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• W2024652333 abstract "Histidine-rich glycoprotein (HRG) is an α2-glycoprotein found in mammalian plasma at high concentrations (∼150 μg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn2+-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn2+-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding. Histidine-rich glycoprotein (HRG) is an α2-glycoprotein found in mammalian plasma at high concentrations (∼150 μg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn2+-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn2+-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding. Histidine-rich glycoprotein (HRG) 1The abbreviations used are: HRG, histidine-rich glycoprotein; HRR, histidine-rich region; GAG, glycosaminoglycan; ELISA, enzyme-linked immunosorbent assay; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BSA, bovine serum albumin; CHO, Chinese hamster ovary.1The abbreviations used are: HRG, histidine-rich glycoprotein; HRR, histidine-rich region; GAG, glycosaminoglycan; ELISA, enzyme-linked immunosorbent assay; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BSA, bovine serum albumin; CHO, Chinese hamster ovary. is an ∼75-kDa single polypeptide chain α2-plasma glycoprotein synthesized in the liver and found in the plasma of most vertebrates at a relatively high concentration of 100–200 μg/ml (∼2 μm). The most distinctive feature of HRG arises from its high content of histidine and proline residues, which each account for ∼13% of the total amino acids (1Koide T. Foster D. Yoshitake S. Davie E.W. Biochemistry. 1986; 25: 2220-2225Crossref PubMed Scopus (111) Google Scholar). HRG is predicted to be a multidomain protein consisting of two cystatin-like domains at the N terminus (termed N1 and N2), a central histidine-rich region (HRR) flanked by two proline-rich sequences, and a C-terminal domain. HRG is known to bind a variety of ligands, including heme and Zn2+ (2Guthans S.L. Morgan W.T. Arch Biochem. Biophys. 1982; 218: 320-328Crossref PubMed Scopus (77) Google Scholar, 3Morgan W.T. Biochemistry. 1981; 20: 1054-1061Crossref PubMed Scopus (108) Google Scholar, 4Morgan W.T. Biochemistry. 1985; 24: 1496-1501Crossref PubMed Scopus (97) Google Scholar), plasminogen (5Lijnen H.R. Hoylaerts M. Collen D. J. Biol. Chem. 1980; 255: 10214-10222Abstract Full Text PDF PubMed Google Scholar, 6Ichinose A. Mimuro J. Koide T. Aoki N. Thromb. Res. 1984; 33: 401-407Abstract Full Text PDF PubMed Scopus (27) Google Scholar), fibrinogen (7Leung L.L. J. Clin. Investig. 1986; 77: 1305-1311Crossref PubMed Scopus (118) Google Scholar), thrombospondin and IgG (8Gorgani N.N. Parish C.R. Altin J.G. J. Biol. Chem. 1999; 274: 29633-29640Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9Gorgani N.N. Parish C.R. Easterbrook Smith S.B. Altin J.G. Biochemistry. 1997; 36: 6653-6662Crossref PubMed Scopus (71) Google Scholar), C1q (9Gorgani N.N. Parish C.R. Easterbrook Smith S.B. Altin J.G. Biochemistry. 1997; 36: 6653-6662Crossref PubMed Scopus (71) Google Scholar), and heparin (10Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Abstract Full Text PDF PubMed Google Scholar, 11Lijnen H.R. Hoylaerts M. Collen D. J. Biol. Chem. 1983; 258: 3803-3808Abstract Full Text PDF PubMed Google Scholar, 12Burch M.K. Blackburn M.N. Morgan W.T. Biochemistry. 1987; 26: 7477-7482Crossref PubMed Scopus (38) Google Scholar), and can interact with various cell-surface receptors, including Fcγ receptors (13Gorgani N.N. Smith B.A. Kono D.H. Theofilopoulos A.N. J. Immunol. 2002; 169: 4745-4751Crossref PubMed Scopus (46) Google Scholar, 14Gorgani N.N. Altin J.G. Parish C.R. Int. Immunol. 