A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex
2006; Elsevier BV; Volume: 282; Issue: 2 Linguagem: Inglês
10.1074/jbc.m605684200
ISSN1083-351X
AutoresMarianne Jensby Nielsen, Steen V. Petersen, Christian Jacobsen, Søren Thirup, Jan J. Enghild, Jonas Heilskov Graversen, Søren K. Moestrup,
Tópico(s)Neonatal Health and Biochemistry
ResumoHaptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (α-chain) and a serine protease domain (β-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the β-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin β-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin. Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (α-chain) and a serine protease domain (β-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the β-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin β-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin. Haptoglobin (Hp) 2The abbreviations used are: Hp, haptoglobin; Hb, hemoglobin; proHp, Hp precursor; Hpr, haptoglobin-related protein; WT, wild type; MS, mass spec-trometry; MALDI-MS, matrix-assisted laser desorption/ionization-MS; SPR, surface plasmon resonance; HPLC, high pressure liquid chromatography. 2The abbreviations used are: Hp, haptoglobin; Hb, hemoglobin; proHp, Hp precursor; Hpr, haptoglobin-related protein; WT, wild type; MS, mass spec-trometry; MALDI-MS, matrix-assisted laser desorption/ionization-MS; SPR, surface plasmon resonance; HPLC, high pressure liquid chromatography. is an acute phase protein that circulates in high amounts (0.45–3 mg/ml) in plasma. The primary function of Hp is to capture hemoglobin (Hb) leaking into plasma from ruptured erythrocytes and precursors during intravascular hemolysis. The resulting high affinity complex between Hp and Hb is cleared from the circulation by receptor-mediated endocytosis via the acute phase-regulated monocyte/macrophage-specific protein CD163 (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1262) Google Scholar), which binds Hp-Hb by its scavenger receptor cysteine-rich domain region (2Madsen M. Moller H.J. Nielsen M.J. Jacobsen C. Graversen J.H. van den Berg T. Moestrup S.K. J. Biol. Chem. 2004; 279: 51561-51567Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This Hb clearance mechanism protects against inexpedient toxic effects caused by the oxidative iron-containing heme prosthetic group present in Hb (3Sadrzadeh S.M.H. Graf E. Panter S.S. Hallaway P.E. Eaton J.W. J. Biol. Chem. 1984; 259: 14354-14356Abstract Full Text PDF PubMed Google Scholar).Hp is synthesized as a single polypeptide precursor (proHp) that is proteolytically processed to form separate α- and β-chains representing a complement control repeat and a serine protease domain, respectively. The α- and β-chains are linked by a single disulfide bond. The Hp protein of most mammals is composed of two (αβ)-monomers linked together via a disulfide bond between the two α-chains generating an (αβ)2 structure (termed Hp1-1 in humans). However, three Hp phenotypes exist in humans due to the presence of two Hp gene alleles, designated Hp1 and Hp2. The Hp2 allele, which arose by an intragenic duplication of the Hp1 allele, encodes a slightly larger α-chain but is otherwise identical to the Hp1 allele. Because the cysteine residue connecting the α-chains is duplicated in the encoded Hp2 protein, the Hp2-1 and Hp2-2 phenotypes display a spectrum of various Hp (αβ)-multimers (4Delanghe J.R. Langlois M.R. Clin. Chem. Lab. Med. 2002; 40: 212-216Crossref PubMed Scopus (63) Google Scholar, 5Langlois M.R. Delanghe J.R. Clin. Chem. 1996; 42: 1589-1600Crossref PubMed Scopus (796) Google Scholar).Due to an Hp gene triplication in the late evolution, primates also express the haptoglobin-related protein (Hpr), which shares 91% sequence identity with the Hp1 protein (6Bensi G. Raugei G. Klefenz H. Cortese R. EMBO J. 1985; 4: 119-126Crossref PubMed Scopus (115) Google Scholar, 7Maeda N. J. Biol. Chem. 1985; 260: 6698-6709Abstract Full Text PDF PubMed Google Scholar). Hpr circulates in plasma as a component of two complexes, a subclass of high density lipoprotein particles (∼500 kDa) known as trypanosome lytic factor 1 and a high molecular mass (≥1000 kDa), lipid-poor protein complex termed trypanosome lytic factor 2 (8Hajduk S.L. Moore D.R. Vasudevacharya J. Siqueira H. Torri A.F. Tytler E.M. Esko