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The Human Hyaluronan Receptor for Endocytosis (HARE/Stabilin-2) Is a Systemic Clearance Receptor for Heparin

2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês

10.1074/jbc.m710360200

ISSN

1083-351X

Autores

Edward N. Harris, Janet A. Weigel, Paul H. Weigel,

Tópico(s)

Platelet Disorders and Treatments

Resumo

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The Kd for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and 23.4 ± 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized 125I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of ∼12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations. The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The Kd for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and 23.4 ± 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized 125I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of ∼12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations. Heparin (Hep) 2The abbreviations used are: Hep, heparin; Ab, antibody; AP, alkaline phosphatase; aa, amino acids; BSA, bovine serum albumin; EV, empty vector; GAG, glycosaminoglycan; HA, hyaluronic acid, hyaluronate, hyaluronan; HARE, hyaluronan receptor for endocytosis; HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; LDL, low density lipoprotein; PBS, phosphate-buffered saline; s190-HARE, soluble 190-kDa HARE ectodomain; s315-HARE, soluble ectodomain of the 315-kDa HARE; SA, streptavidin; TBS, Tris-buffered saline; 190-HARE, the 190-kDa HA receptor for endocytosis; 315-HARE, the 315-kDa HA receptor for endocytosis; b-Hep, biotin-heparin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay. 2The abbreviations used are: Hep, heparin; Ab, antibody; AP, alkaline phosphatase; aa, amino acids; BSA, bovine serum albumin; EV, empty vector; GAG, glycosaminoglycan; HA, hyaluronic acid, hyaluronate, hyaluronan; HARE, hyaluronan receptor for endocytosis; HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; LDL, low density lipoprotein; PBS, phosphate-buffered saline; s190-HARE, soluble 190-kDa HARE ectodomain; s315-HARE, soluble ectodomain of the 315-kDa HARE; SA, streptavidin; TBS, Tris-buffered saline; 190-HARE, the 190-kDa HA receptor for endocytosis; 315-HARE, the 315-kDa HA receptor for endocytosis; b-Hep, biotin-heparin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay. is the most anionic proteoglycan, due to the extensive sulfation of its glycosaminoglycan (GAG) chains, and contains many different sulfated disaccharide isomers of N-acetylgalactosamine and glucuronic acid or iduronic acid. Hep binds to many different soluble, matrix, and cell surface proteins and receptors, and has many functions, including its roles as an anti-coagulant and as a co-receptor for some growth factors (1Norrby K. APMIS. 2006; 114: 79-102Crossref PubMed Scopus (116) Google Scholar, 2Mahtouk K. Hose D. Reme T. De Vos J. Jourdan M. Moreaux J. Fiol G. Raab M. Jourdan E. Grau V. Moos M. Goldschmidt H. Baudard M. Rossi J.F. Cremer F.W. Klein B. Oncogene. 2005; 24: 3512-3524Crossref PubMed Scopus (82) Google Scholar). Hep is a highly prescribed drug in surgical patients and those at risk for thromobosis. The genes and metabolic pathway for Hep biosynthesis in mast cells are understood reasonably well, and many of the biological and clinical activities of Hep have been well studied for several decades (3Sugahara K. Kitagawa H. IUBMB Life. 2002; 54: 163-175Crossref PubMed Scopus (214) Google Scholar, 4Rabenstein D.L. Nat. Prod. Rep. 2002; 19: 312-331Crossref PubMed Scopus (691) Google Scholar, 5Lindahl U. Biochem. Soc. Trans. 