1999; 11: 1275-1282Crossref PubMed Scopus (29) Google Scholar) and an undefined T-cell receptor (15Saigo K. Shatsky M. Levitt L.J. Leung L.K. J. Biol. Chem. 1989; 264: 8249-8253Abstract Full Text PDF PubMed Google Scholar). Thus, HRG may potentially regulate numerous biological processes such as hemostasis, fibrinolysis, thrombosis, angiogenesis, leukocyte migration, and cancer metastasis.Despite considerable interest in HRG, many of the fundamental characteristics regarding HRG cell-surface binding remain undefined. For example, despite the identification of numerous soluble ligands for HRG, the cell-surface ligands are not well characterized. Indirect evidence suggests that negatively charged glycosaminoglycans (GAGs) may mediate HRG cell-surface binding, as heparin is a potent inhibitor of HRG binding to cells (16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17Brown K.J. Parish C.R. Biochemistry. 1994; 33: 13918-13927Crossref PubMed Scopus (71) Google Scholar). Zn2+ is also known to interact with HRG, most probably with the proposed metal chelation sites located within the HRR (2Guthans S.L. Morgan W.T. Arch Biochem. Biophys. 1982; 218: 320-328Crossref PubMed Scopus (77) Google Scholar, 3Morgan W.T. Biochemistry. 1981; 20: 1054-1061Crossref PubMed Scopus (108) Google Scholar, 4Morgan W.T. Biochemistry. 1985; 24: 1496-1501Crossref PubMed Scopus (97) Google Scholar), with this interaction enhancing the binding of HRG to cells (18Olsen H.M. Parish C.R. Altin J.G. Immunology. 1996; 88: 198-206Crossref PubMed Scopus (39) Google Scholar). Similarly, the location of the cell surface-binding domain within HRG is unclear. The ability of Zn2+ to enhance cell-surface binding and the assumption that heparin binds to the HRR and inhibits binding have led to the hypothesis that the HRR interacts with cells surfaces (10Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Abstract Full Text PDF PubMed Google Scholar, 12Burch M.K. Blackburn M.N. Morgan W.T. Biochemistry. 1987; 26: 7477-7482Crossref PubMed Scopus (38) Google Scholar, 16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Crossref PubMed Scopus (76) Google Scholar). In contrast, it has also been suggested that a heparin-binding sequence may be located in the N1N2 domain, which raises the possibility that this domain of HRG may interact with cells (17Brown K.J. Parish C.R. Biochemistry. 1994; 33: 13918-13927Crossref PubMed Scopus (71) Google Scholar, 20Koide T. Odani S. Ono T. J. Biochem. (Tokyo). 1985; 98: 1191-1200Crossref PubMed Scopus (24) Google Scholar, 21Koide T. Odani S. Ono T. FEBS Lett. 1982; 141: 222-224Crossref PubMed Scopus (39) Google Scholar).In this study, we have characterized the cell surface-binding properties of HRG using recombinant full-length HRG and the recombinant N1N2 domain together with various approaches to remove GAGs from cell surfaces. We provide clear evidence that heparan sulfate is the dominant cell-surface ligand for HRG, with the interaction being mediated though the N1N2 domain of HRG. Indeed, enzyme-linked immunosorbent assay (ELISA) studies confirmed that the N1N2 domain specifically binds to immobilized heparin with comparable affinity to full-length HRG. Furthermore, cell-surface binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by the presence of physiological concentrations of Zn2+. Based on these data, we propose a model whereby HRG binds to cell-surface heparan sulfate via its N1N2 domain with low affinity, which is enhanced following Zn2+ binding to the HRR. Thus, HRG may play an important physiological and/or pathological role by binding to cell surfaces in local environments that contain high levels of Zn2+ such as sites of inflammation or during tumor metastasis