FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1968656979> ?p ?o ?g. }

• W1968656979 endingPage "36209" @default.
• W1968656979 startingPage "36201" @default.
• W1968656979 abstract "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 ∼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 125I-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 ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 °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 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 190hHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-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. 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 ∼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 125I-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 ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 °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 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 190hHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-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. Multiple extracellular binding proteins and cell surface receptors for HA 1The abbreviations used are: HA, hyaluronic acid, hyaluronate, or 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; hHARE, human HARE; 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; rHARE, rat HARE; SK-HARE, stable SK-Hep-1 cell lines expressing recombinant rat HARE; Tris, trishydroxymethylamino methane; TBS, Tris-buffered saline; TBST, Tris-buffered saline containing 0.05% Tween 20; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; Chon, chondroitin.1The abbreviations used are: HA, hyaluronic acid, hyaluronate, or 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; hHARE, human HARE; 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; rHARE, rat HARE; SK-HARE, stable SK-Hep-1 cell lines expressing recombinant rat HARE; Tris, trishydroxymethylamino methane; TBS, Tris-buffered saline; TBST, Tris-buffered saline containing 0.05% Tween 20; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; Chon, chondroitin. facilitate many biological activities that are important during complex cellular processes, including development, wound healing, and invasion and metastasis of some cancers (1Evered D. Whelan J. CIBA Found. Symp. 1989; 143: 1-288Google Scholar, 2Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar, 3Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (596) Google Scholar, 4Toole B.P. J. Intern. Med. 1997; 242: 35-40Crossref PubMed Scopus (277) Google Scholar, 5Abatangelo G. Weigel P.H. New Frontiers in Medical Sciences: Redefining Hyaluronan. Elsevier Science B. V., Amsterdam.2000Google Scholar). Due to the rapid turnover of HA in many tissues (6Laurent T.C. Fraser J.R.E. Henriksen J.H. Degradation of Bioactive Substances: Physiology and Pathophysiology. CRC Press, Boca Raton, FL1991: 249-265Google Scholar), very high HA levels could occur locally, in lymph fluid, or in plasma. Therefore, efficient mechanisms for HA uptake and degradation are present in mammals to regulate the amount of HA present in a variety of physiological conditions (reviewed in Refs. 2Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar and 7Weigel P.H. Yik J.H.N. Biochim. Biophys. Acta. 2002; 1572: 341-363Crossref PubMed Scopus (186) Google Scholar). The endocytic clearance receptor for HA, which is designated HARE (HA receptor for endocytosis), is abundant in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (8Zhou B. Weigel J.A. Fauss L. Weigel P.H. J. Biol. Chem. 2000; 275: 37733-37741Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 9Zhou B. Weigel J.A. Saxena A. Weigel P.H. Mol. Biol. Cell. 2002; 13: 2853-2868Crossref PubMed Scopus (65) Google Scholar, 10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar). The biological activity of this clearance receptor was discovered in rodents in 1981 (11Fraser J.R. Laurent T.C. Pertoft H. Baxter E. Biochem. J. 1981; 200: 415-424Crossref PubMed Scopus (293) Google Scholar, 12Fraser J.R. Appelgren L.E. Laurent T.C. Cell Tissue Res. 1983; 233: 285-293Crossref PubMed Scopus (103) Google Scholar, 13Eriksson S. Fraser J.R. Laurent T.C. Pertoft H. Smedsrod B. Exp. Cell Res. 1983; 144: 223-228Crossref PubMed Scopus (260) Google Scholar), and the protein was finally purified by Zhou et al. in 1999 (14Zhou B. Oka J.A. Weigel P.H. J. Biol. Chem. 1999; 274: 33831-33834Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Earlier studies using isolated rat LECs indicated that HARE mediates the binding and endocytosis of HA via the coated-pit pathway and that this receptor also recognizes and internalizes chondroitin sulfates (15Smedsrod B. Malmgren M. Ericsson J. Laurent T.C. Cell Tissue Res. 1988; 253: 39-45Crossref PubMed Scopus (32) Google Scholar, 16Raja R.H. McGary C.T. Weigel P.H. J. Biol. Chem. 1988; 263: 16661-16668Abstract Full Text PDF PubMed Google Scholar, 17McGary C.T. Raja R.H. Weigel P.H. Biochem. J. 1989; 257: 875-884Crossref PubMed Scopus (125) Google Scholar). Unlike the rat HARE proteins, which have been studied extensively in isolated rat LECs, there have been no cellular studies of the human HARE proteins. Human LECs are not available commercially, and to date, no cell lines have been identified that express either the 190- or 315-kDa hHARE isoforms. Consequently, very little is known about the GAG specificity or function of human HARE. In both rat (14Zhou B. Oka J.A. Weigel P.H. J. Biol. Chem. 1999; 274: 33831-33834Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and human (10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar), two isoforms of HARE are present with molecular masses of 175 or 300 kDa and 190 or 315 kDa, respectively. The human ∼315-kDa HARE is a complex composed of two disulfide-bonded subunits of about 250 and 220 kDa, in a ratio of ∼2:1 (10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar). In contrast, the small hHARE isoform contains a single subunit. All subunits in both hHARE isoforms, although they are different sizes, appear to be derived by proteolysis from the same precursor protein (9Zhou B. Weigel J.A. Saxena A. Weigel P.H. Mol. Biol. Cell. 2002; 13: 2853-2868Crossref PubMed Scopus (65) Google Scholar), which is Stabilin 2 (18Politz 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. Goerdt S. Biochem. J. 2002; 362: 155-164Crossref PubMed Scopus (238) Google Scholar). Full-length Stabilin 2 is a very large putative protein (2551 amino acids), whose synthesis and processing have not yet been fully elucidated. For example, it is not known if the largest subunit in the 315-kDa hHARE corresponds to full-length Stabilin 2 or to a truncated form of the protein. The small rat and human HARE proteins are not encoded directly by mRNA. We recently demonstrated (9Zhou B. Weigel J.A. Saxena A. Weigel P.H. Mol. Biol. Cell. 2002; 13: 2853-2868Crossref PubMed Scopus (65) Google Scholar) that the native small rHARE isoform contains the C-terminal 1431 residues of the predicted full-length protein and that this smaller HARE, when expressed in SK-Hep-1 cells in the absence of the large isoform, colocalizes with clathrin as expected for a coated-pit-coupled endocytic HA receptor (19Mellman I. Annu. Rev. Cell Biol. 1996; 12: 575-625Crossref Scopus (1325) Google Scholar). The two rat HARE species, therefore, appear to be functionally independent isoreceptors for HA. In the present study, we have created an artificial spleen cDNA for the 190-kDa hHARE to assess its functionality and GAG specificity in stable cell lines. In addition to demonstrating that this smaller hHARE isoform can function as an endocytic, recycling receptor in the absence of the larger hHARE, we found that the human protein has slightly different specificity for various GAGs than the highly related rat 175-kDa HARE isoform. Unexpectedly, several anti-HARE mAbs showed very different reactivity with the rat and human recombinant HARE proteins and were able to block partially the internalization of HA by cells expressing the 190-kDa hHARE. Materials and Buffers—Na125I was from Amersham Biosciences, and 125I-HA was prepared as described previously (20Raja R.H. LeBoeuf R. Stone G. Weigel P.H. Anal. Biochem. 