Chapter 26 Purification of C1q Receptors and Functional Analysis Berhane Ghebrehiwet and Ellinor I.B. Peerschke Abstract The recognition subunit of C1, C1q, has emerged as an important player in various pathophysiologic conditions largely in part due to its ability to interact with pathogen-associated or cell surface expressed ligands and receptors. Identification and purification of these molecules is therefore of paramount importance if we are to procure valuable information with regards to the structure, function, and cell surface distribution. Since the interaction of C1q is better served when the receptors are purified from homologous species, we discuss here a simple guideline for the purification and characterization of the two C1q receptors, cC1qR (calreticulin) and gC1qR, from human cell lines. Key words C1q receptor, Collagen C1q receptor, calreticulin, globular C1q receptor
Abbreviations cC1q cC1qR CRT gC1q gC1qR
1
The collagen domain of C1q Receptor for cC1q Calreticulin another name for cC1qR The globular heads of C1q Receptor for gC1q
Introduction Human C1q circulates in plasma as part of a multimolecular complex comprising of C1q and a Ca2+-dependent tetramer, C1r2– C1s2. The traditional role of C1q within this complex is to serve as a unit through which C1 recognizes immune complexes or unique patterns on pathogen-associated molecular ligands. This interaction, in turn, is subsequently translated into a highly specific and orderly intramolecular rearrangement resulting in a sequential proteolytic activation—first of C1r and then of C1s—thereby triggering activation of the classical pathway [1–3]. However, information accumulated to date show that the role of C1q is not restricted to recognition of immune complexes or other molecules that activate
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_26, © Springer Science+Business Media New York 2014
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Fig. 1 Chain structure of C1qRs. Purified gC1qR (A) and cC1qR (B) were subjected to SDS-PAGE under reducing conditions
the classical pathway, but is also a key player in a rapidly expanding list of pathophysiological conditions as well as cellular functions, which contribute significantly to a plethora of inflammatory processes. These functions in turn are, by and large, mediated through the interaction of C1q with cell-associated membrane glycoproteins and surface receptors [4–7]. Because C1q is a chimeric protein comprising of a collagenlike region (cC1q) and a globular domain (gC1q), it is capable of interacting with cell surface molecules via either of its two regions. Hence, although we are witnessing an ever-increasing diversity of biologic functions mediated by C1q by virtue of its versatility to bind an increasingly long list of cell-associated molecules, there are presently two, well-characterized surface molecules that bind to either the cC1q or gC1q domains hence designated cC1qR (calreticulin) and gC1qR (p33 or p32), respectively (Fig. 1). Since receptors are involved in the control and modulation of a wide range of cellular processes, their purification from cell membranes will not only help us identify their structure and biochemical composition but also allow us to generate antibodies so that their function, cellular distribution, and structure could be assessed. In this paper, we describe a simple method for the purification of C1q receptors from cell membranes. The two C1q receptors are ubiquitously distributed, and virtually every cell type expresses these receptors, although some cells (e.g., lymphocytes and monocytes) may express more copies per cell than quiescent endothelial cells, which need to be activated by inflammatory cytokines (e.g., LPS, INFγ, and TNFα) [9]. Therefore almost any cell type can be used as a source of C1q receptors and there appears to be no structural difference between receptors isolated from fresh circulating human cells or cultured cell lines. However since a large number of cells is required to generate a reasonable amount of membrane proteins from which quantifiable amount of protein could be recovered, it is more pragmatic to use
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a cultured cell line. We have successfully used both Raji cells, (a B cell line) and U937 (a monocyte-like cell line), to generate cell membrane proteins from which C1qRs were purified [4]. The purpose of purifying membrane proteins is to isolate a specific membrane receptor(s) for functional analysis and biochemical characterization. Therefore, one of the first and fundamental steps to be considered is the choice of a solubilization buffer that will keep the desired receptor in its functional state. This includes the choice of a detergent such as octyl glucoside, CHAPS, Emulphogene BC 720 (polyoxyethylene-10-tridecyl ether), and a suitable buffer medium with phosphate buffer for example being more effective than Tris buffer [10]. In addition, since the breakup of membranes releases enzymes that could potentially digest the very protein of interest, the addition of sufficient cocktail of enzyme inhibitors becomes of paramount importance although even then, one could never be sure that every enzyme released would be completely inhibited. Membrane solubilization buffers as well as cocktail mix of enzyme inhibitors are now commercially available from various sources. The example presented here uses U937 cells to isolate C1q receptors and could be adapted for the purification of other cell surface molecules as well.