1990; 18: 803-805Crossref PubMed Scopus (16) Google Scholar, 6Kusche M. Lindahl U. J. Biol. Chem. 1990; 265: 15403-15409Abstract Full Text PDF PubMed Google Scholar). In contrast, we know less about the catabolism of Hep and how total body homeostasis of this multifunctional proteoglycan is maintained. In particular, the mechanisms controlling systemic turnover of Hep, whether as endogenous proteoglycan or free chain drugs, are not known. Although receptors for Hep have been characterized on a variety of cell types (e.g. macrophages, vascular smooth muscle cells), none of these mediate substantial clearance of Hep (7Savage J.M. Gilotti A.C. Granzow C.A. Molina F. Lowe-Krentz L.J. J. Cell. Physiol. 2001; 187: 283-293Crossref PubMed Scopus (22) Google Scholar, 8Delputte P.L. Vanderheijden N. Nauwynck H.J. Pensaert M.B. J. Virol. 2002; 76: 4312-4320Crossref PubMed Scopus (148) Google Scholar, 9Mentz S. de Lacalle S. Baerga-Ortiz A. Knauer M.F. Knauer D.J. Komives E.A. J. Neurochem. 1999; 72: 980-987Crossref PubMed Scopus (17) Google Scholar). A few reports implicated a role for Kupffer cells in Hep clearance, but were not followed up (10Nakamura T. Yuasa H. Watanabe J. Biol. Pharm. Bull. 2000; 23: 743-747Crossref PubMed Scopus (6) Google Scholar, 11Praaning-van Dalen D.P. Brouwer A. Knook D.L. Gasterenterology. 1981; 81: 1036-1044Abstract Full Text PDF PubMed Scopus (153) Google Scholar). The possible contribution of liver sinusoidal endothelial cells to Hep uptake in these primary cell preparations was not examined. Because Hep is a widely prescribed drug, it is even more important to understand the factors that control its clearance and, therefore, pharmokinetics. Animal studies of Hep clearance, except by renal function, have been difficult to perform, because Hep binds to so many soluble, cell surface or matrix proteins and become widely distributed after injection. Unfractioned Hep (3000–30000 Da), low molecular mass Hep (300–8000 Da), and the pentasaccharide, Fondaparinux, are the three classes of Hep drugs used to treat venous thrombosis, acute myocardial infarction, trauma, obesity, and coronary and peripheral vascular procedures; all situations wherein patients need immediate platelet anti-coagulation therapy (12Jacobs L.G. J. Am. Geriatr. Soc. 2003; 51: 1472-1478Crossref PubMed Scopus (22) Google Scholar). Following an intravenous bolus, unfractioned Hep has a half-life of ∼1 h and is cleared from the circulation by the liver and kidney (13Dinwoodey D.L. Ansell J.E. Clin. Geriatr. Med. 2006; 22 (vii): 1-15Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Low molecular mass Hep and the pentasaccharide, subcutaneously injected, have half-lives of ∼3–6 and ∼17 h, respectively (14Hirsh J. Raschke R. Chest. 2004; 126: 188S-203SAbstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar). Larger more structurally diverse Hep is more readily cleared than the lower molecular weight fragments. Although clinical handbooks declare that Hep is metabolized and cleared by the reticuloendothelial system, the mechanisms for Hep clearance are not known. The primary scavenger receptor for systemic turnover of HA and most types of chondroitin sulfate, but not HS, is HARE/Stab-2, which mediates most of the total body HA turnover per day (15Laurent T.C. Fraser J.R.E. Henriksen J.H. Degradation of Bioactive Substances: Physiology and Pathophysiology. CRC Press, Boca Raton, FL1991: 249-264Google Scholar, 16Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar, 17Falkowski M. Schledzewski K. Hansen B. Goerdt S. Histochem. Cell Biol. 2003; 120: 361-369Crossref PubMed Scopus (106) Google Scholar). HARE is found primarily in the sinusoidal endothelial cells of the lymph nodes, liver, and spleen (18Zhou B. Weigel J.A. Fauss L.A. Weigel P.H. J. Biol. Chem. 2000; 275: 37733-37741Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 19Zhou B. Weigel J.A. Saxena A. Weigel P.H. Mol. Biol. Cell. 2002; 13: 2853-2868Crossref PubMed Scopus (65) Google Scholar, 20Zhou B. McGary C.T. Weigel J.A. Saxena A. Weigel P.H. Glycobiology. 