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• W2035965514 abstract "CD163 is the macrophage receptor for endocytosis of haptoglobin·hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteinerich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin·hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin·hemoglobin complexes. CD163 is the macrophage receptor for endocytosis of haptoglobin·hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteinerich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin·hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin·hemoglobin complexes. The haptoglobin (Hp) 1The abbreviations used are: Hp, haptoglobin; Hb, hemoglobin; CHO, Chinese hamster ovary; SRCR, scavenger receptor cysteine-rich; SPR, surface plasmon resonance; aa, amino acid; IL, interleukin.·hemoglobin (Hb) receptor CD163 is a 130-kDa macrophage protein belonging to a group of proteins constituting a subfamily of scavenger receptor cysteine-rich (SRCR) domain proteins (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar, 2Law S.K.A. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (218) Google Scholar, 3Graversen J.H. Madsen M. Moestrup S.K. Int. J. Biochem. Cell Biol. 2002; 34: 309-314Crossref PubMed Scopus (190) Google Scholar). These proteins, including CD5, CD6, CD163, CD163b, and WC1, are membrane proteins with a short cytoplasmic tail, a transmembrane segment, and an extracellular domain consisting exclusively of a variant number of consecutive class B SRCR domains (3Graversen J.H. Madsen M. Moestrup S.K. Int. J. Biochem. Cell Biol. 2002; 34: 309-314Crossref PubMed Scopus (190) Google Scholar, 4Gronlund J. Vitved L. Lausen M. Skjodt K. Holmskov U. J. Immunol. 2000; 165: 6406-6415Crossref PubMed Scopus (49) Google Scholar). The overall structure of the ∼100-aa class B SRCR domain is suggested to resemble the class A SRCR domain, which based on its crystal structure (5Hohenester E. Sasaki T. Timpl R. Nat. Struct. Biol. 1999; 6: 228-232Crossref PubMed Scopus (141) Google Scholar, 6Sarrias M.R. Gronlund J. Padilla O. Madsen J. Holmskov U. Lozano F. Crit. Rev. Immunol. 2004; 24: 1-37Crossref PubMed Google Scholar) is described as a six-stranded β-sheet cradling an α-helix. The class B domains differ from the class A domains by having four disulfide bridges instead of three. Furthermore, the class B domains are most common in multidomain mosaic proteins containing single SRCR domains associated to other functional domains, whereas the class A domains are most common in proteins exclusively composed of tandem repeats of SRCR domains. The SRCR domain structure is compatible with a role in molecular recognition, as shown for the membrane-proximal region of the three SRCR domains of CD6, which interacts with the activated leukocyte cell adhesion molecule (CD166) (7Bowen M.A. Aruffo A.A. Bajorath J. Proteins Struct. Funct. Genet. 2000; 40: 420-428Crossref PubMed Scopus (77) Google Scholar), and for two of the SRCR domains in bovine WC1 that bind to an unknown counter receptor (8Ahn J.S. Konno A. Gebe J.A. Aruffo A. Hamilton M.J. Park Y.H. Davis W.C. J. Leukocyte Biol. 2002; 72: 382-390PubMed Google Scholar). CD163 is the only protein of the class B SRCR domain protein subfamily identified as a receptor for an extracellular ligand. It binds with high affinity to the Hp·Hb complex (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar) that instantly forms in plasma when Hb is released from ruptured erythrocytes and is exposed to plasma Hp (0.3-2 g/liter) (9Dati F. Schumann G. Thomas L. Aguzzi F. Baudner S. Bienvenu J. Blaabjerg O. Blirup-Jensen S. Carlstrom A. Hyltoft-Petersen P. Johnson A.M. MilfordWard A. Ritchie R.F. Svendsen P.J. Whicher J. Eur. J. Clin. Chem. Clin. Biochem. 