1984; 139: 168-177Crossref PubMed Scopus (66) Google Scholar) using HA oligosaccharides (MW = 133,000 based on gel permeation chromatography coupled to multiangle laser light scattering analysis), modified only at their reducing ends to contain covalently attached hexylamine. Male Sprague-Dawley rats (200 g) were from Charles River Labs. BSA Fraction V and fetal bovine serum were from Intergen Co. Collagenase was from Roche Applied Science. The preparation and characterization of mouse mAbs raised against the rat 175-kDa HARE were described previously (8Zhou B. Weigel J.A. Fauss L. Weigel P.H. J. Biol. Chem. 2000; 275: 37733-37741Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Tris, SDS, ammonium persulfate, N,N′-methylenebisacrylamide, and SDS-PAGE standards were from Bio-Rad. Digitonin, from ACROS Organics, was prepared as a 25% (w/v) stock solution in Me2SO and then diluted into medium as required. 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. The weight-average molecular masses (determined by gel permeation chromatography coupled to multiangle laser light scattering analysis) for all but two of the GAGs were 14.2–38.8 kDa. The two exceptions were Chon (7.4 kDa) and CS-E (187.7 kDa). Nitrocellulose membranes were from Schleicher & Schuell. HBSS and PBS were prepared according to the Invitrogen catalog formulations. Z-buffer contains 60 mm dibasic sodium phosphate and 40 mm monobasic sodium phosphate, pH 7.0, 10 mm KCl, 1.0 mm MgSO4, and 50 mm 2-mercaptoethanol. TBS contains 20 mm Tris-HCl, pH 7.0, and 150 mm NaCl. TBST is TBS containing 0.05% (v/v) Tween 20. Construction of hHARE Expression Vector—The 190-kDa hHARE coding region was amplified from pooled lymph node cDNAs (Marathon system; Clontech) using Advantage 2 polymerase (Clontech), and gene-specific forward (5′-GGATCCTCCTTACCAAACCTGCTCATGCGG-3′) and reverse (5′-GGATCCCAGTGTCCTCAAGGGGTCATTG-3′) primers. The PCR product representing the artificial cDNA was then purified by agarose gel electrophoresis using a 0.8% gel containing 0.002% crystal violet for visualization. The band was excised, gene-cleaned, ligated into pCR-XL-TOPO, and transformed into TOP10 Escherichia coli cells (Invitrogen). Clones were selected and screened for the full-length insert by restriction digestion and PCR analysis, and a correct clone was used to prepare and isolate the expression vector. The hHARE cDNA was then cut out of the pCR-XL-TOPO plasmid and inserted into the BamHI site of pSecTag/FRT/V5-His-TOPO (Invitrogen). This vector provides a κ light chain secretion signal fused at the N terminus of the hHARE reading frame and two epitope tags (V5 and His-6) fused at the C terminus of the gene product. After transformation into TOP10 E. coli cells, several clones were selected and size and orientation of the cDNA insert were verified. The complete sequences of promoter, fusion, and cDNA regions of the final clones were determined and confirmed to be correct. Selection and Characterization of Stable Transfectants Expressing the 190-kDa hHARE—Flp-In 293 cells (3 × 106; from Invitrogen) were plated in 100-mm tissue culture dishes the day prior to transfection. Cells in 10 ml of antibiotic-free medium were transfected by addition of 750 μl of serum-free DMEM containing 9 μg of pOG44 (which encodes the Flp-In recombinase), 1 μg of pSecTag-190hHARE, and 20 μl of LipofectAMINE 2000 (Invitrogen). Two days post-transfection the medium was replaced with DMEM containing 100 μg/ml hygromycin B (Invitrogen). Due to the build-up of dead cells, the medium was changed every 2–3 days. Visible colonies were observed at days 10–14 and then isolated using cloning rings or collected directly with a plastic pipette tip. Isolated colonies were grown to confluence in 24-well dishes in 1.0 ml of DMEM with 100 μg/ml hygromycin B. After a monolayer of cells had developed from each clone, the cells were scraped and suspended in 1.0 ml of fresh medium. One portion (100 μl) of cells was re-seeded and allowed to grow for subsequent procedures. Another 100 μl of cells was resuspended in DMEM plus 100 μg/ml Zeocin and allowed to grow for 1 week to test for Zeocin sensitivity. A