and angiogenesis.EXPERIMENTAL PROCEDURESCell Lines—B16F1, COS-7, MT4, HT1080, and Jurkat cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum. CHO-K1 and pgsA-745 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 21 μg/ml l-proline and 10% fetal calf serum. Mammalian cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The Spodoptera frugiperda-derived insect cell line Sf9 was cultured in Sf-900 II serum-free medium (Invitrogen) at 27 °C.Purification of HRG—Native human HRG was purified from fresh human plasma as described previously (22Rylatt D.B. Sia D.Y. Mundy J.P. Parish C.R. Eur. J. Biochem. 1981; 119: 641-646Crossref PubMed Scopus (52) Google Scholar). Briefly, a phosphocellulose column was equilibrated with loading buffer (0.5 m NaCl, 10 mm sodium phosphate, and 1 mm EDTA (pH 6.8)) for 24 h. Fresh human plasma was provided by Red Cross House of Canberra Hospital (Canberra, Australia) and mixed with NaCl and EDTA to final concentrations similar to those of the loading buffer and with the protease inhibitors aprotinin (2 μg/ml), phenylmethylsulfonyl fluoride (100 μg/ml), and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (100 μg/ml). The plasma was passed through the equilibrated column; unbound protein was removed by extensive washing with loading buffer; and bound HRG was eluted with 2.0 m NaCl, 10 mm sodium phosphate, and 1 mm EDTA (pH 6.8).Synthesis of L1–L5 Peptides—The HRR within human HRG is composed of 12 tandem repeats, with the dominant consensus repeat sequence being the 5-amino acid motif GHHPH. Synthetic peptides composed of one to five repeats of this 5-amino acid motif (termed L1–L5, respectively) were produced. L1–L3 peptides biotinylated at the N terminus were also prepared. The L1–L5 peptides were provided as a kind gift by Dr. Joe Altin (School of Biochemistry and Molecular Biology, Australian National University).Plasmid Constructs—Using the pUC9-HRG plasmid construct as a template (1Koide T. Foster D. Yoshitake S. Davie E.W. Biochemistry. 1986; 25: 2220-2225Crossref PubMed Scopus (111) Google Scholar), PCR with oligonucleotides ALJ-1 and ALJ-5 was carried out to amplify a cDNA encoding full-length human HRG comprising the leader, first cystatin (N1) domain, second cystatin (N2) domain, HRR, and C-terminal domain coding regions. The N1N2 coding region cDNA was generated by amplification with oligonucleotides ALJ-1 and ALJ-3. N1N2 was engineered to contain a hexahistidine tag at the 3′-end of the molecule through inclusion of six tandem histidine codons in oligonucleotide ALJ-3. The oligonucleotide primer sequences used are as follows: ALJ-1, 5′-TTGAATTCTAAAGGGATGGTTTAACAAAATG-3′; ALJ-3, 5′-AAGGTACCTTAGTGATGGTGATGGTGATGCTGAGGGTCGAAGACTTCAC-3′; and ALJ-5, 5′-AAGGTACCTTATTTTGGAAATGTATGTGTAAAAAAC-3′. The amplified HRG and N1N2 cDNAs were cloned into the EcoRI/KpnI sites in the donor vector construct pFastBac (Invitrogen), generating the pFastBac-HRG and pFastBac-N1N2 constructs, respectively. The nucleotide integrity of each clone was confirmed by automated sequencing using an Applied Biosystems 3730 DNA analyzer.Transfections and Recombinant Protein Production Using the Baculovirus Expression System—Recombinant proteins of full-length human HRG and the N2N2 domain of human HRG were produced using the “Bac-to-Bac” baculovirus expression system (Invitrogen). Recombinant bacmid constructs were generated using the pFastBac-HRG or pFast-Bac-N1N2 construct according to the manufacturer's instructions. Briefly, Sf9 cells were transfected using Cellfectin reagent (Invitrogen), with 1–2 μg of recombinant bacmid DNA being transfected per 9 × 105 cells for 5 h at 27 °C. Supernatant containing recombinant baculovirus