1996; 34: 517-520PubMed Google Scholar). This captor-receptor system controlling the metabolic route of Hb during limited hemolysis thereby protects against heme-mediated oxidative damages, in particular in the kidneys, that readily filtrate non-complexed Hb and take up Hb in the proximal tubules (10Gburek J. Verroust P.J. Willnow T.E. Fyfe J.C. Nowacki W. Jacobsen C. Moestrup S.K. Christensen E.I. J. Am. Soc. Nephrol. 2002; 13: 423-430PubMed Google Scholar). CD163 has no measurable affinity for noncomplexed Hp or Hb (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). This Hp·Hb complex-specific recognition by CD163 explains the decrease in Hp concentration in plasma during accelerated hemolysis (11Bunn H.F. Rosse W. Braunwald E. Fauci A.S. Kasper D.L. Hauser S.L. Longo J.L. Jameson J.L. Harrison's Principles of Internal Medicine. McGraw-Hill, New York2001: 681-691Google Scholar). Besides having a role in clearance of Hb from plasma, CD163 is suggested to have an immunomodulatory role (12Van den Heuvel M.M. Tensen C.P. van As J.H. van den Berg T.K. Fluitsma D.M. Dijkstra C.D. Dopp E.A. Droste A. Van Gaalen F.A. Sorg C. Hogger P. Beelen R.H.J. J. Leukocyte Biol. 1999; 66: 858-866Crossref PubMed Scopus (190) Google Scholar, 13Ritter M. Buechler C. Kapinsky M. Schmitz G. Eur. J. Immunol. 2001; 31: 999-1009Crossref PubMed Scopus (62) Google Scholar, 14Moestrup S.K. Moller H.J. Ann. Med. 2004; 36: 347-354Crossref PubMed Scopus (340) Google Scholar, 15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar). Two distinct mechanisms may account for this. First, clearance of the hemoglobin results in conversion of the heme molecule to CO, bilirubin, and Fe, which overall are suggested to have an anti-inflammatory effect (15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar, 16Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar). Second, ligand binding to CD163 is reported to induce tyrosine kinase- and calcium-dependent signaling and increased secretion of IL-6 and IL-10 (12Van den Heuvel M.M. Tensen C.P. van As J.H. van den Berg T.K. Fluitsma D.M. Dijkstra C.D. Dopp E.A. Droste A. Van Gaalen F.A. Sorg C. Hogger P. Beelen R.H.J. J. Leukocyte Biol. 1999; 66: 858-866Crossref PubMed Scopus (190) Google Scholar, 15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar). CD163 also circulates in plasma (∼1-3 mg/liter) as a soluble protein with a size identical to that of the nine extracellular SRCR domains (17Moller H.J. Peterslund N.A. Graversen J.H. Moestrup S.K. Blood. 2002; 99: 378-380Crossref PubMed Scopus (187) Google Scholar, 18Sulahian T.H. Hintz K.A. Wardwell K. Guyre P.M. J. Immunol. Methods. 2001; 252: 25-31Crossref PubMed Scopus (49) Google Scholar). During sepsis and other conditions affecting macrophage activity, the level of soluble CD163 may raise many-fold (19Moller H.J. de Fost M. Aerts H. Hollak C. Moestrup S.K. Eur. J. Haematology. 2004; 72: 135-139Crossref PubMed Scopus (96) Google Scholar, 20Schaer D.J. Schleiffenbaum B. Kurrer M. Imhof A. Bächli E. Fehr J. Møller H.J. Moestrup S.K. Schaffner A. Eur. J. Haematology. 2004; (in press)Google Scholar). The function of the extracellular domain is unknown, but it has been claimed to have an anti-inflammatory role (21Frings W. Dreier J. Sorg C. FEBS Lett. 2002; 526: 93-96Crossref PubMed Scopus (88) Google Scholar). It is not reported whether soluble CD163 binds any ligands. To delineate ligand binding properties of the membrane-associated and soluble CD163 protein and to identify the SRCR domains involved in ligand and antibody binding leading to endocytosis and signaling, we have performed a comprehensive analysis of a panel of recombinant CD163 variants and proteolytic fragments of CD163, including soluble CD163 circulating in plasma. Purified CD163, Ligand, and Antibodies—Full-length CD163 was purified from solubilized human