third portion (400 μl) of cells was pelleted and resuspended in 4× Laemmli sample buffer (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) to test for HARE protein expression by SDS-PAGE and Western analysis. The Western blot was probed with anti-V5 antibody (Bethyl Laboratories; 1:5000 dilution) in TBST. The remaining 400 μl of cell suspension was pelleted and assayed for β-galactosidase activity. Both the Zeocin and β-galactosidase tests indicate whether pSecTag-190-kDa hHARE was inserted correctly and uniquely into the Flp-In recombination site by the Flp-In recombinase encoded by pOG44. The recombinase is lost during subsequent cell divisions, because the encoding plasmid lacks an antibiotic selection gene. For the β-galactosidase assay, a clonal cell pellet was resuspended in 250 μl of 0.5% Triton X-100 in PBS, and 10 μl of cell lysate per well (in a 96-well plate) was combined with 20 μl of distilled deionized H2O, 70 μl of Z-buffer, and 20 μl of 4 mg/ml o-nitrophenyl-β-d-galactoside in Z-buffer. After 15 min at 37 °C, the enzyme reaction was terminated by the addition of 0.1 ml 1 m sodium bicarbonate, and absorbance values were determined at 420 nm. Human embryonic kidney Flp-In 293 cells and 293 cells were included in each assay set as positive and negative controls, respectively. Stable clones with a single plasmid integrated into the correct, unique chromosomal site were those that demonstrated and maintained no detectable β-galactosidase expression, poor or no growth in DMEM containing 100 μg/ml Zeocin, normal cell morphology, and good HARE protein expression. Suitable clones were maintained in DMEM containing 100 μg/ml hygromycin B and 8% fetal bovine serum. Western and Ligand Blot Assays—Western blotting was performed as described by Burnette (22Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5847) Google Scholar) with minor modifications. Cell lysates were mixed with equal volumes of 2× SDS sample buffer (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), 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 (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 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 (23Yannariello-Brown J. Zhou B. Ritchie D. Oka J.A. Weigel P.H. Biochem. Biophys. Res. Commun. 1996; 218: 314-319Crossref PubMed Scopus (20) Google Scholar). The nitrocellulose membrane was washed with TBST five times for 5 min each and dried at room temperature. Bound 125I-HA was detected by autoradiography using Kodak BioMax MS or MR film exposed at –85 °C for 6–48 h. Nonspecific binding in this assay is typically <5%. For Western analysis, 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 Ig-alkaline phosphatase conjugate (1:1500 dilution) for 1 h at room temperature. The nitrocellulose was washed with TBST five times for 5 min each 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 Assays—Stably transfected 190-kDa hHARE cell lines were grown to confluence in DMEM containing 8% fetal calf serum and 100 μg/ml hygromycin B in tissue culture multiwell dishes (usually 24-well plates). The cells were washed with HBSS and incubated at 37 °C in fresh medium without serum for 30–60 min, the plates were then placed on ice, and the cells washed once or twice with HBSS prior to the experiment. 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. To assess HA binding by the total cell receptor population, 0.055% (w/v) digitonin was added to the medium to permeabilize the cells (24Weigel P.H. Ray D.A. Oka J.A. Anal. Biochem. 1983; 133: 437-449Crossref PubMed Scopus (80) Google Scholar, 25Oka J.A. Weigel P.H. J. Biol. Chem. 1983; 258: 10253-10262Abstract Full Text PDF PubMed Google Scholar). At the noted times, the medium was removed by aspiration, the cells were washed three times with HBSS and lysed in 0.3 n NaOH, and radioactivity and protein content were determined. Values were normalized for cell protein content per well and are presented as cpm/μg of protein. In some cases, HA data are expressed as cpm or femtomoles per million cells. For Flp-In 293 cells, the mean protein content was determined to be 398 ± 85 μg of protein/106 cells (n = 6). 