form, August 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the 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). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained ~25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass ~ 133 kDa) to the 175-kDa HARE at 4 °C was 4.1 nM with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 °C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 °C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In a typical 70-kg adult, the total HA1 content is ~15 g, and about one-third of this turns over every day (1). HA is a structural component in all vertebrate tissue matrices and plays key roles in cell proliferation and adhesion, morphogenesis, differentiation, inflammation, and wound healing (2–6). HA and other GAGs, in particular the various CSs, are synthesized and degraded continuously in tissues throughout the body. For example, the HA in skin has a metabolic half-life of only ~1.5 days (7). Some turnover of extracellular CS likely occurs in a coordinated way and by a similar mechanism with HA turnover, because some CS chains are covalently attached to PGs, which in turn are bound to HA chains released from the ECM. Coordinated turnover of HA and some of the body's large CS pool makes sense physiologically, because HA, CS, and other GAGs in the ECM could be released simultaneously after the cleavage of HA. CS could also be released from different ECMs by other mechanisms, such as regulated proteolysis of various aggregating or nonaggregating PGs. In this case, the final turnover (i.e. uptake and degradation) of such fragments that enter the lymph or blood could be mediated either by HARE or other CS-recognizing receptors or by receptors specific for the PG protein.

The present model for the high turnover of HA and CS in ECMs throughout the mammalian body is that very large native HA molecules (approaching 107 Da) are partially digested to produce large HA fragments (~106 Da) that are then released from the matrix (8–12). These released HA fragments could still be bound to Link proteins and aggregating PGs (i.e. aggrecan, versican, neurocan, and brevican), which can contain covalently attached CS chains; all these components, therefore, would be released concurrently from ECM networks. These HA-PG fragments would also likely contain other proteins associated with particular PGs, such as growth factors. The released ECM fragments then enter lymphatic vessels and flow to regional lymph nodes, which are initial sites for the clearance and degradation of the HA and CS. Lymph nodes are the primary clearance sites, accounting for ~85% of the HA turnover. The second clearance site is the liver, which accounts for ~15% of the total body HA, and presumably CS, turnover. The HA/CS clearance and degradation in liver and lymph nodes is mediated by the same endocytic receptor (now designated HARE), which is expressed in the sinusoidal endothelial cells of these tissues (13, 14).

Although humans turn over ~5 g of HA per day, the two HA/CS clearance systems utilizing HARE in lymph node and liver maintain a very low steady-state concentration of HA in blood (i.e. 10–100 ng/ml). Presumably, the removal of HA from lymph fluid and blood is very important for normal health. In particular, if the concentration of high molecular mass HA increased, then the viscosity of these latter fluids could increase to dangerous levels. For example, the passage of blood cells through narrow microcapillary beds would be impaired if blood viscosity increased. Coagulation homeostasis could also be perturbed, because HA specifically binds to human fibrinogen (15) and stimulates fibrin clot formation (16). Elevated levels of serum HA occur in several diseases, including some cancers (17), psoriasis (18), scleroderma (19), rheumatoid arthritis (20), and liver cirrhosis (21, 22). Hepatic clearance of HA is such an important function that elevated serum HA is often used as a diagnostic tool to detect or monitor liver failure (23).

In previous studies, we used a specific mAb and a ligand blot assay to purify two membrane-bound HA-binding proteins from rat LECs (24) and human spleen (25). The rHARE isoforms are 175 and 300 kDa and the hHARE isoforms are 190 and ~315 kDa. These HARE isoforms are expressed in the liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes (25, 26). The small HARE isoforms contain a single subunit, whereas the large isoforms contain multiple disulfide-bonded subunits, e.g. the ~315-kDa hHARE has two subunits (250 and 220 kDa) in a ratio of ~3:1 (25). Although they are different sizes, all of the subunits in both HARE isoforms appear to be derived from the same large (2551-amino acid) precursor protein, which has also been called Stabilin 2 (27). When expressed in SK-Hep-1 cells in the absence of the large isoform, the small rHARE isoform co-localized with clathrin as expected for a coated pit-coupled endocytic HA receptor (28). The two HARE species, therefore, appear to be functionally independent isoreceptors for HA.

In this study, we functionally characterized the 175-kDa rHARE in stable cell lines. Previous cellular studies have used rat LECs, which express both the 175- and ~300-kDa HARE isoforms. This study is the first examination of the ligand specificity and endocytic activity of a single HARE isoform in the absence of the other.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Buffers—Na125I was from Amersham Pharmacia Corp. 125I-HA was prepared as described previously (29) using a hexylamine derivative of HA oligosaccharides (number-average molecular mass = 133,000 based on gel permeation chromatography coupled to multiangle light scattering analysis), modified only at the reducing ends. Male Sprague-Dawley rats (200 g) were from Charles River Laboratories. BSA Fraction V was from Intergen Co. Collagenase was from Roche Applied Science. The preparation and characterization of mouse mAbs against the rat HARE were described previously (26). Tris, SDS, ammonium persulfate, N,N'-methylenebisacrylamide, and SDS-PAGE standards were from Bio-Rad. Digitonin was from ACROS Organics. Unless noted otherwise, other chemicals and reagents were from Sigma Chemical Co. All GAGs (with the exception of heparin, which came from Sigma) were obtained from Seikagaku Corp. Nitrocellulose membranes were from Schleicher & Schuell. Goat anti-mouse (polyvalent) IgG-alkaline phosphatase conjugate was from Sigma. HBSS and PBS were formulated according to the Invitrogen catalog formulations. Medium 1 is Eagle's Basal Medium (Invitrogen) supplemented with 100 mg/liter succinic acid sodium salt, 75 mg/liter succinic acid, 2.4 g/liter HEPES, and 0.22 g/liter NaHCO3. Medium 1/BSA is Medium 1 supplemented with 0.1% BSA (w/v). TBS contains 20 mM Tris-HCl, pH 7.0, and 150 mM NaCl.

Preparation of LECs—Rat LECs were prepared by a collagenase liver perfusion procedure and purified by differential and Percoll gradient centrifugation as previously described (13, 24). Contaminating Kupffer cells were allowed to adhere to a glass Petri dish, and nonadherent LECs were then collected, cultured on fibronectin-coated 24-well plates and used the same day.