spleen membranes as previously described (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). Amino-terminal sequencing of CD163 fragments was carried out by the Edman degradation procedure as previously described (22Moestrup S.K. Kozyraki R. Kristiansen M. Kaysen J.H. Rasmussen H.H. Brault D. Pontillon F. Goda F.O. Christensen E.I. Hammond T.G. Verroust P.J. J. Biol. Chem. 1998; 273: 5235-5242Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Human Hp (Hp1-1 and Hp2-2 phenotypes) and Hb were purchased from Sigma. Hp·Hb complexes were made by mixing equimolar concentration of Hp and Hb in phosphate-buffered saline. The following anti-CD163 monoclonal antibodies were used: Ber-MAC3 (DakoCytomation, Copenhagen, Denmark), 5C6-FAT (BMA Biomedicals AG, Augst, Switzerland), EDhu1 (12Van den Heuvel M.M. Tensen C.P. van As J.H. van den Berg T.K. Fluitsma D.M. Dijkstra C.D. Dopp E.A. Droste A. Van Gaalen F.A. Sorg C. Hogger P. Beelen R.H.J. J. Leukocyte Biol. 1999; 66: 858-866Crossref PubMed Scopus (190) Google Scholar), Ki-M8 (BMA Biomedicals AG), D11 (kind gifts from Dr. N. Petrovichev, Moscow), Mac2-48, Mac2-158, and R-20 (a kind gift from Dr. B. Davis, Trillium Diagnostics, Scarborough, ME), GHI/61 (BD PharMingen), and RM 3/1 (BMA Biomedicals AG). A polyclonal rabbit antibody has previously been described (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). Construction of Plasmids for Expression of Recombinant Human CD163 Fragments—CD163 cDNA fragments extended with enzyme restriction sites were amplified by polymerase chain reaction (PCR) with the Pfu turbo polymerase (Stratagene, La Jolla, CA) and purified with the QIAEX II gel extraction kit (Qiagen). The PCR products were subcloned into the expression vector (pcDNA5/FRT or pSecTag2B from Invitrogen) by use of the appropriate restriction enzymes (HindIII and XhoI, New England BioLabs, Beverly, MA) and the T4 DNA ligase (New England BioLabs). Plasmids were transformed using DH5α-competent cells (Clontech, Palo Alto, CA), and plasmid DNA was isolated by the Qiagen Maxiprep method (Qiagen) and sequenced before transfection. The following eleven constructs designated according to their SRCR domain composition were subcloned and expressed: r-CD163SRCR1-3 (aa 1-362); r-CD163SRCR1-4 (aa 1-478); r-CD163SRCR1-5 (aa 1-574); r-CD163SRCR1-6 r-CD163SRCR1-8 (aa (aa 1-679); 1-922); r-CD163SRCR1-7 (aa 1-815); r-CD163SRCR1-9 (aa 1-1025), r-CD163SRCR2-9 (aa 149-1025), r-CD163SRCR3-9 (aa 256-1025), r-CD163SRCR4-9 (aa 363-1025), and r-CD163SRCR5-9 (aa 470-1025). Expression of Recombinant Human CD163 Fragments—Chinese hamster ovary (CHO) K1 cells (BioWhittaker) were transfected using DOSPER liposomal transfection reagent or FuGENE™ 6 transfection reagent (Roche Applied Science). Stable transfected CHO clones were established by limited dilution and selection with 500 μg/ml Hygromycin B (Invitrogen) for constructs in the pcDNA5/FRT vector or 500 μg/ml Zeocin™ (Invitrogen) for constructs in the pSecTag2B vector. Clones were grown in serum-free medium for CHO cells (HyQ-CCM®5 from HyClone®, Logan, Utah) with 300 μg/ml Hygromycin B or 300 μg/ml Zeocin™. Cells were lysed in a solution of 10 mm NaH2PO4, 150 mm NaCl, 0.6 mm CaCl2, 1% Triton X-100 (Merck), 1 mm phenylmethylsulfonyl fluoride (Sigma), pH 7.4. The expression products were detected by Western blotting of growth medium and cell lysate using a rabbit polyclonal anti-CD163 antibody (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). Ligand Affinity Precipitation—The conditioned medium of the transfected cells secreting CD163 fragments into the medium, cell lysate of the cells expressing non-secreted CD163 fragments, and cleaved CD163 were incubated with human Hp·Hb coupled to CNBr-activated Sepharose 4B beads (Amersham Biosciences). Beads were washed with