2
Materials 1. Dulbecco’s PBS: 0.37 M NaCl, 0.002 M KCl, 0.008 M Na2HPO4, 0.001 M KH2PO4, pH 7.4. 2. Solubilization buffer: PBS-containing enzyme inhibitors and 1 % Emulphogene BC720. 3. Coating buffer: 35 mM NaHCO3, 15 mM Na2CO3, pH 9.6. 4. Blocking reagent: 1 % BSA in Tris-buffered saline (20 mM Tris–HCl, 100 mM NaCl, pH 7.5). 5. Alkaline-phosphatase-conjugated NeutrAvidin. 6. pNPP (ρ-nitrophenyl phosphate). 7. PMSF (phenyl-methylsulfonyl fluoride). 8. EACA, (ε-amino-n-caproic acid). 9. EDTA (ethylenediamine-tetraacetic acid). 10. Pepstatin. 11. Chymostatin. 12. Sulfo-NHS-LC-biotin Hexanoate].
[Sulfosuccinimidyl
13. Surface labeling and isolation kit (Pierce). 14. Serum-free culture medium. 15. Anti-cC1qR (calreticulin).
6-(biotinamido)
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16. Anti-gC1qR. 17. 96-Well ELISA plates. 18. ELISA reader. 19. FPLC(fast-performance liquid chromatography). 20. Mono-Q column. 21. HPLC(high-performance liquid chromatography). 22. TSK-gel DEAE-NPR column.
3 3.1
Methods Cell Growth
1. Seed U937 cells at an initial density of 2 × 105 cells in either multiple 75 cm2, 150 cm2 flasks (or 1 L roller bottles) in RPMI 1640 containing 10 % heat inactivated fetal bovine serum. 2. After 3 days pour the contents onto 50 ml sterile conical test tubes and centrifuge for 10 min, at 800 × g, 4 ºC. 3. Remove supernatants and resuspend the cell pellet from each flask in the same volume of fresh, serum-free culture medium. Transfer to 150 cm2 flasks and incubate overnight (see Note 3). 4. After incubation, remove the flasks from the incubator and place them in BSL-2 safety hood. Remove 1 ml sample from each flask and place into corresponding sterile test tubes containing 100 μl of 0.4 % Trypan blue stain (GIBCO). 5. Mix the cells and dye, and after 1–2 min take 100 μl sample and apply it to a hemocytometer for estimation of cell density and viability. Count the cells (see Note 4). 6. Place the cells from each flask in several 50 ml sterile conical test tubes and centrifuge for 10 min at 800 × g, 4 ºC). 7. Discard the supernatants from each tube and resuspend the cell pellets by adding 5 ml of sterile PBS pH 7.4 containing 10 mM EACA, 10 mM EDTA, 20 mM iodoacetamide, 2 mM PMSF, 1 mM pepstatin, 10 mM chymostatin, and 0.02 % NaN3 to each tube. Pool the cells and centrifuged for an additional 10 min at 800 × g, 4 ºC. 8. Resuspend the final cell pellet in the same buffer so that the final cell density is 1 × 108/ml. If the starting total volume was 1 L, then the final cell yield should be 1,000 ml × 106 or 109 cells. 9. Remove 108 cells for surface labeling with sulfo-NHS-LC-biotin, a membrane impermeable reagent that labels accessible primary amino groups on surface exposed proteins (see Note 5). 10. The rest of the cells are kept frozen at −80 ºC until use.
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3.2 Surface Biotinylation
323
The surface biotinylation procedure described below can be done more efficiently using a commercially available biotinylation and purification kit (e.g., Pierce). The rationale for surface biotinylation is to ensure that the target membrane protein(s) of interest is surface expressed and binds to its target protein once purified. In addition, the surface biotinylated fraction can be mixed with the unlabeled portion and used as a tracer during large-scale receptor purification. 1. Centrifuge the cells for surface labeling for 10 min at 800 × g, 4 ºC, discard the supernatant, and resuspend the pellet in 1 ml of PBS. 2. Biotinylation is initiated by addition of freshly prepared SulfoNHS-LC-biotin to a final concentration of 5 mM, and the reaction is left for 1 h at 4 ºC with gentle tumble mixing. 3. After incubation, wash the labeled cells 3× in cold PBS and after the final wash lyse the cell pellet by addition of solubilization buffer, incubate on ice for 2 h to complete solubilization. 4. Centrifuge the mixture for 1 h at 45,000 × g, carefully collect the supernatant containing the solubilized proteins, and keep the samples frozen (−80 ºC) until needed after testing the efficiency of biotinylation (see below). 5. To test the efficiency of biotinylation by ELISA, coat the wells of the microtiter plate with 100 μl of membrane protein dilutions (1:10, 1:100, and 1:1,000) in Coating buffer for 1 h, 37 ºC. 6. Block for 1 h, 37 ºC with Blocking reagent. 7. Visualize the bound proteins by sequential incubation with alkaline-phosphatase-conjugated NeutrAvidin for 1 h at 37 ºC), followed by incubation with pNPP until color development, which is usually 10–30 min. After the final incubation, the color developed is read on a suitable ELISA reader (see Note 6).