Selection of Stable Transfectants Expressing the 175-kDa HARE— SK-Hep-1 cells (from American Type Culture Collection, Manassas, VA) were transfected with purified p175HARE-{kappa} DNA using FuGENE 6 and subjected to selection using G418 as described by Zhou et al. (28). Because the protein is not directly encoded by an mRNA species, the 4708-bp cDNA sequence in this construct contains, at the 5'-end, the membrane insertion (leader) sequence of the mouse immunoglobulin {kappa} chain, so that the resulting protein is inserted in the plasma membrane in the correct orientation. The 46-amino acid {kappa} chain sequence was derived from pSecTag2, and the final construct was assembled in pcDNA3.1 (both from Invitrogen). Cloning rings were used to isolate individual colonies of antibiotic-resistant transfected cells after 2–3 weeks. Cells were detached by treatment with 0.05% trypsin and 0.53 mM EDTA for 5 min at room temperature, collected, and grown in 12-well plates to assess HARE protein expression and function by enzyme-linked immunosorbent assay, Western blot, and 125I-HA binding assays. Positive cultures were further purified by dilution cloning, and the final cloned cell lines were designated by numbers, e.g. SK-HARE-36.

Western and Ligand Blot Assays—Cell lysates were mixed with equal volumes of a 2x SDS sample buffer (30), without reducing agent, to give final concentrations of 16 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% glycerol (v/v), and 0.01% bromphenol blue. After SDS-PAGE, the contents of the gel were electrotransferred to a nitrocellulose membrane overnight at 10 V at 4 °C using 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol, and 0.01% SDS in a Genie blotter apparatus from Idea Scientific. For the ligand blot assay, the nitrocellulose membrane was treated first with TBS containing 0.1% Tween 20 at 4 °C for 2 h, or TBST (Tris-buffered saline containing 0.05% Tween 20) overnight, and then incubated with 1–2 µg/ml 125I-HA in 150 mM NaCl, 10 mM HEPES, pH 7.4, and 5 mM EDTA without, or with, a 100- to 150-fold excess of nonlabeled HA (as competitor) to assess total and nonspecific binding, respectively (31). The nitrocellulose membrane was washed five times for 5 min each with TBST and dried at room temperature. The bound 125I-HA was detected by autoradiography using Kodak BioMax MS film exposed at –85 °C for 6–48 h. Nonspecific binding in this assay is typically <5%. For Western blot assays, the nitrocellulose membranes were blocked with 1% BSA in TBS at 4 °C overnight either after the ligand blot assay (the membranes were rewet with TBST first) or directly after SDS-PAGE and electrotransfer. The membrane was then incubated with anti-rat HARE mAbs (e.g. 1 µg/ml IgG) at 22 °C for 1 h, washed three times for 5 min each with TBST, and incubated with goat anti-mouse IgG-alkaline phosphatase conjugate (1:1500 dilution) for 1 h at room temperature. The nitrocellulose was washed five times for 5 min each with TBST and incubated with p-nitro blue tetrazolium and sodium 5-bromo-4-chloro-3-indolyl phosphate p-toluidine for color development (Bio-Rad), which was stopped by washing the membrane with distilled water.

125I-HA Binding or Endocytosis—Stably transfected SK-HARE cell lines were grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing 0.4 mg/ml G418 in tissue culture multi-well dishes (usually 24-well plates). The cells were washed, incubated at 37 °C in fresh medium without serum for 1 h, the plates were then placed on ice, and the cells washed two times with HBSS prior to all experiments. Medium containing 1–2 µg/ml 125I-HA with or without the noted concentration of IgG or other GAG (as competitor) was added to each well, and the cells were incubated either on ice for 60 min to assess cell surface binding or at 37 °C to allow internalization of ligand. At the noted times the medium was aspirated, the cells were washed three times with HBSS and lysed in 0.3 N NaOH, and radioactivity and protein content were determined. All values were normalized for cell protein per well and are presented as cpm/µg of protein.

Degradation of 125I-HA—Degradation of 125I-HA was measured by a cetylpyridinium chloride precipitation assay as described by McGary et al. (32). 50-µl samples of medium were mixed with 250 µl of 1 mg/ml HA in 1.5-ml microcentrifuge tubes. Alternatively, 100-µl samples of cell lysate (in 0.3 N NaOH) were mixed with 47 µl of 0.6 N HCl, 28 µl of distilled water, and 125 µl of 2 mg/ml HA. After mixing, 300 µl of 6% (w/v) cetylpyridinium chloride in distilled water was added, and the tubes were mixed by vortexing. After 10 min at room temperature, the samples were centrifuged at 9000 rpm in an Eppendorf model 5417 microcentrifuge, using a swinging bucket rotor, at 22 °C for 5 min. A sample (300 µl) of the supernatant was taken for determination of radioactivity, and the remainder was removed by aspiration. The tip of the tube containing the precipitated pellet was cut off and put into a gamma counter tube, and radioactivity was determined. Degradation was measured as the time-dependent increase of nonprecipitable radioactivity. About 80% of the total radioactivity was precipitable at the beginning of each experiment. HA fragments that are smaller than ~50 monomers do not precipitate with cetylpyridinium chloride (32), and because radioautolysis continuously generates smaller fragments, the background in this cetylpyridinium chloride precipitation assay increases with age of the radioiodinated HA.

General—Protein content was determined by the method of Bradford (33) using BSA as a standard. SDS-PAGE was performed according to the method of Laemmli (30). Western blotting was performed as described by Burnette (34) with minor modifications (26). 125I radioactivity was measured using a Packard Auto-Gamma Counting system. Digital images were captured using an Alpha Innotech Fluorochem 8000 or a Molecular Dynamics Personal Model densitometer. Images were taken in Corel Photo Paint (version 9.0) as JPG files, cropped and processed identically and then transferred to Corel Draw (version 9.0) for annotation. N-terminal amino acid sequence analysis was performed by the University of Oklahoma Health Sciences Center Molecular Biology Resource facility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of the 175-kDa rHARE Protein in Stable Cell Lines—The 175-kDa rHARE protein is not directly encoded by a distinct mRNA, but rather is generated by proteolysis from a larger precursor (28). Therefore, to create stable cell lines expressing only this HARE isoform, we used a synthetic cDNA coding for the 1431-amino acid protein fused at the N terminus to the leader sequence of the mouse immunoglobulin {kappa} light chain. This leader sequence serves as a membrane insertion signal that allows correct orientation of the protein and trafficking to the cell surface. This vector was used to transfect SK-Hep-1 cells, after which multiple stable cell lines expressing HARE were cloned using antibiotic selection. The SK-Hep-1 cell line has been used by us and by others for similar studies, and it does not display specific 125I-HA binding or endocytosis activity. Additionally, it has no endogenous surface HA receptors and no cross-reactivity with the anti-HARE mAbs.