was harvested 72 h post-transfection and was amplified for 3–4 days at 27 °C by infecting Sf9 cells with a multiplicity of infection of ∼0.01–0.1. Typically, recombinant baculovirus was amplified three times before being used to produce recombinant protein. Harvested Sf9 supernatant containing recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA)-agarose (QIAGEN Inc., Hilden, Germany). Recombinant full-length HRG and hexahistidine-tagged N1N2 were eluted from Ni-NTA-agarose with 200 mm cold imidazole.Western Blotting—Recombinant proteins were boiled for 10 min in 20 μl of SDS reducing sample buffer (125 mm Tris-HCl (pH 6.8), 20% glycerol, 10% dithiothreitol, and 4% SDS) and then subjected to electrophoresis on a 4–20% (w/v) gradient precast polyacrylamide minigel (Gradipore, Sydney, Australia). Proteins were transferred electrophoretically using a Mini-Protean II apparatus (Bio-Rad) onto a nitrocellulose membrane using a transfer buffer containing 48 mm Tris and 39 mm glycine in 20% (v/v) methanol. The membrane was blocked overnight with 5% (w/v) skim milk powder diluted in phosphate-buffered saline (PBS). Full-length HRG and the N1N2 domain were detected using the HRG-specific monoclonal antibody (mAb) HRG-4 (AGEN, Brisbane, Australia) and by chemiluminescence using ECL Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, United Kingdom).Immunofluorescence Flow Cytometry—Cell lines were analyzed for HRG binding or cell-surface heparan sulfate expression by immunofluorescence flow cytometry. Typically, plasma-purified or recombinant HRG or the recombinant N1N2 domain (100 μg/ml) was added to 5 × 105 cells in PBS and 0.1% bovine serum albumin (BSA) with or without 20 μm Zn2+ for 60 min at 4 °C and washed three times with PBS and 0.1% BSA. Cell-bound HRG or N1N2 was detected using the HRG-specific mAb HRG-4, and cell-surface heparan sulfate was detected by mAb F58-10E4 (Seikagaku Corp., Tokyo, Japan), followed by secondary detection with fluorescein isothiocyanate-labeled sheep anti-mouse Ig (Amrad Biotech, Melbourne, Australia). Cells were analyzed by immunofluorescence flow cytometry using an LSR flow cytometer (BD Biosciences), with forward scatter, side scatter, and Fl-1 data being collected. Flow cytometry data were analyzed using CellQuest Pro software (BD Biosciences). Each treatment condition was typically repeated in triplicate, and each experiment was repeated two to three times unless stated otherwise. In some experiments, cells were treated with mammalian heparanase (2 units/ml), bacterial heparinase III (2 units/ml; Sigma), or chondroitinase ABC (2 units/ml; Sigma) diluted in PBS and 0.1% BSA for 2 h at 37 °C. In other experiments, human HRG (100 μg/ml) was co-incubated with different GAGs (0.5–100 μg/ml), including bovine lung heparin (3.1, 4.5, 10.6, 12.5, and 16.7 kDa) and chondroitin sulfates A, B, C, and E (Sigma). Also, in some cases, cells were incubated with the biotinylated L1–L3 peptides (100 μm), with peptide binding to the cells being detected by R-phycoerythrin-conjugated streptavidin (Caltag Laboratories, Burlingame, CA).ELISAs—ELISAs were performed by coating 96-well polyvinyl chloride microtiter plastic plates (Dynex Technologies Inc., Chantilly, VA) overnight at 4 °C with recombinant full-length HRG or N1N2 (50 μl/well, ∼5 μg/ml) in 0.05 m Na2CO3/NaHCO3 buffer (pH 9.6) (Sigma) or with streptavidin (50 μl/well, 10 μg/ml; Sigma) diluted in PBS. Plates were then washed with PBS and 0.02% Tween 20 and blocked for 120 min at room temperature with 3% (w/v) BSA diluted in PBS. In some experiments, biotinylated heparin (10 μg/ml) diluted in PBS and 1% BSA was added to the streptavidin-coated plates for 60 min at