2003; 13: 339-349Crossref PubMed Scopus (47) Google Scholar, 21Politz O. Gratchev A. McCourt P.A.G. Schledzewski K. Guillot P. Johansson S. Svineng G. Franke P. Kannicht C. Kzhyshkowska J. Longati P. Velten F.W. Goerdt S. Biochem. J. 2002; 362: 155-164Crossref PubMed Scopus (238) Google Scholar), and also in specialized tissues such as corneal and lens epithelium, mesenchymal cells of heart valves, prismatic epithelial cells covering the renal papillae (17Falkowski M. Schledzewski K. Hansen B. Goerdt S. Histochem. Cell Biol. 2003; 120: 361-369Crossref PubMed Scopus (106) Google Scholar), and in oviduct (22Ulbrich S.E. Schoenfelder M. Thoene S. Einspanier R. Mol. Cell. Endocrinol. 2004; 214: 9-18Crossref PubMed Scopus (32) Google Scholar). Partially degraded HA perfuses from extracellular matrices in tissues and enters the lymphatic and hepatic vascular circulation systems where it is endocytosed by HARE and catabolized. The partially degraded HA perfusing from tissues first encounters HARE in lymph nodes, which mediates ∼85% of the daily HA turnover. The remaining 15% of HA drains from the lymphatics into the circulatory system and is removed by HARE in liver sinusoidal endothelium. The large full-length human HARE, encoded by the STAB2 gene (accession number NM_017564 on chromosome 12q23.3 (NCBI data base), is a ∼315-kDa 2551-aa glycoprotein (21Politz O. Gratchev A. McCourt P.A.G. Schledzewski K. Guillot P. Johansson S. Svineng G. Franke P. Kannicht C. Kzhyshkowska J. Longati P. Velten F.W. Goerdt S. Biochem. J. 2002; 362: 155-164Crossref PubMed Scopus (238) Google Scholar, 23Harris E.N. Kyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The smaller 190-kDa HARE is not a splice variant, but rather it is an isoform derived from the full-length 315-kDa HARE by proteolysis (23Harris E.N. Kyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This 190-kDa HARE is, in fact, a functional endocytic recycling receptor that mediates GAG uptake in the absence of the larger 315-kDa HARE (24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A primary function of these two HARE isoforms is to bind and internalize HA and most of the chondroitin sulfate types (24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25Laurent T.C. Fraser J.R.E. Pertoft H. Smedsrod B. Biochem. J. 1986; 234: 653-658Crossref PubMed Scopus (130) Google Scholar, 26Tzaicos C. Fraser J.R.E. Tsotsis E. Kimpton W.G. Biochem. J. 1989; 264: 823-828Crossref PubMed Scopus (18) Google Scholar, 27Smedsrod B. Malmgren M. Ericsson J. Laurent T.C. Cell Tissue Res. 1988; 253: 39-45Crossref PubMed Scopus (32) Google Scholar, 28Weigel J.A. Weigel P.H. J. Biol. Chem. 2003; 278: 42802-42811Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar); advanced glycation end products are also ligands (29Tamura Y. Adachi H. Osuga J. Ohashi K. Yahagi N. Sekiya M. Okazaki H. Tomita S. Iizuka Y. Shimano H. Nagai R. Kimura S. Tsujimoto M. Ishibashi S. J. Biol. Chem. 2003; 278: 12613-12617Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Hansen B. Longati P. Elvevold K. Nedredal G.-I. Schledzewski K. Olsen R. Falkowski M. Kzhyshkowska J. Carlsson F. Johansson S. Smedsrod B. Goerdt S. Johansson S. McCourt P. Exp. Cell Res. 2005; 303: 160-173Crossref PubMed Scopus (101) Google Scholar). The internalized HARE-ligand complexes traffic through the early endocytic pathway. After dissociation, the receptor recycles back to the cell surface, whereas the GAG ligand is delivered to lysosomes for degradation (19Zhou B. Weigel J.A. Saxena A. Weigel P.H. Mol. Biol. Cell. 2002; 13: 2853-2868Crossref PubMed Scopus (65) Google Scholar, 24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Hansen B. Longati P. Elvevold K. Nedredal G.