Seven independent SK-HARE clones were obtained, all of which had similar characteristics with respect to 175-kDa rHARE expression and function. Based on Western analyses, all cell lines expressed comparable levels of HARE protein and showed similar HA-binding activity in ligand blots (Fig. 1). Each of the previously described anti-175-kDa rHARE mAbs that recognize the native nonreduced protein (26) also individually recognizes the recombinant rHARE in Western blots (not shown). Untransfected cells and SK-Hep-1 cells, transfected with the same vector containing an unrelated cDNA insert, displayed only a low level of nonspecific 125I-HA uptake at 37 °C and showed no bands in similar Western or ligand blots (not shown). In experiments examining multiple cell lines, the molecular mass of the recombinant 175-kDa HARE was 182 ± 3 kDa (n = 10), compared with 180 ± 4 kDa (n = 6) for the native rat LEC protein. Two bands were apparent in each SK-HARE clone (Fig. 1), a major larger protein and a minor smaller protein. Amino acid sequence analysis revealed multiple amino acids released at each cycle, indicating that neither protein contains a unique N terminus, but rather is a mixture of proteins with many different N termini. The sequence data for the smaller minor HARE band were consistent with at least two proteins, one starting at Asp80 (DL) and another at Gly121 (GVI). The latter HARE variant corresponds well to a minor sequence found in the native rat 175-kDa HARE that begins at Val122 (28). The sequence data for the major recombinant 175-kDa HARE band were consistent with cleavage of the signal peptide, followed by variable proteolysis within a region of ~40 amino acids starting from this cleavage site at Ala–25 (AAQPA) to Ser16 (SIFR). The recombinant rHARE, therefore, is processed by SK-Hep-1 cells and LECs in a similar manner to yield a variety of proteolytic cleavage products.



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  		FIG. 1.
Expression of the recombinant 175-kDa rHARE in stable SK-Hep-1 cell lines. Extracts of the indicated SK-HARE and vector-alone (–) clones were made by scraping the cells from the tissue culture plastic into PBS and then treating the cell pellets with 0.1% (w/v) digitonin in PBS for 10 min at 4 °C. After centrifugation, the resulting cell pellets were solubilized in Laemmli sample buffer (30) and subjected to nonreducing SDS-PAGE using 5% gels. Western blotting (left panel, using a mixture of mAbs 159, 174, 235, and 467) and ligand blotting (right panel, using 125I-HA) were performed as described under "Experimental Procedures." The HA-binding analysis is from a separate experiment with clone #35; specificity of binding was assessed in the presence (+HA) or absence (–HA) of a 100-fold excess of unlabeled HA.

 

The native and recombinant rHARE proteins contained 20–25 kDa of N-linked oligosaccharides that could be released by treatment with endoglycosidase-F (Fig. 2). The size of the apparent HARE core protein from SK-HARE cells was ~155 kDa; this is consistent with the predicted mass of the protein derived from the synthetic cDNA (minus the 21-residue signal peptide) and is essentially identical in size to the deglycosylated native rat liver protein. As with the native HARE, the deglycosylated recombinant 175-kDa HARE retained its HA-binding activity. Glycosylation of the protein is not required for its ability to bind ligand (Fig. 2, left panel).



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  		FIG. 2.
The de-N-glycosylated 175-kDa rHARE retains HA-binding activity. LECs and cells from SK-HARE clones #27 and #35 were extracted in 0.5% Nonidet P-40 and incubated with mAb-30-Sepharose for 60 min at 4 °C. The resin was washed twice, and the HARE proteins were eluted with 0.5% SDS in 0.1 M Tris, pH 7.4, by heating at 100 °C for 5 min. The resin was removed by centrifugation, and half of each supernatant from each cell type was incubated with or without N-glycosidase F (EC 3.5.1.52 [EC] from Calbiochem) at 37 °C overnight. Laemmli sample buffer (4x) was added, and the samples were heated at 100 °C for 3 min and then subjected to SDS-PAGE and electrotransfer. The nitrocellulose membranes were incubated in TBS with 0.1% Tween 20 for 2.5 h at 22 °C and then for 2 h at 4 °C. Ligand blotting and autoradiography (left panel) were performed as described under "Experimental Procedures." The same membrane was then wetted in TBST then blocked with 1% BSA in TBS at room temperature for 2 h, and Western analysis (right panel) was performed using a mixture of eight anti-HARE mAbs (1 µg/ml each) in TBS containing 1% BSA.

 

The binding of 125I-HA to SK-HARE cells at 4 °C showed a typical hyperbolic increase that plateaued after 1 h (Fig. 3A). 125I-HA binding curves in the presence of increasing concentrations of unlabeled HA (Fig. 3B) were essentially identical among the SK-HARE cell lines. Based on the midpoint of these binding curves from multiple experiments with several SK-HARE clones, the apparent Kd for HA binding was 4.3 ± 1.1 nM (n = 6). Two independent equilibrium binding experiments (as in Fig. 3C), each using SK-HARE cell lines #26 and #36, were performed and analyzed by the method of Scatchard (35). In each experiment, we observed a single class of binding sites with identical Kd values of 4.1 nM, as calculated by first order linear regression analyses (cc <= –0.95). Bmax values ranged from 160,000 to 220,000 HA-binding sites/cell, which is similar to the number of binding sites observed previously on rat LECs (36).



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  		FIG. 3.
Kinetic and Scatchard analyses of 125I-HA binding to cells expressing the recombinant rat 175-kDa HARE. SK-HARE cell lines #26 and #36 were cultured in 4- or 6-well plates until confluent, and the cells were incubated for 60 min in medium without serum and then washed. At the end of each experiment, cells were washed and cell-associated radioactivity and cell protein were determined as described under "Experimental Procedures." The data in A and B are presented as the mean ± S.D. of duplicates for each of the two clones (n = 4). A, after washing the cells, medium containing 1 µg/ml 125I-HA with ({blacksquare}) or without (•) 100 µg/ml unlabeled HA was added to each well, and the cells were allowed to bind the HA on ice for the indicated times. Specific binding, assessed in the presence of 100 µg/ml HA, was ~70%. B and C, after washing, medium (0.9 ml) containing 0.1 µg/ml 125I-HA with the indicated increasing amount of unlabeled HA was added, and the cells were allowed to bind HA on ice for 90 min. Medium was removed to determine free 125I-HA, and the cells were washed to determine cell-associated 125I-HA (B) as a function of increasing unlabeled HA. The specifically bound HA was calculated, and the results are presented in the format of a Scatchard plot (35) in panel C. Each point is the average of duplicates for the two cell lines (n = 4). The average protein content for six different SK-HARE cell lines was 1.6 ± 0.18 mg per million cells.