room temperature before addition of HRG or the N1N2 domain (50 μl/well, ∼0.1–100 nm) diluted in PBS in the absence or presence of 20 μm Zn2+ and/or 1 mm EDTA. Bound HRG and N1N2 were detected using the HRG-specific mAb HRG-4, followed by secondary antibody detection with horseradish peroxidase-conjugated sheep anti-mouse Ig (Amrad Biotech). Plate-bound peroxidase was detected using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) peroxidase substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) and measuring the absorbance at 405 nm (reference wavelength of 490 nm) on a Thermomax microplate reader. Data were analyzed using SoftMax Pro software (Molecular Devices, Sunnyvale, CA).RESULTSHRG Binding to Cell Surfaces Is Zn2+-dependent—Previous studies have suggested that the binding of HRG to cell surfaces is highly Zn2+-dependent, with physiological concentrations of Zn2+ (∼20 μm) being particularly efficacious (18Olsen H.M. Parish C.R. Altin J.G. Immunology. 1996; 88: 198-206Crossref PubMed Scopus (39) Google Scholar). Initial experiments used immunofluorescence flow cytometry to analyze the effects of 20 μm Zn2+ on the binding of plasma-derived HRG to six different mammalian cell lines with widely differing tissue and species origins, viz. mouse melanoma cells (B16F1), human T-cells (MT4 and Jurkat), monkey kidney fibroblasts (COS-7), human fibrosarcoma cells (HT1080), and human umbilical vein endothelial cells (Fig. 1A). HRG bound to five of the six lines tested in a highly Zn2+-dependent manner, with the exception of human umbilical vein endothelial cells, which exhibited negligible HRG binding in both the presence and absence of Zn2+.Human HRG Interacts with Cell-surface Heparan Sulfate—It has been postulated previously that cell-surface GAGs such as heparan sulfate are able to interact with HRG (16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17Brown K.J. Parish C.R. Biochemistry. 1994; 33: 13918-13927Crossref PubMed Scopus (71) Google Scholar). To test this hypothesis directly, advantage was taken of Chinese hamster ovary (CHO) cell lines that either express cell-surface GAGs (CHO-K1) or lack cell-surface GAGs (pgsA-745) due to a deficiency in xylosyltransferase (23Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (483) Google Scholar, 24Esko J.D. Elgavish A. Prasthofer T. Taylor W.H. Weinke J.L. J. Biol. Chem. 1986; 261: 15725-15733Abstract Full Text PDF PubMed Google Scholar). We found that GAG-expressing (GAG+ve) CHO cells bound HRG in a Zn2+-dependent manner, but that GAG-deficient (GAG–ve) CHO cells did not bind HRG in either the presence or absence of 20 μm Zn2+ (Fig. 1, B and C). These results indicate that HRG binds to cell-surface GAGs and that this interaction is enhanced by physiological concentrations of Zn2+ (20 μm). Previous studies have reported that HRG interacts with the GAG heparin (10Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Abstract Full Text PDF PubMed Google Scholar, 11Lijnen H.R. Hoylaerts M. Collen D. J. Biol. Chem. 1983; 258: 3803-3808Abstract Full Text PDF PubMed Google Scholar, 12Burch M.K. Blackburn M.N. Morgan W.T. Biochemistry. 1987; 26: 7477-7482Crossref PubMed Scopus (38) Google Scholar). Thus, ELISA studies were carried out to analyze the effect of Zn2+ on the binding of full-length HRG to plastic immobilized heparin. Consistent with previous results (16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), HRG interacted with immobilized heparin, and this interaction was enhanced ∼4-fold in the presence of 20 μm Zn2+ (Fig. 1D). Cell-surface GAGs are composed of a mixture of heparan sulfate, chondroitin sulfates A and C, dermatan sulfate (chondroitin sulfate B), and hyaluronic