-I. Schledzewski K. Olsen R. Falkowski M. Kzhyshkowska J. Carlsson F. Johansson S. Smedsrod B. Goerdt S. Johansson S. McCourt P. Exp. Cell Res. 2005; 303: 160-173Crossref PubMed Scopus (101) Google Scholar, 31McGary C.T. Raja R.H. Weigel P.H. Biochem. J. 1989; 257: 875-884Crossref PubMed Scopus (125) Google Scholar). In this report, we identify HARE as the receptor that specifically binds, internalizes, and mediates degradation of Hep. We demonstrate specific and high affinity HARE-Hep binding under a variety of conditions and active HARE-mediated endocytosis of Hep in stable cell lines. We conclude that Hep clearance by the body is mediated, probably predominantly, by HARE in the sinusoidal endothelium of the liver and lymph nodes. Materials, Solutions, and Buffers—Hep (free chains) was from Celsus (Cincinnati, OH) and Sigma. Flp-In 293 cells, fetal bovine serum, high glucose Dulbecco's modified Eagle's medium (DMEM), hygromycin B, Zeocin, glutamine, plasmid expression vectors, super-competent TOP10 Escherichia coli, LysoTracker DND-99, SA-Alexa 488, and Lipofectamine 2000 were from Invitrogen. 125Iodine (100 mCi/ml; specific activity of >0.6 TBq/mg) in NaOH and Sepharose 6 Fast Flow (nickel-nitrilotriacetic acid) resin were from GE Healthcare. PolySorP strips were from Nalgene Nunc, Int. (Rochester, NY). EpranEx plates were from Plasso, LLC (UK). Streptavidin (SA) was from Pierce. 125I-SA was prepared as described previously (32Raja R.H. LeBoeuf R.D. Stone G.W. Weigel P.H. Anal. Biochem. 1984; 139: 168-177Crossref PubMed Scopus (66) Google Scholar, 33McGary C.T. Weigel J.A. Weigel P.H. Methods Enzymol. 2003; 363: 354-366Crossref PubMed Scopus (13) Google Scholar). The protocol of Yu and Toole (34Yu Q. Toole B.P. BioTechniques. 1995; 19: 122-129PubMed Google Scholar) was used to make biotin-Hep (b-Hep), and the number of biotins/chain was quantified using a QuantTag™ Biotin Kit (Vector Labs, Burlingame, CA). Size exclusion multiangle laser light scattering was performed to measure Hep molar mass and concentration, and to verify the addition of 1–2 biotins per Hep chain, without degradation (23Harris E.N. Kyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Western blot analysis was by colorimetric or enhanced chemiluminescence detection of blotted protein. Other materials, reagents, and kits were obtained as described recently (23Harris E.N. Kyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). TBS contains 20 mm Tris-HCl, pH 7.0, 150 mm NaCl; TBST is TBS with 0.1% Tween 20. TBST/BSA is TBST with 1.0% (w/v) BSA. Coating Buffer for ELISA contains 15 mm Na2CO3, 36 mm NaHCO3, pH 9.5. PBS contains 137 mm NaCl, 8 mm Na2HPO4, 1.5 mm KH2PO4, 2.7 mm KCl, pH 7.2. Hanks' balanced salts solution contains 5 mm KCl, 0.4 mm KH2PO4, 0.8 mm MgSO4, 137 mm NaCl, 0.3 mm Na2HPO4, 5.5 mm glucose, 1.26 mm CaCl2, 0.5 mm MgCl2, and 28 μm phenol red; at the time of use, 3.5 g/100 ml of NaHCO3 was added and the pH was adjusted to 7.2 with HCl. Endocytosis medium contains DMEM with 0.05% BSA. Constructs and Cell Lines Expressing Membrane-anchored or Soluble 190-HARE, 190-HARE(ΔLink), or 315-HARE—Preparation of cDNA constructs and vectors for the creation of stably transfected 293 Flp-In cell lines expressing, the full-length 315-HARE, 190-HARE, or the secreted ectodomains of the 315-HARE or 190-HARE (lacking transmembrane and cytoplasmic domains) were described by Harris et al. (23Harris E.N. Kyosseva S.V. Weigel J.A. Weigel P.H. J. Biol. Chem. 2007; 282: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Creation of 190-HARE(ΔLink) cDNA constructs and stable cell lines was described recently (35Kyosseva S.V. Harris E.N. Weigel P.H. J. Biol. Chem. 2008; 283 (April 2)10.1074/jbc.M709921200Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). All recombinant HARE proteins contain C-terminal V5 and His6 epitope tags. Final clones were selected based on criteria of normal growth and morphology, good HARE expression, and that a single cDNA had