 

Endocytosis and Degradation of HA Mediated by the 175-kDa rHARE—All stable cell lines mediated the specific and continuous endocytosis of 125I-HA for many hours at 37 °C (Fig. 4). Specificity of HA endocytosis ranged from 67% to 79%, as assessed in the presence of excess unlabeled HA, and the cells internalized an average of 4.2-times their number of surface HA receptors within 3 h (Table I). Western analysis confirmed that HARE protein content was not decreased during this period, indicating that HARE was not delivered to lysosomes for degradation following internalization of HA-HARE complexes (not shown). The constant cellular HARE content despite the high ratio of HA molecules taken up per HARE indicates that the recombinant 175-kDa HARE is a recycling endocytic receptor, as expected based on earlier studies with rat LECs (14). Endocytosis of 125I-HA mediated by HARE in these stable cell lines led to degradation of the internalized ligand (Fig. 4). Degradation products first accumulated intracellularly (presumably in lysosomes) and then, after a long lag time, appeared in the medium. We previously found in rat LECs (32) that the HA-hexylamine derivative, which is modified at the reducing end to contain a hydroxyphenol group, behaves as a residualizing label in that degradation products first reach high levels in lysosomes before appearing in the medium (37). As expected for a coated pit-mediated receptor, the uptake and degradation of 125I-HA by SK-HARE cells was completely sensitive to hyperosmolarity (Fig. 5). Hyperosmolar conditions disrupt clathrin recycling and coated pit formation (38, 39), thus inhibiting continuous endocytosis mediated by recycling receptors such as the low density lipoprotein, asialoglycoprotein, and HARE receptors (40).



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  		FIG. 4.
Endocytosis and degradation of 125I-HA by SK-HARE cells. Confluent SK-HARE #27 cells, cultured in 4-well tissue culture plates, were incubated at 37 °C in medium without serum for 1 h. The plates were then placed on ice, and the cells were washed twice with 1 ml of HBSS. Medium containing 2 µg/ml 125I-HA with or without 200 µg/ml unlabeled HA was added to each well, and the cells were incubated at 37 °C to allow internalization. At the noted times, the medium was removed, and the cells were washed three times with 1 ml of HBSS and then lysed in 0.3 N NaOH. Cell protein content, cell-associated total and degraded 125I-HA, and degraded 125I-HA in the medium were determined as described under "Experimental Procedures." The total amount of 125I-HA processed by the cells ({circ}) is the sum of the cell-associated radioactivity, and the radioactivity representing degraded HA in the medium. The degraded 125I-HA (•) is the sum of the degraded products secreted into the medium ({blacksquare}) and still intracellular.

 

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  		TABLE I
Cell surface binding and endocytosis of 125I-HA in multiple SK-HARE clones

After a 1-h serum-free incubation at 37 °C, the cells were allowed to bind 125I-HA on ice for 1 h or to internalize 125I-HA at 37 °C for 3 h. Medium was aspirated, and the cells were washed three times with 1 ml of HBSS. Cells were lysed in 0.3 N NaOH, and radioactivity and protein were determined. Because there was considerable variability among the number of cells per well, the data are expressed as cpm/µg of protein. The results of replicate values from 3–9 experiments (N) for each clone are shown as the mean ± S.E. The degree of HARE reutilization was estimated as the ratio of the values for 125I-HA endocytosis at 37 °C divided by the initial surface binding of 125I-HA at 4 °C.

 


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  		FIG. 5.
Endocytosis of 125I-HA mediated by the 175-kDa rHARE is blocked by hyperosmolarity. Cells from SK-HARE clones #27 ({circ}, •) and #36 ({square}, {blacksquare}) were cultured in 4-well plates and treated as in Fig. 4. Medium containing 2 µg/ml 125I-HA without ({square}, {circ}) or with (•, {blacksquare}) 0.4 M sucrose was added, and the plates were incubated at 37 °C for the noted times. The plates were processed, and cell protein, cell-associated radioactivity (A), and total 125I-HA degradation (B) were determined as described under "Experimental Procedures." The points are the average of duplicates. Specific 125I-HA binding at 4 °C was ~55% as assessed in the presence of 200 µg/ml unlabeled HA.

 

mAb-174 Inhibits 125I-HA Endocytosis by SK-HARE Cells— mAb-174, which is one of the eight mAbs previously raised against the 175-kDa rHARE (24, 26), completely blocks uptake in LECs at 37 °C and almost completely inhibits the clearance of HA by perfused isolated rat liver (41). The endocytic function of the recombinant rHARE in SK-HARE cells was effectively inhibited by mAb-174, although the kinetics of total cell HA accumulation remained linear (Fig. 6A). In previous studies with rat LECs, mAb-235 was the only other mAb that could block HA uptake, although the extent of inhibition leveled off at ~50% (26). However, the effect of mAb-235 on the recombinant rHARE was only marginally inhibitory and appeared variable, e.g. among different SK-HARE cell lines (Fig. 6, A and B). Only a slight inhibition by mAb-235 was evident at longer times (Fig. 6A). None of the other anti-HARE mAbs caused significant, reproducible inhibition (Fig. 6B).



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  		FIG. 6.
MAb-174 inhibits 125I-HA uptake by SK-HARE cells at 37 °C. SK-HARE cell lines were cultured and processed as described in Fig. 4. After the additions noted in panels A and B, the dishes were incubated at 37 °C for the indicated times, the media were aspirated, and the cells were washed three times with HBSS. The cells were solubilized, and radioactivity and protein content were measured as described under "Experimental Procedures." A (Clones #26 and #34), incubation medium contained 2 µg/ml 125I-HA and 5 µg/ml mAb-235 ({blacksquare}), 5 µg/ml mAb-174 ({square}), 100 µg/ml HA (•), or no addition ({circ}). The values shown are the average of four wells (two from each clone) ± S.D. The zero-time values (at 4 °C) with unlabeled HA were lower because of effective competition for 125I-HA binding. In contrast, the zero-time values with mAb-174 were not lower, because this antibody does not block HA binding at 4 °C (26). B (Clones #27 and #35), incubation medium contained 1 µg/ml 125I-HA and 10 µg/ml of the indicated IgG or 100 µg/ml HA. The dishes were incubated at 37 °C for 3 h. The values shown are the average of four wells from each clone (two each from two different experiments). The values in the presence of 10 µg/ml mouse IgG were 112 and 86% of the no-addition controls, respectively, for clones #27 and #35.

 

Specific 125I-HA uptake by SK-HARE cells was inhibited about 80% by 5 µg/ml mAb-174, whereas control mouse IgG had no effect (Fig. 7, top panel). In this experiment, complete inhibition of specific 125I-HA uptake occurred at 25 µg/ml (not shown). By comparison, specific endocytosis of 125I-HA by rat LECs was completely inhibited at 1.5 µg/ml (Fig. 7, top panel, inset). An interesting and unusual feature of the action of mAb-174 should be noted. As seen in Fig. 6A, this antibody blocks HA binding and endocytosis at 37 °C but does not block HA binding at 4 °C (compare the different y-intercepts, i.e. the 4 °C values representing zero time, for 125I-HA binding in the presence of excess unlabeled HA or in the presence of mAb-174). The 125I-HA-binding activity of recombinant 175-kDa rHARE in a ligand blot assay was also inhibited by mAb-174 but not by mouse IgG or other anti-HARE antibodies, including mAb-235 (Fig. 7, bottom panel). As observed earlier with the native rat 175-kDa HARE protein (26), mAb-174 only partially inhibited HA binding to the recombinant HARE in the ligand blot format (~30%). Although the cellular HA-binding activity of both native proteins can be completely blocked by mAb-174 (at least at higher temperatures), this is apparently not the case after the proteins have been subjected to SDS-PAGE and electrotransfer.