acid (25Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 390-412Crossref Scopus (1523) Google Scholar). To define the specific cell-surface GAGs that interact with HRG, we enzymatically removed heparan sulfate and chondroitin sulfates A, B, and C from GAG+ve CHO cells. Initially, using a heparan sulfate-specific mAb and immunofluorescence flow cytometry, we verified that GAG+ve CHO cells express high levels of cell-surface heparan sulfate and that GAG–ve CHO cells do not express heparan sulfate (Fig. 2A). The enzymatic activity of mammalian heparanase, bacterial heparinase III, and chondroitinase ABC was verified by demonstrating by fast protein liquid chromatography that the enzymes could cleave heparan sulfate or chondroitin 6-sulfate chains, respectively, into smaller fragments (data not shown). GAG+ve CHO cells that were treated with either mammalian heparanase or bacterial heparinase III were completely depleted of surface heparan sulfate (Fig. 2, B and C). Subsequently, HRG binding to these mammalian heparanase- and heparinase III-treated cells was found to be markedly reduced (∼85–90%) (Fig. 2, D and E). In contrast, chondroitinase ABC treatment had no effect on HRG binding (Fig. 2, D and E). These results indicate that HRG binds specifically to cell-surface heparan sulfate and not chondroitin sulfates and that heparan sulfate is the principal cell-surface receptor for HRG on cells. Results from additional binding inhibition experiments carried out with a range of soluble GAGs were consistent with heparan sulfate being the GAG receptor for HRG on cells. Thus, various sized fragments of bovine lung heparin (4.5–16.7-kDa preparations) were potent inhibitors of the interaction of HRG with GAG+ve CHO cells, although very low molecular mass heparin (3.5 kDa) was a relatively ineffectual inhibitor (Table I). In contrast, chondroitin sulfates A, B, C, and E were totally inactive as inhibitors (Table I).Fig. 2Human HRG interacts with cell-surface heparan sulfate and not chondroitin sulfate. A, cell-surface expression of heparan sulfate by the GAG-expressing (GAG+ve) and GAG-deficient (GAG–ve) CHO cell lines assessed by immunofluorescence flow cytometry using a heparan sulfate-specific mAb. B, effect of mammalian heparanase or bacterial heparinase III treatment on heparan sulfate expression by the GAG-expressing CHO cell line. C, -fold change in heparan sulfate expression in GAG-expressing CHO cells following mammalian heparanase or bacterial heparinase III treatment. Data are means ± S.E. of three determinations. D and E, effect of mammalian heparanase and chondroitinase ABC treatment on HRG binding to GAG+ve CHO cells. D shows representative flow cytometry histograms (filled histograms, background fluorescence; empty histograms, heparan sulfate expression or HRG binding as detected by the appropriate mAb), and E depicts HRG binding (-fold increase in binding above background) following the different treatments. Data are means ± S.E. of three determinations. Cells were incubated with 100 μg/ml plasma-derived HRG in the presence of 20 μm Zn2+.View Large Image Figure ViewerDownload (PPT)Table IAbility of different GAGs to inhibit cell-surface binding of HRG to CHO-K1 cellsGAGIC50aConcentration of GAG that inhibited HRG binding to GAG+ve CHO cells by 50% as measured by immunofluorescence flow cytometry.μg/mlHeparin3.1 kDa504.5 kDa610.6 kDa312.5 kDa316.7 kDa3Chondroitin sulfate A>100Chondroitin sulfate B>100Chondroitin sulfate C>100Chondroitin sulfate E>100a Concentration of GAG that inhibited HRG binding to GAG+ve CHO cells by 50% as measured by immunofluorescence flow cytometry. Open table in a new tab The HRR of HRG Does Not Bind to Cells—It has been suggested previously that the HRR of HRG interacts with heparin/heparan sulfate (12Burch M.K. Blackburn M.N. Morgan W.T. Biochemistry. 