3.3 Preparation of Cell Membranes
1. The frozen cells from Subheading 3.1, step 4 above are quickly thawed and 1 % Emulphogene BC720 and a fresh supply of enzyme inhibitors is added. The mixture is tumbled (1–2 min), and left on ice for 2 h to complete solubilization. 2. Then, the solution is centrifuged for 1 h at 45,000 × g, and the supernatant containing the solubilized proteins collected. The protein concentration is then determined using the detergent compatible BCA (bicinchoninic acid) protein assay. 3. To this unlabeled solubilized protein solution is then added the biotinylated membrane proteins (Subheading 3.2, step 4) and the mixture dialyzed overnight at 4 ºC against 20 mM
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sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene and enzyme inhibitors. 3.4 Purification of C1q Receptors on FPLC Using Mono-Q Column
1. Apply the dialyzed membrane solution from Subheading 3.3, step 3 to FPLC fitted with a 1 ml Mono-Q column that had been equilibrated with the same buffer (10 mM sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene BC720) as described [11]. 2. Wash the column with 3× the column volume buffer (10 mM sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene BC720), and elute the bound proteins with a linear NaCl concentration (0–1,000 mM), and collecting 1 ml fractions. 3. Under these conditions, the C1q binding activity should elute at approximately 450–500 mM NaCl (see Notes 7 and 8).
3.5 Purification of C1q Receptors on HPLC Using DEAE Column Procedure
1. The C1q binding proteins from the Mono-Q column are pooled, dialyzed against 50 mM Tris–HCl, pH 8.0 and then subjected to HPLC purification fitted with a 1 ml TSK-gel DEAE-NPR column that has been equilibrated with the same buffer (50 mM Tris–HCl, pH 8.0). 2. Elute the bound proteins using a 0–500 mM NaCl concentration gradient in equilibrating buffer (50 mM Tris–HCl, pH 8.0) for ~10 min and then a 500–1,000 mM–1 M gradient in 50 mM Tris–HCl, pH 8.0 for an additional 5 min). 3. Test all fractions for C1q activity by the ELISA method described above. 4. Under these conditions, the 60-kDa cC1qR, which is identical to calreticulin, elutes at ~450 nM NaCl, whereas the 33-kDa gC1qR elutes at ~700 nM. 5. Concentrate each receptor fraction by ultrafiltration using a molecular weight cutoff (MWCO) of 3,000–10,000, determine the protein concentration by BCA (bicinchoninic acid) protein assay (Subheading 3.3, step 2), and the purity and chain structure assessed by SDS-PAGE analysis as well as by Western blot analysis using the available anti-cC1qR and antigC1qR antibodies. 6. Aliquot each receptor fraction and keep frozen at −80 ºC.
4
Notes 1. All cell work and handling should be conducted in a sterile BSL-2 safety hood. 2. Bovine serum is more resistant to heat inactivation and therefore must be heated for at least 90 min at 56 ºC.
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3. This step is a precautionary measure to remove residual bovine monomeric or aggregated C1q that may interfere with receptor purification. 4. Since dead or dying cells become leaky, they will take up the dye and will appear dark blue under the microscope. Cell density is estimated as: n × 104/ml, where n = average number of cells counted in four large quadrants. The cell density after 4 days of culture, should reach approximately 1 × 106 and viability in each flask should be ≥95 %. Flasks containing less than 90 % viability should be eliminated. 5. Surface labeling and isolation kit is available from several commercial sources (e.g., Pierce Chem Co, Rockford, IL). 6. Once the efficiency of biotinylation has been established, the biotinylated proteins can be tested by antigen-capture ELISA against C1q to detect the presence of biotinylated C1q binding protein(s) or receptor(s). Alternatively, the proteins captured by C1q can be detected using available antibodies to cC1qR/CR and/or gC1qR. 7. Fractions containing C1q binding activity are then identified by solid phase binding assay (ELISA) using C1q-coated microtiter plates and alkaline-phosphatase-conjugated streptavidin as described above. 8. The C1q-containing fractions are also tested with anti-C1q receptor antibodies to localize the position of each receptor protein. 9. Although the above described methods were those that were used to purify the known receptors, there are now alternative and much simpler approaches to these protocols. Of these affinity purification using immobilized antibodies to the desired receptor should be the most straightforward. Both monoclonal and polyclonal antibodies to cC1qR (Pierce Chem Co) and gC1qR (Abcam) are commercially available. Another approach would be to use immobilized C1q and to elute the bound receptors using NaCl concentration gradient [4]. 10. Interaction of C1q with cell surface molecules induces a diversity of biological responses some of which include stimulation of leukocyte oxidative response [12], suppression of B and T cell proliferation [13], fibroblast and endothelial cell adhesion [8, 14], trophoblast cell migration [15], regulation of dendritic cells [16, 17], angiogenesis [18], and chemotaxis of eosinophils, mast cells, neutrophils, and dendritic cells [19–22]. Another interesting aspect of the C1qRs is that in addition to their cell surface expression, both are also found inside the cell including, in the case of gC1qR, in the mitochondria [23]. Since most of the surface-bound C1q can be internalized, within 30–60 min, it is believed that the receptor bound C1q
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is internalized and then relayed to the intracellular receptors to induce intracellular signaling. Furthermore, both receptors are released into the pericellular milieu, by proliferating, activated, or proinflammatory cells. Because both soluble molecules are capable of activating the classical pathway, it is believed that the secreted molecules may exacerbate or even initiate the local inflammatory process. Thus, functional analysis of receptor function will depend on the availability of the F(ab’2) fragment of inhibitory antibodies or receptor peptides that block the interaction between C1q and the receptor [24].