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  		FIG. 7.
Inhibition of 125I-HA uptake by anti-HARE monoclonal antibodies. SK-HARE cell lines were cultured and processed as described in Figs. 4 and 6. Top panel (Clone #26), medium was added containing 2 µg/ml 125I-HA and the indicated amounts of mAb-174 IgG ({circ}) or mouse IgG (•). The dishes were incubated at 37 °C for 3 h, the medium was aspirated, and the cells were washed three times with HBSS. The values shown are the average of duplicate wells. The inset shows the same type of experiment using rat LECs. Bottom panel, cells from two SK-HARE clones were scraped from the tissue culture plastic in PBS, and the cell pellets were treated with 0.1% digitonin in PBS for 10 min at 4 °C. The washed cell pellets were then solubilized in Laemmli sample buffer, subjected to nonreducing SDS-PAGE in 5% gels and electrotransferred to nitrocellulose. Strips were cut, treated overnight in TBST, washed, and incubated with 1 µg/ml 125I-HA at 4 °C for 2 h with no additions, with 100 µg/ml of unlabeled HA or with 1 µg/ml mouse nonimmune, mAb-174, or mAB-235 IgG. After autoradiography, densitometric analyses of the HARE protein bands were performed using an Alpha Innotech Fluorochem 8000. The values are the mean ± S.D. for two strips from each of the two clones exposed for 18 and 25 h (n = 8). Based on a Student's t test analysis, the HA-binding value for mAb-174 was significantly different (p < 0.05) than those with mAb-235 or control IgG.

 

mAb-30 has proven useful for affinity purification, Western blot, and immunopurification procedures of each of the rat and human HARE proteins. We, therefore, wanted to determine if mAb-30 would also be useful for ligand-independent measurements of HARE protein expression in live cells. To assess this, mAb-30 was iodinated and used in direct binding experiments to detect cell surface or intracellular HARE protein in SK-HARE cell lines at 4 °C (Fig. 8). The cell surface binding of 125I-mAb-30 was specific as assessed by competition with unlabeled mAb-30 (Table II). No similar competition was seen with mouse IgG or most of the other anti-HARE mAbs tested, including numbers 54, 141, 159, 174, or 235 (not shown). This result indicates that these latter mAbs recognize different, non-interfering epitopes within the 175-kDa HARE protein. As expected, HA did not inhibit 125I-mAb-30 binding (Fig. 8).



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  		FIG. 8.
HA does not inhibit the binding of 125I-mAb-30 to the recombinant 175-kDa HARE. Cells from SK-HARE clones #26 and #35 were cultured and processed as in Fig. 4 and then incubated for 1 h in medium containing 1 µg/ml 125I-mAb-30 and 0–100 µg/ml of either HA ({blacksquare}) or unlabeled mAb-30 IgG (•). The cells were solubilized, and radioactivity and protein content were measured as described under "Experimental Procedures." The values shown are the average of four wells (two for each of two clones).

 

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  		TABLE II
Specific binding of 125I-HA and 125I-mAb-30 to LECs and SK-HARE cells

Cells were grown in 24-well tissue culture plates, washed, and incubated for 60 min at 37 °C in medium without serum. The cells were then washed and allowed to bind 1 µg/ml 125I-mAb-30 or 125I-HA for 60 min on ice in the presence or absence of 0.055% (w/v) digitonin (as a permeabilizing agent) to assess total cellular or only cell surface binding, respectively. Specific binding was assessed by including 100 µg/ml of unlabeled mAb or HA. Digitonin was prepared as a 25% (w/v) stock solution in Me2SO, and all samples included the same concentration of Me2SO. The cpm values are the average of duplicates normalized to the initial protein content per well.

 

Studies of 125I-mAb-30 binding to intact or permeabilized SK-HARE cells, however, revealed a difference compared with rat LECs, in that intracellular HARE proteins were present but apparently not active. Cells were treated with the mild nonionic detergent digitonin under conditions that release cytoplasmic and early endosomal proteins but not lysosomal or mitochondrial proteins (42). When LECs are permeabilized in this manner, one detects roughly twice as much specific 125I-HA binding per cell, indicating that ~50% of the total cellular receptors are on the cell surface and ~50% are intracellular (14, 36). Such a distribution has been well documented for a variety of endocytic, recycling receptors that are present in multiple internal pathways as they move through their recycling itinerary (40, 43). When SK-HARE cells were permeabilized, the specific 125I-mAb-30 binding increased as expected if HARE was also present intracellularly (Table II). However, there was no parallel increase in specific 125I-HA binding, indicating that intracellular HARE in these cells is inactive and unable to bind ligand.

The Recombinant175-kDa rHARE Also Binds to Multiple CSs But Not Other GAGs—Studies by us and others (36, 44) have shown that isolated rat LECs internalize HA and CS by the same endocytic receptor. However, because LECs contain both the 175- and 300-kDa HARE species, it has not been possible to assess the ligand specificity of just one of these isoreceptors in the absence of the other. The SK-HARE cell lines provide the first opportunity to determine if the single small rHARE is able to recognize multiple GAGs. All of the CS types tested (CS-A, CS-C, CS-D, and CS-E) and DS (also formerly designated as CS-B) competed for the ability of SK-HARE cells to internalize 125I-HA at 37 °C (Fig. 9). CS-E was a somewhat better competitor, in fact, than HA itself. In multiple experiments, the curves for CS-A and HA were virtually identical, with apparent Ki values of ~1–2 µg/ml for the inhibition of 125I-HA endocytosis at 37 °C. In contrast, the other CS types displayed higher apparent Ki values and lesser extents of competition. No competition for 125I-HA endocytosis was observed with chondroitin, heparin, HS, or KS. The GAG specificity of the 175-kDa rHARE is, therefore, surprisingly broad yet still specific. Even though many CS types are recognized, a high density of negative charge (such as in HS or heparin) is not sufficient for binding by HARE. Other negatively charged carbohydrate polymers, such as DNA, RNA, or polygalacturonic acid, also do not compete for HA binding or endocytosis by rat LECs (36).



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  		FIG. 9.
Only some non-HA GAGs compete for 125I-HA endocytosis by the recombinant 175-kDa rHARE. Cells from SK-HARE clone #26 or #35 were incubated at 37 °C for 3 h in medium containing 1 µg/ml 125I-HA with or without 1.5 to 30 µg/ml of the indicated GAG. The values for competition with unlabeled GAGs or HA (expressed as a percentage of the no addition control) are the mean of replicates for each clone from three independent experiments (n = 18 for HA; n = 6 for the other GAGs). A, chondroitin (•), chondroitin sulfate C ({circ}), chondroitin sulfate E ({blacktriangledown}), heparin ({triangledown}), and HA ({blacksquare}). B, heparan sulfate ({diamondsuit}), keratan sulfate ({diamond}), chondroitin sulfate A ({triangleup}), chondroitin sulfate D ({square}), and dermatan sulfate ({blacktriangleup}).