a solution of 10 mm NaH2PO4, 150 mm NaCl, 0.6 mm CaCl2, pH 7.4, before and after incubation with the different fragments. Supernatants were removed upon centrifugation at 5000 × g for 2 min. Large scale purification of the r-CD163SRCR1-5, r-CD163SRCR1-6, r-CD163SRCR1-7, r-CD163SRCR1-8, and r-CD163SRCR1-9 constructs from conditioned media was carried out by Hp·Hb affinity chromatography (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). Surface Plasmon Resonance (SPR)—SPR analysis of the binding of Hb in complex with Hp (both the 1-1 and 2-2 phenotypes) to CD163 was carried out on a Biacore 2000 instrument (Biacore, Uppsala, Sweden). The Biacore sensor chips (type CM5) were activated with a 1:1 mixture of 0.2 mN-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and 0.05 mN-hydroxysuccimide in water. Purified recombinant proteins (r-CD163SRCR1-5, r-CD163SRCR1-6, r-CD163SRCR1-7, r-CD163SRCR1-8, r-CD163SRCR1-9) were immobilized in 10 mm sodium acetate, pH 4.0, and the remaining binding sites were blocked with 1 m ethanolamine, pH 8.5. The SPR signal generated from immobilized recombinant CD163 proteins corresponded to 40-70 fmol of protein/mm2. Sensorgrams were generated using Hp (2-2)·Hb concentrations ranging from 5-1000 nm. The flow cells were regenerated with 1.6 m glycine-HCl, pH 3. For the reverse experiment, Hp (2-2)·Hb was immobilized as described above. The SPR signal generated from immobilized Hp (2-2)·Hb complex corresponded to 40-50 fmol of ligand/mm2. Data were recorded with a concentration of the recombinant proteins (r-CD163SRCR1-5, r-CD163SRCR1-6, r-CD163SRCR1-7, r-CD163SRCR1-8, r-CD163SRCR1-9) of 100 nm each diluted in the flow buffer. All binding data were analyzed using the Biamolecular Interaction Analysis evaluation program version 3.1. Analyses with a Panel of Monoclonal Anti-CD163 Antibodies—Immunoblots to a complete set of the CD163 truncation variants listed in Fig. 1 were probed with each monoclonal anti-CD163 antibody, and the domain location of the antibody epitope was defined on basis of the band pattern. SPR analyses were performed on a Biacore 2000 instrument as described above. Purified human full-length CD163 was immobilized to a sensor chip, and the SPR signal generated from immobilized CD163 corresponded to 36-60 fmol receptor/mm2. Each of the monoclonal anti-CD163 antibodies was injected and bound to the CD163 chip prior to incubation with the ligand (either Hp (2-2)·Hb or Hp (1-1)·Hb complex). Identification of Hp·Hb Binding Activity in Serum—Erythrocytes from EDTA-stabilized blood were washed three times in phosphate-buffered saline, centrifuged, and hemolyzed by freezing. Hemolyzed erythrocytes were added to serum (1:100 v/v) from individuals with different Hp phenotypes (Hp (1-1), Hp (2-1), and Hp (2-2), respectively), incubated for 1 h at room temperature, and diluted 1:2 with 0.05 m Tris, 0.3 m NaCl, 0.1% human serum albumin, 0.02% sodium-azide, pH 7.2, containing either 5 mm CaCl2 or 5 mm EDTA. Diluted serum samples (250 μl) were applied to a Superdex™ 200 HR 10/30 column on a SMART™ chromatography system (Amersham Biosciences) for gel filtration with a flow rate of 0.5 ml/min. Tris buffer (with CaCl2 or EDTA) was used to equilibrate the column and elute the sample. Fractions of 0.5 ml were collected and analyzed for soluble CD163 in enzyme-linked immunosorbent assay as described (23Moller H.J. Hald K. Moestrup S.K. Scand. J. Clin. Lab. Investig. 