Acknowledgment This work was supported in part by grants from the National Institutes of Health (R01 AI 060866 and R01 AI-084178). References 1. Cooper NR (1985) The classical complement pathway: activation and regulation of the first complement component. Adv Immunol 37:151–216 2. Schumaker VN, Zavodsky P, Poon PH (1987) Activation of the first component of complement. Annu Rev Immunol 5:21–42 3. Arlaud GJ, Gaboriaud C, Thielens NM, Rossi V, Bersch B, Hernandez J-F, Fontecilla-Camps JC (2001) Structural biology of C1: dissection of a complex molecular machinery. Immunol Rev 180:136–145 4. Ghebrehiwet B, Silvestri L, McDevitt C (1984) Identification of the Raji cell membranederived C1q-inhibitor as a receptor for human C1q. Purification and immunochemical characterization. J Exp Med 160:1375–1389 5. Malhotra R, Reid KBM, Sim RB (1988) Studies on the isolation of C1q-receptor. Biochem Soc Trans 16:735–736 6. Malhotra R, Lu J, Thiel S, Jensenius J-C, Willis AC, Sim RB (1992) C1q receptor (collectin receptor): primary structure, homology, and interaction with ligands. Immunobiology 184:437–443 7. Ghebrehiwet B, Lim B-L, Peerschke EIB, Willis AC, Reid KBM (1994). Isolation cDNA cloning, and overexpression of a 33-kDa cell surface glycoprotein that binds to the globular ‘heads’ of C1q. J Exp Med 179:1809–1821 8. Ghebrehiwet B, Peerschke EIB (2004) cC1qR (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection. Mol Immunol 41:173–183
9. Guo W-X, Ghebrehiwet B, Weksler B, Schweizer K, Peerschke EIB (1999) Upregulation of endothelial cell binding proteins/receptors for complement C1q by inflammatory cytokines. J Lab Clin Med 133:541–550 10. Hjelmeland LM, Chrambach A (1984) Solubilization of functional membrane-bound receptors. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology, vol 1. Alan R Liss, New York 11. Peerschke EIB, Malhotra R, Ghebrehiwet B, Reid KBM, Willis AC, Sim RB (1993) Isolation of human endothelial cell C1q receptor (C1qR). J Leukoc Biol 53:179–184 12. Tenner AJ, Cooper NR (1982) Stimulation of a human polymorphonuclear leukocyte oxidative response by the C1q subunit of the first component of complement. J Immunol 128:2547–2552 13. Ghebrehiwet B, Habicht GS, Beck G (1990) Interaction of C1q with its receptors on cultured cell lines induces an antiproliferative response. Clin Immunol Immunopathol 54:148–160 14. Bordin S, Ghebrehiwet B, Page RC (1990) Participation of C1q and its receptor in adherence of human diploid fibroblasts. J Immunol 145:2520–2526 15. Agostinis C, Bulla R, Tripodo C, Gismondi A, Stabile H, Bossi F, Guarnotta C, Garlanda C, De Seta F, Spessotto P, Santoni A, Ghebrehiwet B, Girardi G, Tedesco F (2010) An alternative role of C1q in cell migration and tissue
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16.
17.
18.
19.