J.D. J. Biol. Chem. 1989; 264: 5210-5217Abstract Full Text PDF PubMed Google Scholar, 9Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar, 10Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (143) Google Scholar, 11Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar). Both trypanosome lytic factor 1 and trypanosome lytic factor 2 protect against the African parasite Trypanosoma brucei brucei by inducing lysis of the parasite, and Hpr is suggested to play a vital role in this primate-specific defense mechanism (12Drain J. Bishop J.R. Hajduk S.L. J. Biol. Chem. 2001; 276: 30254-30260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 13Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. J. Biol. Chem. 2005; 280: 32578-32585Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).Recently we identified Hpr as a high affinity Hb-binding protein associating with Hb virtually as efficiently as Hp (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar). Unlike Hp complex formation with Hb, Hpr complex formation with Hb does not increase Hb binding to CD163, thus indicating that some of the differences in the primary structure of Hp and Hpr are indispensable for high affinity receptor binding. In the present study, we have therefore designed and analyzed recombinant Hp/Hpr chimeric proteins as an approach to identify Hp regions/residues essential for the high affinity binding of Hp-Hb to CD163.EXPERIMENTAL PROCEDURESConstruction of Plasmids for Expression of Recombinant Hp, Hpr, and Hp/Hpr Chimeras—The plasmid constructs composed of human Hp1 wild type (WT) or human Hpr WT inserted into the KpnI and XhoI sites of the mammalian expression vector pcDNA5/FRT (Invitrogen, Taastrup, Denmark) were described recently (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar). By means of site-directed mutagenesis, a large panel of plasmid constructs encoding Hp/Hpr chimeras was established. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) using the pcDNA5/FRT-Hp1 WT construct as the initial template. Plasmid DNA was isolated by the Qiagen Maxiprep method (Qiagen, Albertslund, Denmark) and sequenced before transfection. The following constructs designated according to their encoded Hp and Hpr amino acid composition were designed: chimera 1, Hpr(α-chain)/Hp(β-chain) (the chimeric protein consisting of the Hpr α-chain and the Hp β-chain); chimera 2, Hp C33F (the Hp protein with a single mutation of Cys-33 to Phe); chimera 3, Hp(α-chain)/Hpr(β-chain) (the chimeric protein consisting of the Hp α-chain and the Hpr β-chain); chimera 4, Hpr(α-chain)/Hpr(β-chain 104–234)/Hp(β-chain 234–347) (the chimeric protein consisting of the Hpr α-chain, Hpr β-chain residues 104–234, and Hp β-chain residues 234–347); chimera 5, Hpr(α-chain)/Hpr(β-chain 104–253)/Hp(β-chain 253–347) (the chimeric protein consisting of the Hpr α-chain, Hpr β-chain residues 104–253, and Hp β-chain residues 253–347); chimera 6, Hpr(α-chain)/Hpr(β-chain 104–265)/Hp(β-chain 265–347) (the chimeric protein consisting of the Hpr α-chain, Hpr β-chain residues 104–265, and Hp β-chain residues 265–347); chimera 7, Hpr(α-chain)/Hpr(β-chain 104–265, C260V)/Hp(β-chain 265–347) (the chimeric protein consisting of the Hpr α-chain, Hpr β-chain residues 104–259 and 261–265, and Hp β-chain residues 259 and 265–347); chimera 8, Hp C33F, V259C (the Hp protein with mutations of Cys-33 to Phe and Val-259 to Cys); and chimera 9,Hp E261K, K262W, T264A (the Hp protein with mutations of Glu-261 to Lys, Lys-262 to Trp, and Thr-264 to Ala).Establishment of Cell Lines Stably Transfected with Hp/Hpr Chimeras—Flp-In 293 cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium (Cambrex Bioscience Verviers, Verviers, Belgium) supplemented with 10% fetal calf serum, 2 mm glutamine, and 100 μg/ml zeocin (Invitrogen) and transfected using FuGENE 6 (Roche Diagnostics, Hvidovre, Denmark). Stable transfectants were selected with 150 μg/ml hygromycin B (Invitrogen). For visualization of expression products, culture medium harvested from cells grown in serum-free 293 medium (Invitrogen) was subjected to SDS-PAGE and subsequent immunoblotting using a rabbit polyclonal anti-human Hp antibody (DAKOCytomation, Glostrup, Denmark). The generation of Flp-In 293 cells stably transfected with Hp WT and Hpr WT was described previously (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar).Purification of Hp, Hpr, and Hp/Hpr Chimeras by Hb Affinity Chromatography—Purification of Hp, Hpr, and Hp/Hpr chimeras was performed by subjecting the harvested serum-free 293 cell culture medium containing secreted expression products to Hb affinity chromatography, essentially as described (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar, 15Liau C.Y. Chang T.M. Pan J.P. Chen W.L. Mao S.J.T. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003; 790: 209-216Crossref PubMed Scopus (45) Google Scholar). Affinity columns were prepared by coupling 10 mg of Hb A0 (Sigma, Brøndby, Denmark) to 1 g of CNBr-activated Sepharose 4B (Amersham Biosciences, Hørsholm, Denmark) essentially as described by the manufacturer. After allowing the expression products to bind the column material, the columns were washed extensively with phosphate-buffered saline (10 mm NaH2PO4, 0.15 m NaCl, 0.6 mm CaCl2), pH 7.4, and bound proteins subsequently eluted by 5 m urea containing 0.15 m NaCl. The collected material was dialyzed overnight at 4 °C against phosphate-buffered saline, pH 7.4, with one buffer change and analyzed by SDS-PAGE followed by silver staining to visualize the purified protein products.Automated Edman Degradation—Samples were analyzed by automated Edman degradation using an Applied Biosystems Procise 491HT sequencer with on-line phenylthiohydantoin HPLC analysis. The instruments were operated according to instructions by the manufacturer.Mass Spectrometry (MS)—Approximately 40 μg of recombinant Hpr in 30 mm HEPES, pH 8.3, containing 5 m guanidinium hydrochloride was S-carboxyamidomethylated by adding iodoacetamide to a final concentration of 10 mm. The reaction was allowed to proceed for 30 min before the material was acidified and desalted on to a 2.1 × 220 mm Aquapore RP-300 C8 reverse-phase HPLC column (Brownlee Labs) connected to an AKTA explorer (GE Healthcare). Bound proteins were eluted by a linear gradient from 0.1% trifluoroacetic acid (solvent A) to 90% acetonitrile, 0.08% trifluoroacetic acid (solvent B) (1% B min-1). The column was operated at 23 °C at a flow rate of 200 μl min-1. Protein was detected at 220 and 280 nm, and fractions were collected manually. The fraction containing Hpr was lyophilized, redissolved in 50 mm Tris-HCl, 137 mm NaCl, pH 7.4, and digested by the addition of trypsin (sequence grade, Promega) at a 1:20 ratio for 16 h at 37 °C. The tryptic peptides were subsequently separated by reverse-phase HPLC as described above. Fractions were collected manually and analyzed by MALDI-MS before and after reduction using a Q-Tof Ultima Global mass spectrometer (Micromass/Waters Corp.) calibrated using a mixture of polyethylene glycol-200, -600, -1000, -2000, and NaI. The acquired mass spectra were mass-corrected using glu-fibrinopeptide B as an external calibrant. Aliquots of fractions selected for subdigestion using Staphylococcus aureus V8 protease (Sigma) were lyophilized and redissolved in 50 mm NH4HCO3. The digestions were allowed to proceed for 30 min at 37 °C. The generated peptides were recovered by using Stagetips (C18, Proxeon) as recommended by the manufacturer and spotted onto a MALDI sample target using 1 μl of matrix solution (0.4% (w/v) recrystallized α-cyano-4-hydroxy-cinnamic acid in 70% (v/v) acetonitrile, 0.03% (v/v) trifluoroacetic acid) and analyzed by MALDI-MS as described above.Surface Plasmon Resonance (SPR) Analysis—CD163 binding of Hb A0, Hp, Hpr, Hp/Hpr chimeras (all 25 μg/ml) alone or complexes of Hb A0 with Hp, Hpr, or Hp/Hpr chimeras was studied by SPR analysis on a Biacore 3000 instrument (Biacore, Uppsala, Sweden), essentially as described (2Madsen M. Moller H.J. Nielsen M.J. Jacobsen C. Graversen J.H. van den Berg T. Moestrup S.K. J. Biol. Chem. 2004; 279: 51561-51567Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Biacore sensor chips (type CM5) were activated with a 1:1 mixture of 0.2 m N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and 0.05 m N-hydroxysuccimide in water according to instructions by the manufacturer. CD163 purified from human spleen (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1262) Google Scholar) was immobilized in 10 mm sodium acetate, pH 4.0, and remaining binding sites were blocked with 1 m ethanolamine, pH 8.5. A control flow cell was made by performing the