2002; 62: 293-299Crossref PubMed Scopus (138) Google Scholar). Hp phenotyping was performed by Western blotting of serum from healthy individuals. Identification of Hp·Hb Binding SRCR Domains in CD163—A panel of truncation variants of the SRCR domain region of CD163 was expressed (Fig. 1A) in stably transfected CHO cells. Fig. 1B shows Western blotting of the constructs expressed. Except for four of the constructs, the truncation variants were effectively secreted (lanes 3-7 and 10-11) to the cell culture medium. The non-secreted variants, which included the amino-terminal r-CD163SRCR1-3 and r-CD163SRCR1-4 constructs (Fig. 1B, lanes 1-2) and the constructs truncated in the amino-terminal part (r-CD163SRCR2-9 and r-CD163SRCR3-9) (lanes 8-9), accumulated in the cells, indicating abnormal folding and/or incomplete processing of these recombinant products. Binding of the secreted CD163 truncation variants to Hp·Hb as tested by precipitation with Hp·Hb-Sepharose beads revealed binding activity in the r-CD163SRCR1-5 variant and all larger variants (CD163SRCR1-6, CD163SRCR1-7, CD163SRCR1-8, and CD163SRCR1-9) encompassing the SRCR1-5 region. Accordingly, the variants were readily purified from conditioned growth media by Hp·Hb affinity chromatography. Fig. 2 shows SDS-PAGE of the purified r-CD163SRCR1-5 and r-CD163SRCR1-9 variants and their high affinity binding (Kd = 1-5 nm) to Hp·Hb as measured by SPR analyses. No binding to the r-CD163SRCR4-9 or r-CD163SRCR4-9 variants was seen (Fig. 1B), indicating that the amino-terminal third (SRCR1-3) of the extracellular CD163 domain is essential for ligand binding. Hp·Hb precipitation of the four non-secreted constructs (r-CD163SRCR1-3, r-CD163SRCR1-4, r-CD163SRCR2-9, and r-CD163SRCR3-9) accumulating in the cells did not reveal any binding activity (not shown). However, this finding might be due to an incomplete processing of these amino-terminal truncation variants; the finding therefore does not exclude that the corresponding parts of full-length native CD163 contain a binding site for Hp·Hb. Further information on the location of the ligand binding site was obtained by mapping the binding sites and analyzing ligand-inhibiting effects of a panel of ten monoclonal antibodies (Table I). Western blotting of the recombinant CD163 expression products and the proteolytic fragments revealed that four antibodies (Ber-MAC3, 5C6-FAT, Mac 2-48, Mac2-158) bind within SRCR domain 1 and two antibodies (EDhu1 and Ki-M8) bind to SRCR domain 3, whereas R-20, D11, GHI/61, and RM 3/1 bind to SRCR domains 4, 6, 7, and 9, respectively (Fig. 3, Table I). The SPR data revealed that only the antibodies EDhu1 and the Ki-M8 with binding epitopes in SRCR domain 3 of CD163 effectively compete for binding of Hp·Hb complexes to CD163 (Fig. 3, Table I). A partial inhibition was seen by the antibodies binding to domain 1, 4, and 9, suggesting that these antibodies may cause some steric hindrance or conformational change but not a direct blocking of ligand binding (Table I).Table IAnalysis of a panel of monoclonal anti-CD163 antibodiesMac2- 48Mac2-158Ber- MAC35C6- FATEDhu1KI-M8R-20D11GHI/61RM 3/1r-CD163SRCR1-3++++++−−−−r-CD163SRCR1-4+++++++−−−r-CD163SRCR1-5+++++++−−−r-CD163SRCR1-6++++++++−−r-CD163SRCR1-7+++++++++−r-CD163SRCR1-8+++++++++−r-CD163SRCR1-9++++++++++r-CD163SRCR2-9−−−−++++++r-CD163SRCR3-9−−−−++++++r-CD163SRCR4-9−−−−−−++++r-CD163SRCR5-9−−−−−−−+++CD163 amino-terminal fragmentaThe fragments seen after cleavage of CD163 in SRCR domain 3. Major cleavage site at Asp-265.++++−−−−−−CD163 carboxyl-terminal fragmentaThe fragments seen after cleavage of CD163 in SRCR domain 3. Major cleavage site at Asp-265.