125I-HA Degradation Assay—Degradation of 125I-HA was measured by a cetylpyridinium chloride precipitation assay as described by McGary et al. (26McGary C.T. Yannariello-Brown J. Kim D.W. Stinson T.C. Weigel P.H. Hepatology. 1993; 18: 1465-1476Crossref PubMed Scopus (18) Google Scholar). 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 2.0 mg/ml HA. After mixing at room temperature, 300 μl of 6% (w/v) cetylpyridinium chloride in distilled water was added, and the tubes were mixed by vortexing. After 10 min, the samples were centrifuged at 22 °C for 5 min at 9000 rpm in an Eppendorf model 5417 microcentrifuge, using a swinging bucket rotor. 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 then put in a gamma counter tube, and radioactivity was determined. Degradation was measured as the time-dependent increase of nonprecipitable radioactivity. At least 80% of the total radioactivity was precipitable at the beginning of each experiment. General—Protein content was determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using BSA as a standard. SDS-PAGE was performed according to the method of Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). 125I radioactivity was measured using a Packard Auto-Gamma Counting system. Digital images were captured using an Alpha Innotech Fluorochem 8000. Images were taken into 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. Expression of Recombinant Human 190-kDa HARE—We recently purified two hHARE proteins, of 190 and 315 kDa, from spleen extracts and then molecularly cloned a partial cDNA from pooled human lymph node and spleen that encoded part or all of the subunits in these two isoforms (10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar). The 190-kDa hHARE protein is not expressed from a unique mRNA, but rather is encoded by a 4383-bp region (1461 amino acids) at the 3′ end of the full-length Stab 2 coding region. To express the 190-kDa protein, we created an artificial cDNA for a recombinant 190-kDa hHARE in the pSecTag/FRT/V5/His-TOPO expression vector. For proper membrane orientation and trafficking to the cell surface, the pSecTag vector provides an immunoglobulin κ-chain secretion signal sequence fused at the N terminus of the protein. Transiently transfected Flp-In 293 cells expressed sufficiently high levels of the recombinant 190-kDa hHARE to mediate the specific binding and internalization of 125I-HA (Fig. 1). Compared with vector alone, cells transfected with hHARE cDNA internalized ∼4 times the amount of HA, and this uptake was completely blocked by unlabeled HA. Specific HA uptake, therefore, was ∼80% of the total. The potential advantage of using Flp-In 293 cells as the parental cell line for generation of stable cell lines expressing hHARE is that all clones should be virtually identical if the plasmid inserts at only the single unique chromosome site containing the engineered integration site. Correct integration at this site interrupts a β-galactosidase gene and a Zeocin resistance gene in the engineered site. Clones containing a single plasmid insertion at the correct engineered site are, therefore, hygromycin B-resistant, negative for β-galactosidase activity, and Zeocin-sensitive. If plasmid insertion occurs at other chromosome sites, rather than the correct engineered site, then clones will express β-galactosidase and be Zeocin-resistant. Out of 41 stably transfected clones that we selected and characterized, three (#9, #14, and #40) had no detectable β-galactosidase activity, were Zeocin-sensitive, and were judged to contain a plasmid insertion at the unique engineered site. A protein of the correct size for the 190-kDa hHARE was expressed in the three selected stable Flp-In 293 cell lines, and this protein bound 125I-HA with >98% specificity in a ligand blot assay following SDS-PAGE and electrotransfer (Fig. 2A). The 190-kDa hHARE protein expressed in Flp-In 293 cells had the characteristics previously found for native hHARE purified from spleen (10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar), and expression of the 190-kDa hHARE did not alter