 

A surprising result was obtained when we examined the temperature-dependence of GAG recognition by HARE. We noted above that mAb-174 does not block HA binding to the 175-kDa HARE at low temperature (Fig. 6A). In fact, none of the GAGs that effectively block 125I-HA endocytosis at 37 °C were able to block 125I-HA binding alone when measured at 4 °C (Fig. 10). Even CS-E, which inhibited 125I-HA endocytosis by ~75% (compared with 60% inhibition by unlabeled HA), was completely ineffective as a competitor at 4 °C. The differential temperature sensitivity in the ability of the 175kDa HARE to bind other GAGs compared with HA indicates that the HARE protein might have different binding sites for HA and the CSs.



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  		FIG. 10.
Chondroitin sulfates and DS compete for 125I-HA endocytosis by the 175-kDa rHARE at 37 °C but do not compete for 125I-HA binding at 4 °C. SK-HARE cells (clones #26 and #35), grown and processed as in Fig. 4, were incubated in medium containing 1 µg/ml 125I-HA with or without 20 µg/ml of the indicated GAG. The cells were allowed to bind ligand on ice for 60 min to determine the surface binding values (gray bars), or they were allowed to internalize the ligand at 37 °C for 3 h to determine the endocytosis values (black bars). Cells were lysed, and radioactivity and protein were determined as described under "Experimental Procedures." The values shown are the means ± S.E. of duplicate wells from the two clones (n = 4), expressed as a percentage of the control (no GAG competitor added).

 

The relative contributions of the 175- and 300-kDa HARE species to the total HA- or CS-binding capabilities of rat LECs is not known. As an initial attempt to assess this, we compared the ability of the various GAGs to compete for the endocytosis of 125I-HA by isolated rat LECs, which express both HARE isoreceptors, or by SK-HARE cells expressing only the 175-kDa HARE (Fig. 11). The results confirm that the GAG specificity of HARE is very similar in either cell type, but there were several differences. Heparin, HS, and KS were not recognized effectively by LECs, whereas all of the CS types and DS were very good competitors. However, the extents of inhibition by these latter GAGs were greater in LECs than in SK-HARE cells, indicating that the larger HARE isoform may account for more CS binding relative to HA than the smaller HARE isoform. The biggest difference observed was that chondroitin was an effective competitor of 125I-HA endocytosis in LECs but not in SK-HARE cells. Thus, chondroitin may be differentially recognized by the two HARE isoreceptors, i.e. it appears to bind better to the larger HARE isoform.



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  		FIG. 11.
The GAG specificities for endocytosis are different for SK-HARE cells expressing only the 175 kDa compared with LECs expressing both HARE isoforms. Cells from SK-HARE clones #26 and #35 and freshly isolated LECs were cultured and processed as described under "Experimental Procedures." The cells were then incubated at 37 °C for 3 h in medium containing 1 µg/ml 125I-HA with or without 30 µg/ml of the indicated GAG. Cells were lysed, and radioactivity and protein were determined as described under "Experimental Procedures." The values are the average of duplicate wells from the two clones (n = 4) for the SK-HARE cells, or the average of duplicate wells from two separate experiments for the LECs (n = 4), expressed as a percentage of control (no GAG added). The error bars represent the standard error.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HA, which is composed of the repeating disaccharide unit 2-deoxy, 2-acetamido-D-glucopyranosyl-{beta}(1,4)-D-glucuronopyranosyl-{beta}(1,3), is a ubiquitous component of essentially all vertebrate ECMs. HA is the only GAG that is not sulfated and not assembled in an obligatory manner on Ser/Thr residues of a PG core protein. PGs such as aggrecan and versican aggregate in the presence of HA, because they contain discrete HA-binding domains that allow them to bind to a single large HA polymer (45–48). The HA-PG interactions are often stabilized by Link proteins, which bind to both the HA and PG, to create even stronger matrix networks, e.g. in cartilage. HA-PG interactions are very important in matrix assembly and organization (49). In fact, the ability of HA to interact with ECM PGs and to form large stable networks in vivo (or aggregates in vitro) is necessary for normal tissue differentiation (50, 51).

The HA turnover process likely involves many more molecules than just HA, because HA and PGs form networks; in essence ECM particles or fragments. Over 50 cell surface or ECM hyaladherins (4, 52) have been identified, and more will likely be discovered as the human genome and proteome are elucidated. It is very likely that many different hyaladherins remain associated with HA-PG network fragments that are carried by lymph fluid from matrices to lymph nodes during the turnover process described above. Thus, the turnover of HA-PG network fragments may enable the simultaneous uptake by liver, spleen, and lymph node of some proteins for which there may not be specific clearance receptors. Such a "piggy-backing" process would provide an indirect endocytic pathway by which many hyaladherins could be internalized and degraded in lysosomes in a HARE-mediated process. If this indirect clearance function occurs, then the physiological function of HARE would be much broader than presently suspected.

Unlike other cell surface HA receptors such as CD44, LYVE-1, CD168, or ICAM-1 (53–56), HARE has a broader GAG specificity and is an endocytic, recycling receptor that mediates the rapid and efficient endocytosis of its HA/GAG ligands via the clathrin-coated pit pathway. The sensitivity of HARE-mediated HA uptake to hyperosmolarity is a hallmark characteristic of the coated pit endocytosis pathway. Other evidence that the recombinant HARE is a coated pit-targeted recycling receptor comes from confocal microscopy studies demonstrating that internalized HA but not HARE is delivered to lysosomes in SK-HARE cells and that HARE and clathrin are co-localized (28). It is likely that other HA receptors may play a role in some local tissue-specific HA turnover processes, but HARE is responsible for the systemic clearance of HA/GAGs derived from many tissues. Some HA receptors, such as CD44, can mediate HA internalization (57, 58), although these processes are often slower and of lower capacity than expected for coated pit-mediated uptake.

Most cell surface receptors can mediate slow rates of ligand uptake due to general membrane recycling. However, receptors with particular sequence motifs, e.g. {varphi}XXB (where {varphi} is an aromatic residue and B is a bulky hydrophobic residue), in their cytoplasmic domains can be targeted to coated pits and internalized very rapidly. In particular, the clearance receptors (e.g. the mannose, low density lipoprotein, asialoglycoprotein, and liver HA receptors) are endocytic, recycling receptors with such motifs. Although the functional coated pit-targeting motifs in HARE have not yet been identified, the protein contains several good candidate {varphi}XXB sequences similar to those found in other recycling clearance receptors (25, 28, 39). The 175-kDa rHARE protein functions in SK-Hep-1 cells as a recycling, coated pit receptor and mediates the continuous uptake and degradation of HA for at least 28 h. During that time cells internalized an amount of HA equal to their surface HA receptor number about every 40 min. Although this is a slower rate of receptor recycling than observed in LECs or established cell lines, which are ~10–15 min, the results clearly demonstrate the recycling and reutilization of HARE.