activation and blocking procedure only. The SPR signal generated from immobilized recombinant protein corresponded to 60–70 fmol of receptor/mm2. Samples were dissolved in 10 mm Hepes, 150 mm NaCl, 2.0 mm CaCl2, 1.0 mm EGTA, and 0.005% Tween 20, pH 7.4. Sample and running buffers were identical. Regeneration of the sensor chip after each analysis cycle was performed with 10 mm glycine, pH 4.0, containing 20 mm EDTA and 500 mm NaCl. The Biacore response is expressed in relative response units, i.e. the difference in response between protein and control flow channel. Kinetic parameters were determined by BIAevaluation 4.1 software using a Langmuir 1:1 binding model.The effect of the synthetic peptides RHYEGSTVPEKKTPKSPVGVQPILNEHT and THYEGSTVPKWKAPKSPVGVQPILNEHT (KJ ROSS-Petersen ApS, Klampenborg, Denmark) on the binding of Hp-Hb to immobilized CD163 was studied using an Hp (1-1)-Hb A0 complex formed by mixing 8 μg/ml Hb A0 with 8 μg/ml plasma-derived Hp1-1 (Sigma). The synthetic peptides were dissolved in sample buffer, and experiments were performed in a concentration range from 0 to 750 μm of peptides. Sensorgrams were monitored, and the relative response at 600 s was used in calculation of the percentage of binding of the Hp-Hb complex.Modeling of the Hp Structure— The core of the model of human Hp was constructed using the Swiss-Model server (16Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4424) Google Scholar). SwissModel selected three structures of C1r (Protein Data Bank code: 1MD8, 1MD7, 1GPZ) and one structure of C1s (Protein Data Bank code: 1ELV) as templates for the modeling. Alternative suggestions for the structure of Hp loop 1 were identified by locating the nearly conserved sequence pattern PXXXXPXX-PXXXXP found in Hp in the set of non-redundant sequences of the Protein Data Bank using PATTERNSEARCHDB (Biology Workbench). Of 32 occurrences of the pattern, the loop from bovine mitochondrial aconitase (Protein Data Bank code: 1ACO) (residues 320–333) was chosen based on the agreement between endpoints of the loop and the core generated by Swiss-Model. The loop was incorporated into the Hp model using O (17Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A Found. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar).RESULTSComparison of the Primary Structure of Hp and Hpr and Determination of the Disulfide Bridge Pattern of Hpr—Fig. 1A shows an alignment of Hp1 and Hpr with the 27-amino-acid residue differences in the α- and β-chains indicated in black. The differences include 2 cysteine residues, the Cys-260 in the Hpr β-chain, which is a replacement of a Val in Hp, and Cys-33 in Hp, which is replaced by Phe in Hpr. The latter difference abolishes the covalent stabilization of the structure because Cys-33 mediates the disulfide bridging of the α-chains of Hp (18Kurosky A. Barnett D.R. Lee T.H. Touchstone B. Hay R.E. Arnott M.S. Bowman B.H. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3388-3392Crossref PubMed Scopus (206) Google Scholar). To investigate whether Hpr Cys-260 perturbs the disulfide bond pattern relatively to the disulfide arrangement known from Hp (18Kurosky A. Barnett D.R. Lee T.H. Touchstone B. Hay R.E. Arnott M.S. Bowman B.H. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3388-3392Crossref PubMed Scopus (206) Google Scholar), purified recombinant Hpr was digested by trypsin, and the generated peptides were subsequently separated by reverse-phase HPLC. All fractions were analyzed by MALDI-MS, and selected fractions containing disulfide-linked peptides were subsequently subdigested using S. aureus V8 protease. This analysis revealed that the disulfide bridge pattern of Hpr was homologous to that of the Hp (αβ)-unit (Fig. 1B). Furthermore, the analysis showed that Cys-260 was cysteinylated. The latter conclusion was based on the finding that under non-reducing conditions, the peptide encompassing Cys-260 had a mass increase of 119 Da corresponding to the cysteinylation of the cysteine residue. The identification of Cys-260 as the cysteinylated residue was determined by enzymatic subdigestion of the disulfide-linked tryptic peptide. As Hpr contains an uneven number of cysteine residues, the inability to detect any S-carboxyamidomethylated Cys-containing peptides further supports the presence of a cysteinylation.Expression of Recombinant