the morphology of Flp-In 293 cells. The recombinant nonreduced protein was recognized in Western blots by the three anti-rHARE mAbs that cross-reacted with native hHARE (mAbs 30, 154, and 159) but not mAbs 28, 174, 235, and 467 (Fig. 2B, NR). Similarly, the reduced 190-kDa hHARE protein reacted with only mAbs 159 and 174 (Fig. 2B, R). Based on its HA-binding activity in these in vivo and in vitro assays, the recombinant hHARE protein appeared to be folded properly. Consistent with this interpretation, three other characteristics of the recombinant hHARE were identical to those of the native protein (10Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar). Reduction of disulfide bonds resulted in slower migration of the 190-kDa hHARE in SDS-PAGE compared with the nonreduced protein (Fig. 2C, lanes 1 and 3, WB). Reduction of disulfide bonds also caused loss of HA-binding activity (Fig. 2C, lanes 1 and 3, AR). After treatment with endoglycosidase-F to release N-linked oligosaccharides, the recombinant protein migrated at a position corresponding to a loss of ∼25 kDa (Fig. 2C, lanes 3 and 4, WB). The de-N-glycosylated hHARE protein was still able to bind HA in this ligand blot format (Fig. 2C, lane 4, AR). In addition, anti-V5 antibody recognition of the C-terminal epitope provided by the vector was suitable for immunoprecipitation (not shown). HA Binding and Internalization by Cells Expressing the 190-kDa hHARE—The specific binding of 125I-HA at 4 °C by stable cell lines was typical for a membrane-bound receptor; binding kinetics was hyperbolic and saturated after about 90 min (Fig. 3). Essentially no specific binding of 125I-HA occurred in the control cells transfected with empty vector, consistent with the absence of any significant HA receptor activity in 293 cells (Table I). Also, as found for other endocytic, recycling receptors (e.g. the asialoglycoprotein and mannose receptors), about 30–50% of the total cellular hHARE population was on the cell surface, and the remainder was intracellular. The native rHARE in isolated LECs is an active endocytic receptor that recycles so that HA can be continually internalized and delivered to lysosomes for degradation over a period of many hours to days (7Weigel P.H. Yik J.H.N. Biochim. Biophys. Acta. 2002; 1572: 341-363Crossref PubMed Scopus (186) Google Scholar, 17McGary C.T. Raja R.H. Weigel P.H. Biochem. J. 1989; 257: 875-884Crossref PubMed Scopus (125) Google Scholar, 26McGary C.T. Yannariello-Brown J. Kim D.W. Stinson T.C. Weigel P.H. Hepatology. 1993; 18: 1465-1476Crossref PubMed Scopus (18) Google Scholar). To assess the ability of the recombinant 190-kDa hHARE to recycle, cells were allowed to internalize 125I-HA for 4 h, and the amount of specific HA uptake was calculated as the number of cell surface receptor equivalents. This estimates the" @default.
• W1968656979 created "2016-06-24" @default.
• W1968656979 creator A5054631928 @default.
• W1968656979 creator A5065463612 @default.
• W1968656979 creator A5070546753 @default.
• W1968656979 date "2004-08-01" @default.
• W1968656979 modified "2023-10-16" @default.
• W1968656979 title "Endocytic Function, Glycosaminoglycan Specificity, and Antibody Sensitivity of the Recombinant Human 190-kDa Hyaluronan Receptor for Endocytosis (HARE)" @default.
• W1968656979 cites W134859281 @default.
• W1968656979 cites W1483126826 @default.
• W1968656979 cites W1502377225 @default.
• W1968656979 cites W1583037972 @default.
• W1968656979 cites W1955333425 @default.
• W1968656979 cites W1968850370 @default.
• W1968656979 cites W1970368657 @default.
• W1968656979 cites W1974991241 @default.
• W1968656979 cites W1981873159 @default.
• W1968656979 cites W1983649348 @default.
• W1968656979 cites W1985045436 @default.
• W1968656979 cites W2006030099 @default.
• W1968656979 cites W2010479615 @default.
• W1968656979 cites W2010628683 @default.
• W1968656979 cites W2013798488 @default.
• W1968656979 cites W2023326206 @default.
• W1968656979 cites W2026127417 @default.
• W1968656979 cites W2026444939 @default.
• W1968656979 cites W2051514868 @default.
• W1968656979 cites W2061484282 @default.