We also found similar slow receptor recycling times in recent studies of endocytosis mediated by the human asialoglycoprotein receptor in stable SK-Hep-1 cell lines (59–61). These SK-Hep-1 cells, in fact, have a very active transcytosis pathway (62), and about half of the asialoglycoproteins internalized are transferred "across" the cell rather than being delivered to lysosomes for degradation. Consistent with the presence of this amplified transcytosis pathway, the SK-Hep-1 cell line, which was initially considered to be parenchymal, is now known to be of endothelial origin (63). Therefore, the intracellular accumulation of HA in the SK-HARE cell lines is an underestimate, because about 50% of the internalized HA is probably delivered intact back to the medium via transcytosis. Thus, a corrected estimate of the HARE reutilization rate is ~20 min.

The recombinant 175-kDa rHARE, expressed from an artificial cDNA, is an endocytic receptor with essentially identical structural and functional characteristics compared with the native rat protein. Both the recombinant and native175-kDa rHARE are about the same mass, contain ~25 kDa of N-linked oligosaccharides, and retain HA-binding activity after treatment with endoglycosidase-F. The HA-binding activities of both native and recombinant proteins are inhibited by mAb-174 in a ligand blot assay and in live cells. The only difference observed was that the bandwidth after SDS-PAGE was broader for the native protein than the recombinant rHARE. Native 175-kDa rHARE preparations have two major N termini, indicating the protein is sensitive to proteases that produce multiple N termini in the functional protein. A similar pattern of multiple N termini was found with the recombinant protein. Thus, in both cases, the N-terminal ends have been frayed by proteolysis so that multiple related species with different N-terminal sequences are present. In LECs, the 260-kDa subunit (or its precursor) of the 300-kDa HARE is the likely initial gene product, from which both the 230-kDa subunit and 175-kDa protein are then derived by proteolysis. LECs express the proteases necessary to generate the three discrete subunits found in the 175-kDa HARE (which contains one subunit) and 300-kDa HARE (which contains two subunits of 230 and 260 kDa), all of which are derived from the presumed full-length 2551-amino acid product of the STAB2 gene (27). No one has yet identified the proteases involved or determined whether a full-length HARE/Stabilin 2 protein is synthesized and processed to generate the 175-kDa HARE species and the large subunits of the 300-kDa HARE species.

The reason(s) that there are two HARE isoreceptors in mammalian liver, spleen, and lymph node remain unclear. We previously suggested the possibility that two isoreceptors may be needed to mediate the efficient uptake and degradation of HA due to its extremely broad range of sizes (26, 28). Unlike other molecules in the body, HA is relatively unique in being present as species ranging over more than two orders of magnitude in molecular mass (e.g. from <105 to ~107 kDa). The two HARE isoreceptors could have different preferences for the size of the HA with which they interact. Another possible reason for two HARE isoreceptors could be related to differences in their GAG specificities. Although the GAG competition patterns were similar for cells containing both HARE species (LECs) or only the small isoform, there were differences (Fig. 11). In particular, chondroitin might be preferentially recognized by the larger HARE isoreceptor. Several CS types (e.g. CS-C and CS-D) were also more efficient competitors for HA uptake in LECs compared with SK-HARE cells. In contrast, competition by DS and CS-A were more comparable between the two cell types. Despite these indications of specificity differences, it is clearly too early to draw conclusions from these initial data. Stable cell lines expressing only the larger or only the smaller HARE will be needed in future studies to address differences in GAG specificity between the two isoreceptors.

The rat 175-kDa HARE interacted with six of the ten GAGs tested (Figs. 9 and 10), indicating that the GAG specificity of the receptor is surprisingly broad. The extracellular domain of the 175-kDa rHARE is relatively large (~1324 amino acids) and could contain multiple GAG-binding sites (28). Apparent competition of one GAG for HARE binding to another GAG does not necessarily mean they share a common binding site, because the binding of one large GAG could preclude, e.g. by steric hindrance or allosteric modulation, the binding of other GAGs to independent sites. Studies are in progress to map the GAG-binding sites and determine whether they are of unique, common, or overlapping specificity.

The 175-kDa rHARE recognizes GlcUA-GlcNAc disaccharide units in HA but not the GlcUA-GalNAc disaccharide units of chondroitin, which differ only as epimers at the C4 position of the amino sugars. Apparently the equatorial -OH of HA at this position is accepted in the binding site(s), whereas the axial -OH in chondroitin prevents a similar interaction. In contrast, HARE recognized all of the sulfated CS types despite the presence of the axial -OH in the chondroitin backbone (Fig. 9). The position and number of sulfates varies greatly among the set of CS types recognized. Chondroitins sulfated at C4, C6, or C4,6 of GalNAc (i.e. CS-A, CS-C, and CS-E) were effective competitors of HA binding; in fact, CS-E bound with apparently higher affinity than HA. CS-D, which is sulfated at C6 of GalNAc and C2 of GlcUA, bound as well as HA did. KS, which contains Gal rather than a uronic acid in its repeating disaccharide unit, was not bound by HARE. If the presence of a uronic acid in the GAG is important, however, it may not be critical whether this is GlcUA or IdUA, because DS was recognized; although weakly bound, the DS and CS-C competition curves were essentially identical (Fig. 9). The other sulfated, uronic acid-containing GAGs that were not recognized by the 175-kDa rHARE (heparin and HS) contain N-sulfated glucosamine residues that may not be accommodated in binding site(s) that are designed to recognize N-acetyl groups in other GAGs.

A novel finding in the present study is the temperature-dependence for HARE recognition of GAGs other than HA. Similar earlier results on the ability of mAb-174 to block HA binding led us to suggest that HARE undergoes conformational changes between 4 °C and 37 °C. At lower temperatures the HA-binding sites are functional, whereas mAb-174 binding either does not occur or it occurs but the bound mAb no longer precludes HA binding (e.g. a conformational change moves the bound mAb so it no longer blocks access of HA to its binding site). Based on their ability to compete with HA, the five other GAGs that bound to HARE at 37 °C did not bind at 4 °C; HA was the only GAG that bound to HARE at either temperature. If a similar conformational change within HARE can explain this differential temperature sensitivity of GAG binding, then the binding site(s) for HA might be different than those for the CS types and DS. Additional studies will be needed to map the various GAG binding sites in the extracellular domain of HARE and to determine the molecular basis for the novel temperature effect.