inserted at the recombinase-mediated integration site provided in the Flp-In cell lines. SDS-PAGE using 5% gels and Western analysis using anti-V5 Ab were performed as described (24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Preparation of 125I-SA·b-Hep Complexes—For cell-based assays, 2:1 molar ratios of b-Hep and 125I-SA were mixed, at 40–50-fold higher concentrations than in experiments, in 0.5 ml of Endocytosis medium for 30 min on a rotary mixer at room temperature just prior to an experiment. For controls with 125I-SA alone, the same proportional amounts of free biotin and 125I-SA were used. After mixing, the 125I-SA·b-Hep or 125I-SA·biotin complexes were diluted into Endocytosis medium. Final concentrations of b-Hep and 125I-SA complexes in assays were 100 and 96 nm (2.5 μg/ml), respectively. Binding and Endocytosis of 125I-SA·b-Hep—Stably transfected cells, clone 9 (24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), expressing 190-kDa HARE, were plated in 12-well dishes and grown in DMEM with 8% fetal calf serum and 100 μg/ml hygromycin B for at least 2 days prior to experiments. Cells were washed with Hanks' balanced salt solution and incubated at 37 °C for 60 min with Endocytosis medium (no serum) to allow HARE-mediated internalization of any bound serum GAGs. Endocytosis assays with these washed cells were performed at 37 °C in Endocytosis medium containing pre-formed complexes of 125I-SA·b-Hep with or without unlabeled Hep as competitor. Binding of 125I-SA·b-Hep to cells was measured at 4 °C. Specific binding or endocytosis was assessed in the presence of excess unlabeled Hep and, in most cases, in cells incubated in parallel with only 125I-SA-biotin to determine background counts/min values. These values were subtracted from all data points to determine specific 125I-SA·b-Hep endocytosis. To determine the amount of 125I-SA·b-Hep binding by the total cell HARE population, cells on ice were permeabilized with 0.055% (w/v) digitonin, using a 25% stock solution dissolved in anhydrous dimethyl sulfoxide (36Weigel P.H. Ray D.A. Oka J.A. Anal. Biochem. 1983; 133: 437-449Crossref PubMed Scopus (80) Google Scholar). At the indicated times, cells were washed three times with ice-cold Hanks' balanced salt solution, lysed in 0.3 n NaOH, and radioactivity and protein content (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) were determined and expressed as counts/min/μg of protein. Ligand Blot Assay for b-Hep Binding—Cells expressing 315- or 190-HARE were scraped from T-75 flasks in the presence of serum and lysed in 0.5% Nonidet P-40. After rotating for 1 h at 24 °C, cell debris was removed by centrifugation, and the cell lysate was immunoprecipitated with anti-V5 Ab, which recognizes this fusion epitope on both recombinant proteins. Following 5% SDS-PAGE, the proteins were transferred to nitrocellulose membranes and blocked with TBST, 1% BSA at 4 °C for 2 h. The method for detecting b-Hep binding to HARE is a modification of a ligand blot procedure for HA binding (38Yannariello-Brown J. Zhou B. Weigel P.H. Glycobiology. 1997; 7: 15-21Crossref PubMed Scopus (29) Google Scholar). Nitrocellulose membranes were incubated with TBST/BSA for 2 h at 4 °C, washed, and incubated with 200 nm b-Hep in TBST/BSA, with or without 2000 nm unlabeled Hep as competitor to assess nonspecific binding. After incubation for 1.5 h at 4 °C, the membrane was washed with excess TBST at least 5 times over 30 min, and incubated with 2.0 μg/ml 125I-SA for 30 min. After washing with TBST at least 5 times over 30 min, the membrane was dried at 22 °C and bound 125I-SA was detected by autoradiography using Kodak MS film exposed at -80 °C for 1–18 h. Western Blot Analysis—Following the ligand blot procedure, the membranes were rehydrated in TBST/BSA, incubated with 20 ng/ml anti-V5 Ab (Bethyl