remodelling: contribution to trophoblast invasion and placental development. J Immunol 185:4420–4429 Castellano G, Woltman AM, Schena FP, Roos A, Daha MR, van Kooten C (2004) Dendritic cells and complement: at the crossroad of innate and adaptive immunity. Mol Immunol 41:133–140 Hosszu KK, Santiago-Schwarz F, Peerschke EIB, Ghebrehiwet B (2010) Evidence that a C1q/C1qR system regulates monocytederived dendritic cell differentiation at the interface of innate and acquired immunity. Innate Immun 16:115–127 Bossi F, Rizzi L, Bulla R, Tripodo C, Guarnota C, Novati F, Ghebrehiwet B, Tedesco F (2011) C1q induces in vivo angiogenesis and wound healing. Molec Immunol 48:1676 (abstract) Ghebrehiwet B, Gruber B, Kew RR, Marchese MJ, Peerschke EIB, Reid KBM (1995) Murine mast cells express two types of C1q receptors that are involved in the induction of chemotaxis and chemokinesis. J Immunol 155: 2614–2619
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20. Kuna P, Iyer M, Peerschke EIB, Kaplan AP, Reid KBM, Ghebrehiwet B (1996) Human C1q induces eosinophil migration. Clin Immunol Immunopathol 81:48–54 21. Leigh LE, Ghebrehiwet B, Perera TP, Bird IN, Strong P, Kishore U, Reid KBM, Eggleton P (1998) C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms. Biochem J 330:247–254 22. Vegh Z, Kew R, Gruber B, Ghebrehiwet B (2005) Chemotaxis of human monocyte derived dendritic cells toward C1q is mediated by gC1qR and cC1qR. Mol Immunol 43:1402–1407 23. Dedio J, Jahnen-Dechent W, Bachmann M, Müller-Esterl W (1998) The multi-ligandbinding protein gC1q-R, putative C1q receptor, is a mitochondrial protein. J Immunol 160: 3534–35542 24. Ghebrehiwet B, Hosszu KK, Valentino A, Peerschke EI (2012) The C1q family of proteins: insights into the emerging non-traditional functions. Front Immunol 3; doi:pii:52
Abbreviations cC1q cC1qR CRT gC1q gC1qR
1
The collagen domain of C1q Receptor for cC1q Calreticulin another name for cC1qR The globular heads of C1q Receptor for gC1q
Introduction Human C1q circulates in plasma as part of a multimolecular complex comprising of C1q and a Ca2+-dependent tetramer, C1r2– C1s2. The traditional role of C1q within this complex is to serve as a unit through which C1 recognizes immune complexes or unique patterns on pathogen-associated molecular ligands. This interaction, in turn, is subsequently translated into a highly specific and orderly intramolecular rearrangement resulting in a sequential proteolytic activation—first of C1r and then of C1s—thereby triggering activation of the classical pathway [1–3]. However, information accumulated to date show that the role of C1q is not restricted to recognition of immune complexes or other molecules that activate
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_26, © Springer Science+Business Media New York 2014
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Fig. 1 Chain structure of C1qRs. Purified gC1qR (A) and cC1qR (B) were subjected to SDS-PAGE under reducing conditions
the classical pathway, but is also a key player in a rapidly expanding list of pathophysiological conditions as well as cellular functions, which contribute significantly to a plethora of inflammatory processes. These functions in turn are, by and large, mediated through the interaction of C1q with cell-associated membrane glycoproteins and surface receptors [4–7]. Because C1q is a chimeric protein comprising of a collagenlike region (cC1q) and a globular domain (gC1q), it is capable of interacting with cell surface molecules via either of its two regions. Hence, although we are witnessing an ever-increasing diversity of biologic functions mediated by C1q by virtue of its versatility to bind an increasingly long list of cell-associated molecules, there are presently two, well-characterized surface molecules that bind to either the cC1q or gC1q domains hence designated cC1qR (calreticulin) and gC1qR (p33 or p32), respectively (Fig. 1). Since receptors are involved in the control and modulation of a wide range of cellular processes, their purification from cell membranes will not only help us identify their structure and biochemical composition but also allow us to generate antibodies so that their function, cellular distribution, and structure could be assessed. In this paper, we describe a simple method for the purification of C1q receptors from cell membranes. The two C1q receptors are ubiquitously distributed, and virtually every cell type expresses these receptors, although some cells (e.g., lymphocytes and monocytes) may express more copies per cell than quiescent endothelial cells, which need to be activated by inflammatory cytokines (e.g., LPS, INFγ, and TNFα) [9]. Therefore almost any cell type can be used as a source of C1q receptors and there appears to be no structural difference between receptors isolated from fresh circulating human cells or cultured cell lines. However since a large number of cells is required to generate a reasonable amount of membrane proteins from which quantifiable amount of protein could be recovered, it is more pragmatic to use
C1q Receptors
321
a cultured cell line. We have successfully used both Raji cells, (a B cell line) and U937 (a monocyte-like cell line), to generate cell membrane proteins from which C1qRs were purified [4]. The purpose of purifying membrane proteins is to isolate a specific membrane receptor(s) for functional analysis and biochemical characterization. Therefore, one of the first and fundamental steps to be considered is the choice of a solubilization buffer that will keep the desired receptor in its functional state. This includes the choice of a detergent such as octyl glucoside, CHAPS, Emulphogene BC 720 (polyoxyethylene-10-tridecyl ether), and a suitable buffer medium with phosphate buffer for example being more effective than Tris buffer [10]. In addition, since the breakup of membranes releases enzymes that could potentially digest the very protein of interest, the addition of sufficient cocktail of enzyme inhibitors becomes of paramount importance although even then, one could never be sure that every enzyme released would be completely inhibited. Membrane solubilization buffers as well as cocktail mix of enzyme inhibitors are now commercially available from various sources. The example presented here uses U937 cells to isolate C1q receptors and could be adapted for the purification of other cell surface molecules as well.