The present results show that the rat 175-kDa HARE is a bone fide endocytic receptor capable of functioning independently of the 300-kDa HARE to mediate the internalization of HA, as well as DS and a variety of CS types. Although it is possible that the 175-kDa HARE and 300-kDa HARE species could function together as a large complex, it is apparently not necessary for these two HAREs to be present in the same cell to create a specific functional HA/CS receptor targeted to the coated pit pathway.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM35978. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 405-271-1288; Fax: 405-271-3092; E-mail: paul-weigel{at}OUHSC.edu.

1 The abbreviations used are: HA, hyaluronic acid, hyaluronate, hyaluronan; CS, chondroitin sulfate; CS-A, chondroitin 4-sulfate; CS-C, chondroitin 6-sulfate; CS-D, chondroitin 2,6-sulfate; CS-E, chondroitin 4,6-sulfate; DS, dermatan sulfate; ECM, extracellular matrix; GAG, glycosaminoglycan; HARE, HA receptor for endocytosis; HBSS, Hanks' balanced salt solution; Hep, heparin; HS, heparan sulfate; KS, keratan sulfate; LECs, liver sinusoidal endothelial cells; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PG, proteoglycan; SK-HARE; stable SK-Hep-1 cell lines expressing HARE; TBS, Tris-buffered saline; TBST, Tris-buffered saline containing 0.05% Tween 20. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Edward Harris and Robert Raymond for helpful discussions and Dr. Bin Zhou for help with the initial isolation of cell lines.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Laurent, T. C., and Fraser, J. R. E. (1991) in Degradation of Bioactive Substances: Physiology and Pathophysiology (Henriksen, J. H., ed) pp. 249–265, CRC Press, Boca Raton, FL
  2. Abatangelo, G., and Weigel, P. H. (2000) (eds) New Frontiers in Medical Sciences: Redefining Hyaluronan, Elsevier Science B.V., Amsterdam
  3. Toole, B. P. (1997) J. Intern. Med. 242, 35–40[CrossRef][Medline] [Order article via Infotrieve]
  4. Knudson, C. B., and Knudson, W. (1993) FASEB J. 7, 1233–1241[Abstract]
  5. Laurent, T. C., and Fraser, J. R. E. (1992) FASEB J. 6, 2397–2404[Abstract]
  6. Evered, D., and Whelan, J. (eds) (1989) CIBA Found. Symp. 143, 1–288[Medline] [Order article via Infotrieve]
  7. Tammi, R., Saamanen, A. M., Maibach, H. I., and Tammi, M. (1991) J. Invest. Dermatol. 97, 126–130[CrossRef][Medline] [Order article via Infotrieve]
  8. Fraser, J. R., Appelgren, L. E., and Laurent, T. C. (1983) Cell Tissue Res. 233, 285–293[Medline] [Order article via Infotrieve]
  9. Fraser, J. R., Kimpton, W. G., Laurent, T. C., Cahill, R. N., and Vakakis, N. (1988) Biochem. J. 256, 153–158[Medline] [Order article via Infotrieve]
  10. Lebel, L., Smith, L., Risberg, B., Gerdin, B., and Laurent, T. C. (1988) J. Appl. Physiol. 64, 1327–1332[Abstract/Free Full Text]
  11. Tzaicos, C., Fraser, J. R., Tsotsis, E., and Kimpton, W. G. (1989) Biochem. J. 264, 823–828[Medline] [Order article via Infotrieve]
  12. Laurent, U. B., Dahl, L. B., and Reed, R. K. (1991) Exp. Physiol. 76, 695–703[Abstract]
  13. Eriksson, S., Fraser, J. R., Laurent, T. C., Pertoft, H., and Smedsrod, B. (1983) Exp. Cell Res. 144, 223–228[CrossRef][Medline] [Order article via Infotrieve]
  14. McGary, C. T., Raja, R. H., and Weigel, P. H. (1989) Biochem. J. 257, 875–884[Medline] [Order article via Infotrieve]
  15. LeBoeuf, R. D., Raja, R. H., Fuller, G. M., and Weigel, P. H. (1986) J. Biol. Chem. 261, 12586–12592[Abstract/Free Full Text]
  16. LeBoeuf, R. D., Gregg, R. R., Weigel, P. H., and Fuller, G. M. (1987) Biochemistry 26, 6052–6057[CrossRef][Medline] [Order article via Infotrieve]
  17. Thylen, A., Wallin, J., and Martensson, G. (1999) Cancer 86, 2000–2005[CrossRef][Medline] [Order article via Infotrieve]
  18. Lundin, A., Engstrom-Laurent, A., Hallgren, R., and Michaelsson, G. (1985) Br. J. Dermatol. 112, 663–671[Medline] [Order article via Infotrieve]
  19. Freitas, J. P., Filipe P., Emerit I., Meunier, P., Manso, C. F., and Guerra Rodrigo, F. (1996) Dermatology 192, 46–49[Medline] [Order article via Infotrieve]
  20. Manicourt, D. H., Poilvache, P., Nzeusseu, A., van Egeren, A., Devogelaer, J. P., Lenz, M. E., and Thonar, E. J. (1999) Arthritis Rheum. 42, 1861–1869[CrossRef][Medline] [Order article via Infotrieve]
  21. Yamada, M., Fukuda, Y., Nakano, I., Katano, Y., Takamatsu, J., and Hayakawa, T. (1998) Acta Haematol. 99, 212–216[CrossRef][Medline] [Order article via Infotrieve]
  22. Lai, K. N., Szetp, C. C., Lam, C. W. K., Lai, K. B., Wong, T. Y. H., and Leung, J. C. K. (1998) J. Lab Clin. Med. 131, 354–359[CrossRef][Medline] [Order article via Infotrieve]
  23. Bramley, P. N., Rathbone, B. J., Forbes, M. A., Cooper, E. H., and Losowsky, M. S. (1991) J. Hepatol. 13, 8–13[Medline] [Order article via Infotrieve]
  24. Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831–33834[Abstract/Free Full Text]
  25. Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339–349[Abstract/Free Full Text]
  26. Zhou, B., Weigel, J. A., Fauss, L., and Weigel, P. H. (2000) J. Biol. Chem. 275, 37733–37741[Abstract/Free Full Text]
  27. Politz, O., Gratchev, A., McCourt, P. A., Schledzewski, K., Guillot, P., Johansson, S., Svineng, G., Franke, P., Kannicht, C., Kzhyshkowska, J., Longati, P., Velten, F. W., Johansson, S., and Goerdt, S. (2002) Biochem. J. 362, 155–164[CrossRef][Medline] [Order article via Infotrieve]
  28. Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853–2868[Abstract/Free Full Text]
  29. Raja, R. H., LeBoeuf, R., Stone, G., and Weigel, P. H. (1984) Anal. Biochem. 139, 168–177[CrossRef][Medline]