−−−−−−++++Inhibition of Hp·Hb binding*****M409629200M409629200*No inhibitionNo inhibition*a The fragments seen after cleavage of CD163 in SRCR domain 3. Major cleavage site at Asp-265. Open table in a new tab The indicated role of SRCR domain 3 in the Hp·Hb binding was further verified by analyzing cleavage products of affinity-purified native CD163 from spleen membranes. Limited proteolytic cleavage occurred spontaneously after exposure of native receptor to calcium-free condition, as apparent by SDS-PAGE showing the appearance of two major proteins bands of ∼90-95 and ∼35-40 kDa (Fig. 4, lane 1). No cleavage was seen when 5 mm calcium or 1 mm serine proteinase inhibitor (phenylmethyl-sulfonylfluoride) was added (not shown), indicating that calcium protects against proteinase activity. Western blotting using the mapped monoclonal antibodies (Table I), Triton X-114 precipitation (precipitates integral membrane proteins), and amino-terminal sequencing (data not shown) revealed that the 35-40-kDa band represents the amino-terminal part and the larger 90-95-kDa band the remaining carboxyl-terminal part, including the transmembrane segment. The amino-terminal sequencing disclosed cleavage in four close positions (the peptide bond amino-terminal of Asp-265, Arg-277, Ser-329, Ala-356) with Asp-265 being the primary site. The cleavage sites are all located in SRCR domain 3, thus indicating that this domain represents an exposed domain accessible for proteolytic attack. In contrast to the non-cleaved receptor, the cleavage products were not precipitable with Hp·Hb-beads (Fig. 4, lane 2), indicating that the structural integrity of domain SRCR 3 is essential for the ligand receptor interaction. Furthermore, the cleavage virtually abolished recognition of CD163 by the EDhu1 and Ki-M8 antibodies (Table I). This further indicates that the epitopes of these antibodies overlap with the Hp·Hb binding region of CD163. The ligand binding activity of the recombinant CD163 products encompassing SRCR 3 suggests that the soluble form of CD163 circulating in plasma is active in terms of ligand binding. To verify that soluble CD163 binds Hp·Hb complexes formed during hemolysis, we added Hb (lysed erythrocytes) to serum and measured the elution volume for plasma CD163. As seen in Fig. 5, the addition of Hb changed the elution volume of CD163 toward that of a protein of substantially larger size. The change was, as expected, more pronounced using serum from a person with the oligomeric high molecular mass Hp 2-2 and Hp 2-1 phenotypes compared with serum with the lower molecular mass Hp 1-1 phenotype. In accordance with the known calcium dependence of the Hp·Hb binding to CD163, no change in elution was seen when calcium was complexed to EDTA. Finally, we used the purified soluble ligand-binding r-CD163SRCR1-5 protein to characterize the calcium and pH sensitivity of Hp·Hb binding to the ligand binding site of CD163. As shown by SPR analysis (Fig. 6A), concentrations of calcium lower than the plasma concentration of calcium (∼2.2-2.5 mm) resulted in a dose-dependent decrease in affinity. No binding was measured in calcium concentrations below 0.2 mm. Accordingly, low calcium concentrations strongly accelerated dissociation of Hp·Hb bound to CD163 at high calcium concentrations (Fig. 6B). An accelerated dissociation of the ligand was also seen when pH was lowered to 6.0-6.5. (Fig. 6C). In the present study we have analyzed a large spectrum of CD163 truncation variants and used various approaches to identify and characterize the ligand binding site of the CD163 molecule. Fig. 7 summarizes the mapping of the calcium-sensitive ligand binding site and the antibody epitopes. The blocking effect of the EDhu1 and Ki-M8 antibodies as well as the inactivation of ligand binding by cleavage in SRCR domain 3 pinpointed SRCR domain 3 as crucial for the CD163 recognition of Hp·Hb complexes. The position of the Hp·Hb site in the amino-terminal third of the receptor appears biologically meaningful in the sense that this part of the molecule may be spaced a favorable distance from the membrane, thereby facilitating binding of the ligand complexes, which have a size substantially larger than the extracellular part of CD163. The data presented here do not exclude involvement in