Labs, Montgomery, TX), washed, incubated with the appropriate secondary Ab conjugated with horseradish peroxidase, and detected by ECL. The ECL signal was captured using Classic Blue Film BX (MIDSCI, St. Louis, MO). Densitometry on both the Western and ligand blot films was performed with an Alpha Innotech FluorChem 8000. Fluorescence Microscopy—Cells were grown on 18-mm glass coverslips in complete medium for at least 2 days prior to the experiment. B-Hep (100 nm) or unlabeled Hep was combined with 1 μg/ml SA-Alexa 488 in 0.5 ml of DMEM and rotated for 30 min prior to the experiment. Live cells were incubated with SA-Alexa 488·(biotin)-Hep conjugates in Endocytosis medium for 6 h at 37 °C. The medium was replaced with fresh Endocytosis medium containing 50 nm LysoTracker and the cells were incubated for 1 h. Live cells on glass coverslips were washed by dipping slides into PBS 3 times and immediately mounted onto glass slides prior to visualization using a Nikon Diaphot 300 fluorescence microscope. Images were captured with a DXM1200 side-mounted camera operated by Act-1 software version 2.6.3. sHARE Purification—s190-HARE and s315-HARE were purified from pooled growth media by Ni2+ chelate affinity chromatography, followed by SDS-PAGE and electroelution as described by Harris et al. (24Harris E.N. Weigel J.A. Weigel P.H. J. Biol. Chem. 2004; 279: 36201-36209Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Final purified HARE preparations were concentrated to 0.1–1.0 mg/ml using Amicon concentrators (Millipore), washed twice with PBS, and stored at -20 °C until use in subsequent assays. ELISA-like—A set amount of HARE protein in Coating Buffer (200 μl) was placed in each well of a PolySorP strip, sealed to prevent evaporation, and incubated overnight at room temperature. All subsequent steps were carried out at 37 °C. The surfaces of the wells were then blocked by incubation with TBST and 2% BSA for 1.5 h. After washing, increasing concentrations of b-Hep were added and the plates were incubated for 2 h, washed 6 times with TBST, incubated for 1 h with SA-AP, washed 6 times in TBST, and finally incubated with p-nitrophenol phosphate. The A405 values of the strips were determined every 15 min for 1 h using a SpectraMax 340 (Molecular Devices) plate reader. Biotinylation and Iodination of BSA—BSA (2 mg) was biotinylated using Sulfo-NHS-LC-biotin (Pierce) according to the manufacturer's instructions and then dialyzed extensively against PBS to eliminate excess free biotin. A 10:1 molar ratio of biotin-to-BSA molecules was used and the final b-BSA product contained an average of 5 biotins/BSA (a 50% efficient reaction), as assessed with the QuantTag Biotin Kit (Vector Labs). B-BSA (0.4 mg) was then iodinated as described previously using 0.5 mCi of 125I for 15 min at 22 °C (39Weigel P.H. J. Biol. Chem. 1980; 255: 6111-6120Abstract Full Text PDF PubMed Google Scholar). 125I-b-BSA was separated from free iodine on a PD-10 column, and 0.5-ml fractions were collected and assessed for radioactivity using a γ-counter. Fractions in the first void volume peak were pooled, and protein content and radioactivity were determined. Degradation Assay—190-HARE and EV cells were grown to 80–90% confluence in 12-well plates prior to experiments. In initial trials, ternary complexes of 125I-b-BSA·SA·b-Hep were formed using numerous ratios of the three components, and then tested for specific endocytosis by 190-HARE cells. These results indicated an optimal ratio of 100 nm 125I-b-BSA, 200 nm SA, and 100 nm b-Hep. The ternary complexes were allowed to form while mixing by rotation at room temperature for 2 h just prior to an experiment. Similar mixtures containing 125I-b-BSA and b-Hep without the SA linker or the ternary complexes with a 100-fold excess of unlabeled Hep were used as controls for nonspecific endocytosis. One hour