2
Materials 1. Dulbecco’s PBS: 0.37 M NaCl, 0.002 M KCl, 0.008 M Na2HPO4, 0.001 M KH2PO4, pH 7.4. 2. Solubilization buffer: PBS-containing enzyme inhibitors and 1 % Emulphogene BC720. 3. Coating buffer: 35 mM NaHCO3, 15 mM Na2CO3, pH 9.6. 4. Blocking reagent: 1 % BSA in Tris-buffered saline (20 mM Tris–HCl, 100 mM NaCl, pH 7.5). 5. Alkaline-phosphatase-conjugated NeutrAvidin. 6. pNPP (ρ-nitrophenyl phosphate). 7. PMSF (phenyl-methylsulfonyl fluoride). 8. EACA, (ε-amino-n-caproic acid). 9. EDTA (ethylenediamine-tetraacetic acid). 10. Pepstatin. 11. Chymostatin. 12. Sulfo-NHS-LC-biotin Hexanoate].
[Sulfosuccinimidyl
13. Surface labeling and isolation kit (Pierce). 14. Serum-free culture medium. 15. Anti-cC1qR (calreticulin).
6-(biotinamido)
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16. Anti-gC1qR. 17. 96-Well ELISA plates. 18. ELISA reader. 19. FPLC(fast-performance liquid chromatography). 20. Mono-Q column. 21. HPLC(high-performance liquid chromatography). 22. TSK-gel DEAE-NPR column.
3 3.1
Methods Cell Growth
1. Seed U937 cells at an initial density of 2 × 105 cells in either multiple 75 cm2, 150 cm2 flasks (or 1 L roller bottles) in RPMI 1640 containing 10 % heat inactivated fetal bovine serum. 2. After 3 days pour the contents onto 50 ml sterile conical test tubes and centrifuge for 10 min, at 800 × g, 4 ºC. 3. Remove supernatants and resuspend the cell pellet from each flask in the same volume of fresh, serum-free culture medium. Transfer to 150 cm2 flasks and incubate overnight (see Note 3). 4. After incubation, remove the flasks from the incubator and place them in BSL-2 safety hood. Remove 1 ml sample from each flask and place into corresponding sterile test tubes containing 100 μl of 0.4 % Trypan blue stain (GIBCO). 5. Mix the cells and dye, and after 1–2 min take 100 μl sample and apply it to a hemocytometer for estimation of cell density and viability. Count the cells (see Note 4). 6. Place the cells from each flask in several 50 ml sterile conical test tubes and centrifuge for 10 min at 800 × g, 4 ºC). 7. Discard the supernatants from each tube and resuspend the cell pellets by adding 5 ml of sterile PBS pH 7.4 containing 10 mM EACA, 10 mM EDTA, 20 mM iodoacetamide, 2 mM PMSF, 1 mM pepstatin, 10 mM chymostatin, and 0.02 % NaN3 to each tube. Pool the cells and centrifuged for an additional 10 min at 800 × g, 4 ºC. 8. Resuspend the final cell pellet in the same buffer so that the final cell density is 1 × 108/ml. If the starting total volume was 1 L, then the final cell yield should be 1,000 ml × 106 or 109 cells. 9. Remove 108 cells for surface labeling with sulfo-NHS-LC-biotin, a membrane impermeable reagent that labels accessible primary amino groups on surface exposed proteins (see Note 5). 10. The rest of the cells are kept frozen at −80 ºC until use.
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3.2 Surface Biotinylation
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The surface biotinylation procedure described below can be done more efficiently using a commercially available biotinylation and purification kit (e.g., Pierce). The rationale for surface biotinylation is to ensure that the target membrane protein(s) of interest is surface expressed and binds to its target protein once purified. In addition, the surface biotinylated fraction can be mixed with the unlabeled portion and used as a tracer during large-scale receptor purification. 1. Centrifuge the cells for surface labeling for 10 min at 800 × g, 4 ºC, discard the supernatant, and resuspend the pellet in 1 ml of PBS. 2. Biotinylation is initiated by addition of freshly prepared SulfoNHS-LC-biotin to a final concentration of 5 mM, and the reaction is left for 1 h at 4 ºC with gentle tumble mixing. 3. After incubation, wash the labeled cells 3× in cold PBS and after the final wash lyse the cell pellet by addition of solubilization buffer, incubate on ice for 2 h to complete solubilization. 4. Centrifuge the mixture for 1 h at 45,000 × g, carefully collect the supernatant containing the solubilized proteins, and keep the samples frozen (−80 ºC) until needed after testing the efficiency of biotinylation (see below). 5. To test the efficiency of biotinylation by ELISA, coat the wells of the microtiter plate with 100 μl of membrane protein dilutions (1:10, 1:100, and 1:1,000) in Coating buffer for 1 h, 37 ºC. 6. Block for 1 h, 37 ºC with Blocking reagent. 7. Visualize the bound proteins by sequential incubation with alkaline-phosphatase-conjugated NeutrAvidin for 1 h at 37 ºC), followed by incubation with pNPP until color development, which is usually 10–30 min. After the final incubation, the color developed is read on a suitable ELISA reader (see Note 6).