• W1968656979 cites W2061980960 @default.
• W1968656979 cites W2074436878 @default.
• W1968656979 cites W2074462213 @default.
• W1968656979 cites W2083776114 @default.
• W1968656979 cites W2092791644 @default.
• W1968656979 cites W2095432481 @default.
• W1968656979 cites W2095741501 @default.
• W1968656979 cites W2100837269 @default.
• W1968656979 cites W2107258708 @default.
• W1968656979 cites W2111817213 @default.
• W1968656979 cites W2134344512 @default.
• W1968656979 cites W2141223494 @default.
• W1968656979 cites W2151580767 @default.
• W1968656979 cites W2153557573 @default.
• W1968656979 cites W2170425634 @default.
• W1968656979 cites W2411943407 @default.
• W1968656979 cites W2883164299 @default.
• W1968656979 cites W300861030 @default.
• W1968656979 cites W4243231451 @default.
• W1968656979 cites W4245667286 @default.
• W1968656979 cites W4293247451 @default.
• W1968656979 doi "https://doi.org/10.1074/jbc.m405322200" @default.
• W1968656979 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15208308" @default.
• W1968656979 hasPublicationYear "2004" @default.
• W1968656979 type Work @default.
• W1968656979 sameAs 1968656979 @default.
• W1968656979 citedByCount "74" @default.
• W1968656979 countsByYear W19686569792012 @default.
• W1968656979 countsByYear W19686569792013 @default.
• W1968656979 countsByYear W19686569792014 @default.
• W1968656979 countsByYear W19686569792015 @default.
• W1968656979 countsByYear W19686569792016 @default.
• W1968656979 countsByYear W19686569792018 @default.
• W1968656979 countsByYear W19686569792019 @default.
• W1968656979 countsByYear W19686569792020 @default.
• W1968656979 countsByYear W19686569792021 @default.
• W1968656979 countsByYear W19686569792022 @default.
• W1968656979 countsByYear W19686569792023 @default.
• W1968656979 crossrefType "journal-article" @default.
• W1968656979 hasAuthorship W1968656979A5054631928 @default.
• W1968656979 hasAuthorship W1968656979A5065463612 @default.
• W1968656979 hasAuthorship W1968656979A5070546753 @default.
• W1968656979 hasBestOaLocation W19686569791 @default.
• W1968656979 hasConcept C104317684 @default.
• W1968656979 hasConcept C108625454 @default.
• W1968656979 hasConcept C117943116 @default.
• W1968656979 hasConcept C14036430 @default.
• W1968656979 hasConcept C153074725 @default.
• W1968656979 hasConcept C153911025 @default.
• W1968656979 hasConcept C159654299 @default.
• W1968656979 hasConcept C170493617 @default.
• W1968656979 hasConcept C185592680 @default.
• W1968656979 hasConcept C203014093 @default.
• W1968656979 hasConcept C28005876 @default.
• W1968656979 hasConcept C40767141 @default.
• W1968656979 hasConcept C55493867 @default.
• W1968656979 hasConcept C79747257 @default.
• W1968656979 hasConcept C86803240 @default.
• W1968656979 hasConcept C95444343 @default.
• W1968656979 hasConceptScore W1968656979C104317684 @default.
• W1968656979 hasConceptScore W1968656979C108625454 @default.
• W1968656979 hasConceptScore W1968656979C117943116 @default.
• W1968656979 hasConceptScore W1968656979C14036430 @default.
• W1968656979 hasConceptScore W1968656979C153074725 @default.
• W1968656979 hasConceptScore W1968656979C153911025 @default.
• W1968656979 hasConceptScore W1968656979C159654299 @default.
• W1968656979 hasConceptScore W1968656979C170493617 @default.
• W1968656979 hasConceptScore W1968656979C185592680 @default.
• W1968656979 hasConceptScore W1968656979C203014093 @default.
• W1968656979 hasConceptScore W1968656979C28005876 @default.

• next