ligand binding of adjacent regions to SRCR 3, such as SRCR domain 2. However, the fact that there is no blocking of ligand binding of the antibodies binding to domain 1 and 4 suggest that these domains are not parts of the ligand binding site, although they might contribute to the receptor folding and accessibility of the ligand. In addition to identifying the ligand binding region, the present study delineated the CD163 epitopes of a broad spectrum of CD163 antibodies, including the ligand-blocking EDhu-1 and Ki-M8 antibodies, which have been widely described in the literature in studies of the function of CD163 (12Van den Heuvel M.M. Tensen C.P. van As J.H. van den Berg T.K. Fluitsma D.M. Dijkstra C.D. Dopp E.A. Droste A. Van Gaalen F.A. Sorg C. Hogger P. Beelen R.H.J. J. Leukocyte Biol. 1999; 66: 858-866Crossref PubMed Scopus (190) Google Scholar, 15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar). Interestingly, the EDhu-1 antibody has been shown to induce a protein tyrosine kinase-dependent signaling, slowtype calcium mobilization, inositol triphosphate production, and secretion of IL-6 and granulocyte-monocyte colony-stimulating factor (12Van den Heuvel M.M. Tensen C.P. van As J.H. van den Berg T.K. Fluitsma D.M. Dijkstra C.D. Dopp E.A. Droste A. Van Gaalen F.A. Sorg C. Hogger P. Beelen R.H.J. J. Leukocyte Biol. 1999; 66: 858-866Crossref PubMed Scopus (190) Google Scholar), whereas the Ki-M8 antibody has been shown to induce IL-10 secretion (15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar). Uptake of Hp·Hb also induces IL-10 secretion (15Philippidis P. Mason J.C. Evans B. Nadra I. Taylor K.M. Haskard D.O. Landis R.C. Circ. Res. 2004; 94: 119-126Crossref PubMed Scopus (406) Google Scholar), but it is unknown whether it is because of a direct receptor-mediated signal or because of the hemoglobin uptake leading to a heme-mediated cellular response. The effects of EDhu-1 and Ki-M8 antibodies are suggested to involve cross-linkage of CD163 in the membrane. It is therefore tempting to believe that the Hp·Hb complex, which is di- or multivalent in terms of CD163 recognition (depending on the Hp phenotype), also is able to cross-link CD163. In vitro binding of Hp·Hb complexes to purified CD163 does, in fact, support that CD163 cross-linkage can occur (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar). The Hp·Hb complex is the first soluble ligand identified for SRCR domains in a membrane receptor. The known ligands to the canonic type A scavenger receptor bind to the collagenous part of this receptor and not to the SRCR domain (24Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1063) Google Scholar). However, the CD6 protein, which has an extracellular domain consisting of three class B SRCR domains, is known to bind an integral membrane protein, the activated leukocyte adhesion protein, CD166 (7Bowen M.A. Aruffo A.A. Bajorath J. Proteins Struct. Funct. Genet. 2000; 40: 420-428Crossref PubMed Scopus (77) Google Scholar). Interestingly, the SRCR domain 3 of CD6 has also been identified as the ligand-binding domain, and site-directed mutagenesis has shown that a highly variable SRCR region of this domain is important for the CD166 interaction (7Bowen M.A. Aruffo A.A. Bajorath J. Proteins Struct. Funct. Genet. 2000; 40: 420-428Crossref PubMed Scopus (77) Google Scholar). In the present study we further explored the previous observation (1Kristiansen M. Graversen J.H. Jacobsen C. Sonne O. Hoffman H.J. Law S.K.A. Moestrup S.K. Nature. 2001; 409: 198-201Crossref PubMed Scopus (1300) Google Scholar) that calcium is essential for binding of Hp·Hb to CD163. This is, to our knowledge, the only SRCR interaction reported to depend on calcium. However, calcium binding to many other endocytic receptors, including the low density lipoprotein receptor family proteins (25Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar, 26Moestrup S.K. Kaltoft K. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1990; 265: 12623-12628Abstract Full Text PDF PubMed Google Scholar, 27Strickland D.K. Gonias S.L. Argraves W.S. Trends Endocrinol. Met. 2002; 13: 66-74Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), is a well known prerequisite for ligand binding. Interestingly, the present data revealed that a much higher calcium concentration is required to elicit ligand binding activity of CD163 compared with the low density lipoprotein receptor family proteins that bind calcium with very high affinity. It is therefore tempting to speculate that release of calcium from a low affinity calcium binding site in CD163 may cause ligand-receptor segregation in acidifying endosomes (28Gerasimenko J.V. Tepikin A.V. Petersen O.H. Gerasimenko O.V. Curr. Biol. 1998; 8: 1335-1338Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The SPR analysis in the present study showed that Hp·Hb bound to CD163 immediately dissociated when the calcium concentration was lowered to 0.2 mm. Decreasing pH to 6.5 also increased dissociation of Hp·Hb from CD163, although at a slower rate. In cell cultures (28Gerasimenko J.V. Tepikin A.V. Petersen O.H. Gerasimenko O.V. Curr. Biol. 1998; 8: 1335-1338Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), the calcium concentration in endosomes has been measured to be less than 10 μm when pH is 6.5. Under these conditions, the Hp·Hb complex may immediately dissociate from CD163. Such a calcium- and pH-dependent mechanism is different from the biological events causing ligand segregation from the low density lipoprotein receptor. This receptor uncouples its ligand by means of the YWTD propeller repeats that at low pH bind with affinity to ligand binding repeat (25Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar). In accordance with the mapping of the Hp·Hb binding to the SRCR domain 3 region, the present data showed that the circulating soluble form of CD163 representing the extracellular domain of CD163 (17Moller H.J. Peterslund N.A. Graversen J.H. Moestrup S.K. Blood. 2002; 99: 378-380Crossref PubMed Scopus (187) Google Scholar) is able to bind Hp·Hb complexes. The physiological implication of this interaction is not known. However, it is possible that it further prevent toxic effects of Hb or it may influence cellular signal pathways as previously proposed for soluble CD163 (21Frings W. Dreier J. Sorg C. FEBS Lett. 2002; 526: 93-96Crossref PubMed Scopus (88) Google Scholar). Probably, soluble CD163 has little effect on the overall clearance of Hb during accelerated hemolysis because soluble CD163 has a ∼1000-fold lower plasma concentration than Hp, which therefore immediately may saturate soluble CD163 when high amounts of Hb are released into plasma from erythrocytes. In conclusion, the present study has provided new insight into the Hp·Hb-CD163 interaction and the role of the SRCR domains in calcium-sensitive ligand coupling and uncoupling. Future studies may define the structural basis of the calcium dependence and the structural change leading to formation of a receptor binding epitope when Hp and Hb join in a complex. Furthermore, the homologous structure of the SRCR domains in CD163 and related proteins suggests that a number of ligand-receptor interactions are yet to be defined in the family of SRCR receptors. We thank Gitte Fynbo Biller, Gitte Petersen Ratz, Anne Marie Bundsgaard, and Lene Dabelstein for excellent technical assistance." @default.
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• W2035965514 title "Molecular Characterization of the Haptoglobin·Hemoglobin Receptor CD163" @default.
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