3.3 Preparation of Cell Membranes
1. The frozen cells from Subheading 3.1, step 4 above are quickly thawed and 1 % Emulphogene BC720 and a fresh supply of enzyme inhibitors is added. The mixture is tumbled (1–2 min), and left on ice for 2 h to complete solubilization. 2. Then, the solution is centrifuged for 1 h at 45,000 × g, and the supernatant containing the solubilized proteins collected. The protein concentration is then determined using the detergent compatible BCA (bicinchoninic acid) protein assay. 3. To this unlabeled solubilized protein solution is then added the biotinylated membrane proteins (Subheading 3.2, step 4) and the mixture dialyzed overnight at 4 ºC against 20 mM
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sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene and enzyme inhibitors. 3.4 Purification of C1q Receptors on FPLC Using Mono-Q Column
1. Apply the dialyzed membrane solution from Subheading 3.3, step 3 to FPLC fitted with a 1 ml Mono-Q column that had been equilibrated with the same buffer (10 mM sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene BC720) as described [11]. 2. Wash the column with 3× the column volume buffer (10 mM sodium phosphate buffer, pH 7.4 containing 0.1 % Emulphogene BC720), and elute the bound proteins with a linear NaCl concentration (0–1,000 mM), and collecting 1 ml fractions. 3. Under these conditions, the C1q binding activity should elute at approximately 450–500 mM NaCl (see Notes 7 and 8).
3.5 Purification of C1q Receptors on HPLC Using DEAE Column Procedure
1. The C1q binding proteins from the Mono-Q column are pooled, dialyzed against 50 mM Tris–HCl, pH 8.0 and then subjected to HPLC purification fitted with a 1 ml TSK-gel DEAE-NPR column that has been equilibrated with the same buffer (50 mM Tris–HCl, pH 8.0). 2. Elute the bound proteins using a 0–500 mM NaCl concentration gradient in equilibrating buffer (50 mM Tris–HCl, pH 8.0) for ~10 min and then a 500–1,000 mM–1 M gradient in 50 mM Tris–HCl, pH 8.0 for an additional 5 min). 3. Test all fractions for C1q activity by the ELISA method described above. 4. Under these conditions, the 60-kDa cC1qR, which is identical to calreticulin, elutes at ~450 nM NaCl, whereas the 33-kDa gC1qR elutes at ~700 nM. 5. Concentrate each receptor fraction by ultrafiltration using a molecular weight cutoff (MWCO) of 3,000–10,000, determine the protein concentration by BCA (bicinchoninic acid) protein assay (Subheading 3.3, step 2), and the purity and chain structure assessed by SDS-PAGE analysis as well as by Western blot analysis using the available anti-cC1qR and antigC1qR antibodies. 6. Aliquot each receptor fraction and keep frozen at −80 ºC.
4
Notes 1. All cell work and handling should be conducted in a sterile BSL-2 safety hood. 2. Bovine serum is more resistant to heat inactivation and therefore must be heated for at least 90 min at 56 ºC.
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3. This step is a precautionary measure to remove residual bovine monomeric or aggregated C1q that may interfere with receptor purification. 4. Since dead or dying cells become leaky, they will take up the dye and will appear dark blue under the microscope. Cell density is estimated as: n × 104/ml, where n = average number of cells counted in four large quadrants. The cell density after 4 days of culture, should reach approximately 1 × 106 and viability in each flask should be ≥95 %. Flasks containing less than 90 % viability should be eliminated. 5. Surface labeling and isolation kit is available from several commercial sources (e.g., Pierce Chem Co, Rockford, IL). 6. Once the efficiency of biotinylation has been established, the biotinylated proteins can be tested by antigen-capture ELISA against C1q to detect the presence of biotinylated C1q binding protein(s) or receptor(s). Alternatively, the proteins captured by C1q can be detected using available antibodies to cC1qR/CR and/or gC1qR. 7. Fractions containing C1q binding activity are then identified by solid phase binding assay (ELISA) using C1q-coated microtiter plates and alkaline-phosphatase-conjugated streptavidin as described above. 8. The C1q-containing fractions are also tested with anti-C1q receptor antibodies to localize the position of each receptor protein. 9. Although the above described methods were those that were used to purify the known receptors, there are now alternative and much simpler approaches to these protocols. Of these affinity purification using immobilized antibodies to the desired receptor should be the most straightforward. Both monoclonal and polyclonal antibodies to cC1qR (Pierce Chem Co) and gC1qR (Abcam) are commercially available. Another approach would be to use immobilized C1q and to elute the bound receptors using NaCl concentration gradient [4]. 10. Interaction of C1q with cell surface molecules induces a diversity of biological responses some of which include stimulation of leukocyte oxidative response [12], suppression of B and T cell proliferation [13], fibroblast and endothelial cell adhesion [8, 14], trophoblast cell migration [15], regulation of dendritic cells [16, 17], angiogenesis [18], and chemotaxis of eosinophils, mast cells, neutrophils, and dendritic cells [19–22]. Another interesting aspect of the C1qRs is that in addition to their cell surface expression, both are also found inside the cell including, in the case of gC1qR, in the mitochondria [23]. Since most of the surface-bound C1q can be internalized, within 30–60 min, it is believed that the receptor bound C1q
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is internalized and then relayed to the intracellular receptors to induce intracellular signaling. Furthermore, both receptors are released into the pericellular milieu, by proliferating, activated, or proinflammatory cells. Because both soluble molecules are capable of activating the classical pathway, it is believed that the secreted molecules may exacerbate or even initiate the local inflammatory process. Thus, functional analysis of receptor function will depend on the availability of the F(ab’2) fragment of inhibitory antibodies or receptor peptides that block the interaction between C1q and the receptor [24].