1987; 26: 7477-7482Crossref PubMed Scopus (38) Google Scholar, 16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Crossref PubMed Scopus (76) Google Scholar). To test this hypothesis, peptides corresponding to the histidine repeat sequence within the HRR of HRG were synthesized and used in binding studies. The peptides comprised one (L1), two (L2), three (L3), four (L4), or five (L5) repeats of the dominant pentapeptide motif GHHPH that is tandemly repeated in the HRR of human HRG. Immunofluorescence flow cytometry analysis indicated that biotinylated preparations of the L1–L3 peptides did not bind to GAG+ve CHO cells in either the presence or absence of 20 μm Zn2+ (Fig. 3A), with R-phycoerythrin-conjugated streptavidin being used to monitor peptide binding. Furthermore, HRG blocking experiments revealed that high concentrations (100 μm) of the L1–L5 peptides failed to inhibit full-length HRG binding to GAG-expressing CHO cells and B16F1 melanoma cells (Fig. 3B). These data contrast with previous indirect evidence that the HRR binds heparan sulfate (12Burch M.K. Blackburn M.N. Morgan W.T. Biochemistry. 1987; 26: 7477-7482Crossref PubMed Scopus (38) Google Scholar, 16Borza D.B. Morgan W.T. J. Biol. Chem. 1998; 273: 5493-5499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Crossref PubMed Scopus (76) Google Scholar) and imply that the cell surface-binding domain of HRG may be located in another region of the molecule.Fig. 3Peptides corresponding to the histidine-rich repeat sequence of human HRG do not bind to GAG-expressing CHO cells and do not block HRG binding to GAG-expressing CHO cells. A, GAG+ve CHO cells were incubated in the presence or absence of 20 μm Zn2+ with biotinylated peptides that correspond to one (L1), two (L2), or three (L3) repeats of the consensus histidine pentapeptide of the HRR of HRG (GHHPH) (100 μm), with peptide binding being detected using R-phycoerythrin-conjugated streptavidin by immunofluorescence flow cytometry. Results are shown as -fold increase in peptide binding above background, with a value of 1 representing background binding (shown as a dashed line). Binding of HRG (100 μg/ml) as detected by the HRG-specific mAb HRG-4 is presented as a control. B, GAG+ve CHO cells and B16F1 melanoma cells were incubated with HRG (100 μg/ml) and 20 μm Zn2+ in the presence or absence of peptides corresponding to one to five repeats of the consensus histidine pentapeptide (L1–L5; 100 μm) and then analyzed for HRG binding using the HRG-specific mAb HRG-4 by immunofluorescence flow cytometry. Results are shown as percent HRG binding compared with control HRG binding (dashed line). Error bars represent S.E. (n = 3).View Large Image Figure ViewerDownload (PPT)Production of Recombinant Full-length Human HRG and the N-terminal N1N2 Domain—Earlier sequence homology studies suggested that the N1N2 domain of HRG contains a heparinbinding motif and may represent the region of HRG that interacts with cell-surface heparan su" @default.
• W2024652333 created "2016-06-24" @default.
• W2024652333 creator A5025637670 @default.
• W2024652333 creator A5068646165 @default.
• W2024652333 creator A5089817671 @default.
• W2024652333 date "2004-07-01" @default.
• W2024652333 modified "2023-10-06" @default.
• W2024652333 title "Histidine-rich Glycoprotein Binds to Cell-surface Heparan Sulfate via Its N-terminal Domain following Zn2+ Chelation" @default.
• W2024652333 cites W1428197784 @default.
• W2024652333 cites W1543388250 @default.
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