prior to an experiment, cells were incubated in DMEM containing 2% BSA to block nonspecific binding sites and to allow endocytosis of any serum-derived GAGs bound to cell surface HARE. Cells were then incubated in DMEM/BSA with 0.5 ml of the ternary complexes or the control mixtures for 4 h, washed 3 times with 2 ml of cold Hanks' balanced salt solution, and incubated for 15 h in 0.5 ml of fresh DMEM plus 2% serum. The collected chase medium was cleared of cell debris by centrifugation and loaded on a PD-10 (G-25 Sephadex) column and eluted with PBS. Forty-eight 0.5-ml fractions were collected and radioactivity in each fraction was determined using a γ-counter. The possibility that HARE is the major systemic clearance receptor for Hep in the vascular and lymphatic circulatory systems has great physiological significance and would be an important finding. We, therefore, sought to test this using a variety of protein- and cell-based assays. The Purified s315- and s190-HARE Ectodomains Bind B-Hep—To determine whether Hep can bind to both HARE isoforms, we used direct ELISA-like binding assays with the purified recombinant 190-HARE and 315-HARE ectodomains. As the b-Hep concentration increased, the amount of b-Hep bound to s190-HARE increased sharply and saturated above ∼100 nm (Fig. 1A, solid line). These data were readily fit by nonlinear regression analysis to a hyperbolic binding curve, indicating a single class of Hep binding sites with a Kd of 17.4 ± 4.9 nm (p < 0.0001). Binding of b-Hep was specific, because the presence of excess unlabeled Hep eliminated by >95% the binding of SA-AP (not shown). A similar experiment with the 315-HARE ectodomain also showed a hyperbolic binding curve for b-Hep (Fig. 1A, dashed line) with a Kd of 23.2 ± 5.3 nm (p < 0.0001), indicating virtually identical binding kinetics for the s190- and s315-HARE proteins. Thus, the additional N-terminal portion of the s315-HARE, consisting of 1135 aa, is not involved in Hep binding. The results confirm that Hep specifically binds with high affinity to the purified s190-HARE and s315-HARE ectodomains in vitro. A “Reverse” ELISA-like Assay Validates the Specific HARE-Hep Interaction—To ensure that the biotin tag was not producing false positive signals in our in vitro and endocytosis assays, we assessed specific Hep binding using a reverse ELISA-like assay. Unlabeled Hep was coated onto EpranEx plates according to the manufacturer's instructions and then allowed to bind with purified s190-HARE protein. Primary anti-V5 Ab, directed against the HARE C-terminal epitope tag, followed by a secondary Ab-AP conjugate, was used to detect HARE binding with Hep (Fig. 1B). Because Hep is highly anionic, and sticks nonspecifically to many proteins, we included controls to ensure that the primary (1′) or secondary (2′) Abs were not randomly adhering to Hep or to the plastic. All controls showed about the same low values (∼10% of maximum), indicating that the signal output from this assay is not due to nonspecific Hep binding. This assay also validates results from the ligand blot and endocytosis assays that use pre-formed 125I-SA·b-Hep complexes. The Membrane-anchored 315- and 190-HARE Proteins Bind B-Hep—The ability of the intact 190-HARE and 315-HARE isoforms to bind Hep was assessed in membrane extracts after nonreducing SDS-PAGE and transfer to nitrocellulose. The HARE proteins re-nature after electrotransfer and incubation with Tween 20, and can be detected by a ligand blot assay with 125I-HA (38Yannariello-Brown J. Zhou B. Weigel P.H. Glycobiology. 1997; 7: 15-21Crossref PubMed Scopus (29) Google Scholar). Nitrocellulose strips containing 190-HARE or 315-HARE were incubated with b-Hep (Fig. 2), with or without an excess of unlabeled Hep, to determine nonspecific binding, and b-Hep

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