Hp/Hpr Chimeric Proteins—A site-directed mutagenesis strategy was based on the 27 amino acid residues, which are different in Hpr as compared with Hp. Fig. 2 shows the panel of Hp/Hpr chimeric proteins expressed and analyzed to determine the elements important for receptor recognition.FIGURE 2Expression of recombinant Hp, Hpr, and Hp/Hpr chimeras. A, overview of the recombinant Hp, Hpr, and Hp/Hpr chimeric proteins produced in the human 293 cell expression system. The overall structure of the expression products is shown schematically to the right. For clarity, all disulfide bridges, except the inter-α-chain disulfide bridge, have been omitted. B, silver-stained 8–16% SDS-polyacrylamide gel of the purified recombinant Hp, Hpr, and Hp/Hpr chimeric expression products. Expression products were purified by subjecting harvested culture medium to Hb affinity chromatography. Positions of the molecular size standard markers are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In agreement with Hp Cys-33 being responsible for the disulfide-mediated dimerization of (αβ)-monomers to form the Hp (αβ)2 molecule (18Kurosky A. Barnett D.R. Lee T.H. Touchstone B. Hay R.E. Arnott M.S. Bowman B.H. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3388-3392Crossref PubMed Scopus (206) Google Scholar), all expression products containing the C33F mutation primarily formed (αβ)-monomers of approximately half the size of the recombinant Hp WT (αβ)2 molecule as determined by non-reducing SDS-PAGE. The mobility in SDS-PAGE was also influenced by the Hp S184H mutation disrupting a glycosylation motif of the type Asn-X-Ser/Thr. Expression products carrying this amino acid substitution migrated slightly faster than expression products without the mutation.In accordance with recent data showing that only a low degree of cleavage of recombinant proHp to the α- and β-chains takes place in 293 cells (19Wicher K.B. Fries E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14390-14395Crossref PubMed Scopus (50) Google Scholar), amino-terminal sequencing showed that recombinant Hp remained largely uncleaved (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar). However, the recombinant Hp bound to Hb has similar receptor binding activity as Hb in complex with native plasma-derived Hp (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar). Surprisingly, the recombinant Hpr expressed in the 293 cells was fully cleaved into the α- and β-chains (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar), thus suggesting that it is accessible for cleavage by an alternative protease. However, in contrast to the native Hpr found in plasma (9Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar, 10Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (143) Google Scholar), recombinant Hpr was found to lack the hydro-phobic signal peptide (14Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Møller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar).Identification of the Hp β-Chain Region Involved in High Affinity Binding to CD163—As expected, the panel of different Hp/Hpr chimeric expression products all bound Hb as evident by Hb affinity chromatography (Fig. 2B). Using SPR analysis, we subsequently examined the CD163 binding capacity of the purified proteins in complex with Hb (Fig. 3, Table 1). Replacement of the Hp α-chain with that of Hpr had no significant effect on the CD163 binding as seen by comparison of Hp WT and the Hpr(α-chain)/Hp(β-chain) chimera (Fig. 3, A and C). Concordantly, the Hp C33F mutant selectively missing the inter-α-chain disulfide bridge showed high affinity receptor binding (Fig. 3D). Thus, the residues specific for the Hpr α-chain, including the C33F substitution, which prevents the disulfide-mediated (αβ)-dimer formation, do not affect the binding to CD163.FIGURE 3SPR analyses of binding of complexes of Hb and purified recombinant Hp, Hpr, or Hp/Hpr chimeras to CD163. Purified Hb alone, purified recombinant Hp, Hpr, Hp/Hpr chimera 1–9 (C1–9) alone, or the complexes of Hb with Hp, Hpr, and Hp/Hpr chimeras were analyzed for binding to CD163 immobilized on a Biacore sensor chip. The recombinant protein analyzed is indicated above each panel (for designations of the chimeric proteins, refer to Fig. 2). Hb alone displayed low binding to CD163, whereas no binding of Hp, Hpr, or the Hp/Hpr chimeras alone to CD163 could be detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Kinetic parameters derived from SPR analysis of binding of Hb alone or complexes of Hb and purified recombinant Hp, Hpr, and Hp/Hpr chimeras to immobilized CD163 The kinetic parameters were determined under the assumption that Hb alone binds CD163 as a dimer consisting of two globin subunits and that Hpr and Hp/Hpr chimeras harboring the Hp C33F substitution non-covalently dimerize to form the (αβ)2 structure (see discussion). The kinetic parameters were not determined in the cases of CD163-binding curve characteristics indistinguishable from those of Hb alone. Kd, dissociation constant; ka, association rate constant; kd, dissociation rate constant; ND, not determined.LigandKdkakdm1/ms1/sHb3.7 × 10–77.6 × 1032.8 × 10–3Hp wt-Hb1.2 × 10–84.4 × 1045.3 × 10–4Hpr wt-HbNDNDNDChimera 1-Hb1.6 × 10–84.8 × 1047.6 × 10–4Chimera 2-Hb1.1 × 10–85.0 × 1045.5 × 10–4Chimera 3-HbNDNDNDChimera 4-Hb1.3 × 10–85.3 × 1047.1 × 10–4Chimera 5-Hb2.7 × 10–83.8 × 1041.0 × 10–3Chimera 6-HbNDNDNDChimera 7-HbNDNDNDChimera 8-Hb4.7 × 10–82.4 × 1041.1 × 10–3Chimera 9-HbNDNDND Open table in a new tab In striking contrast, substitution of the Hp β-chain with that of Hpr completely abolished the ability to increase Hb binding to CD163 as revealed by binding experiments using the Hp(α-chain)/Hpr(β-chain) chimeric protein (Fig. 3E). In the next step, we analyzed mutant proteins consisting of the Hpr α-chain and chimeric β-chain Hp/Hpr. Substituting the Hp residues 1–233 by the corresponding Hpr residues (1–234) had no significant impact on receptor binding (Fig. 3F). However, introducing the mutations corresponding to the Hpr residues 235–253 and 235–265 had a weak and completely abrogating effect, respectively, on the Hp-inducible increase in Hb binding to CD163 (Fig. 3, G and H). In essence, the triple substitution D248Y, Q249D, and R252T caused partial inhibition of high affinity receptor binding, and combining with the V259C, E261K, K262W, and T264A substitutions entirely eliminated high affinity receptor binding.Introduction of the V259C mutation in Hp (corresponding to Cys-260 of Hpr) appeared interesting because Hpr Cys-260 is cysteinylated (Fig. 1B). However, reintroduction of the original Hp Val at this position failed to rescue high affinity binding (Fig. 3, H and I). On the other hand, introduction of a Cys at Hp position 259, even in the absence of the D248Y, Q249D, R252T, E261K, K262W, and T264A substitutions, caused a substantial decrease in receptor binding as seen by comparing the Hp C33F and Hp C33F, V259C mutant proteins (Fig. 3, D versus J).Finally, the effect of the E261K, K262W, and T264A substitutions on receptor binding was tested in the absence of other residue alterations. Strikingly, as presented in Fig. 3, introduction of these 3 amino acid residue substitutions in Hp was sufficient to obstruct high affinity receptor binding (compare A and K). Taken together, the binding analyses demonstrate that the region encompassing residues 234–264 of the Hp β-chain contains residues that are critical for receptor binding, in particular the residues Val-259, Glu-261, Lys-262, and Thr-264, which in Hpr are replaced by Cys, Lys, Trp, and Ala, respectively.The determination of the structural fold of the complement control repeat and serine protease domains of complement components C1r and C1s by x-ray crystal-lography allows modeling of Hp as depicted in Fig. 4A. Due to the lack of similarity between the templates and the amino-terminal part of Hp, the model only comprises residues 43–346. Interestingly, the region identified as important for receptor binding represents a part of a loop, which has been designated as loop 1 of serine proteases (20Perona J.J. Craik C.S. J. Biol. Chem. 1997; 272: 29987-29990Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). This is a unique part of Hp and Hpr as compared with serine proteases, which all have a considerably shorter loop (Fig. 4B). Due to the length of the loop and the lack of a known template structure, a reliable model for the loop structure cannot be obtained. The presence of 4 proline residues in the loop suggests a limited flexibility.FIGURE 4Computer-modeled three-dimensional structure of Hp showing the r
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