Acknowledgment This work was supported in part by grants from the National Institutes of Health (R01 AI 060866 and R01 AI-084178). References 1. Cooper NR (1985) The classical complement pathway: activation and regulation of the first complement component. Adv Immunol 37:151–216 2. Schumaker VN, Zavodsky P, Poon PH (1987) Activation of the first component of complement. Annu Rev Immunol 5:21–42 3. Arlaud GJ, Gaboriaud C, Thielens NM, Rossi V, Bersch B, Hernandez J-F, Fontecilla-Camps JC (2001) Structural biology of C1: dissection of a complex molecular machinery. Immunol Rev 180:136–145 4. Ghebrehiwet B, Silvestri L, McDevitt C (1984) Identification of the Raji cell membranederived C1q-inhibitor as a receptor for human C1q. Purification and immunochemical characterization. J Exp Med 160:1375–1389 5. Malhotra R, Reid KBM, Sim RB (1988) Studies on the isolation of C1q-receptor. Biochem Soc Trans 16:735–736 6. Malhotra R, Lu J, Thiel S, Jensenius J-C, Willis AC, Sim RB (1992) C1q receptor (collectin receptor): primary structure, homology, and interaction with ligands. Immunobiology 184:437–443 7. Ghebrehiwet B, Lim B-L, Peerschke EIB, Willis AC, Reid KBM (1994). Isolation cDNA cloning, and overexpression of a 33-kDa cell surface glycoprotein that binds to the globular ‘heads’ of C1q. J Exp Med 179:1809–1821 8. Ghebrehiwet B, Peerschke EIB (2004) cC1qR (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection. Mol Immunol 41:173–183
9. Guo W-X, Ghebrehiwet B, Weksler B, Schweizer K, Peerschke EIB (1999) Upregulation of endothelial cell binding proteins/receptors for complement C1q by inflammatory cytokines. J Lab Clin Med 133:541–550 10. Hjelmeland LM, Chrambach A (1984) Solubilization of functional membrane-bound receptors. In: Venter JC, Harrison LC (eds) Receptor Biochemistry and Methodology, vol 1. Alan R Liss, New York 11. Peerschke EIB, Malhotra R, Ghebrehiwet B, Reid KBM, Willis AC, Sim RB (1993) Isolation of human endothelial cell C1q receptor (C1qR). J Leukoc Biol 53:179–184 12. Tenner AJ, Cooper NR (1982) Stimulation of a human polymorphonuclear leukocyte oxidative response by the C1q subunit of the first component of complement. J Immunol 128:2547–2552 13. Ghebrehiwet B, Habicht GS, Beck G (1990) Interaction of C1q with its receptors on cultured cell lines induces an antiproliferative response. Clin Immunol Immunopathol 54:148–160 14. Bordin S, Ghebrehiwet B, Page RC (1990) Participation of C1q and its receptor in adherence of human diploid fibroblasts. J Immunol 145:2520–2526 15. Agostinis C, Bulla R, Tripodo C, Gismondi A, Stabile H, Bossi F, Guarnotta C, Garlanda C, De Seta F, Spessotto P, Santoni A, Ghebrehiwet B, Girardi G, Tedesco F (2010) An alternative role of C1q in cell migration and tissue
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remodelling: contribution to trophoblast invasion and placental development. J Immunol 185:4420–4429 Castellano G, Woltman AM, Schena FP, Roos A, Daha MR, van Kooten C (2004) Dendritic cells and complement: at the crossroad of innate and adaptive immunity. Mol Immunol 41:133–140 Hosszu KK, Santiago-Schwarz F, Peerschke EIB, Ghebrehiwet B (2010) Evidence that a C1q/C1qR system regulates monocytederived dendritic cell differentiation at the interface of innate and acquired immunity. Innate Immun 16:115–127 Bossi F, Rizzi L, Bulla R, Tripodo C, Guarnota C, Novati F, Ghebrehiwet B, Tedesco F (2011) C1q induces in vivo angiogenesis and wound healing. Molec Immunol 48:1676 (abstract) Ghebrehiwet B, Gruber B, Kew RR, Marchese MJ, Peerschke EIB, Reid KBM (1995) Murine mast cells express two types of C1q receptors that are involved in the induction of chemotaxis and chemokinesis. J Immunol 155: 2614–2619
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20. Kuna P, Iyer M, Peerschke EIB, Kaplan AP, Reid KBM, Ghebrehiwet B (1996) Human C1q induces eosinophil migration. Clin Immunol Immunopathol 81:48–54 21. Leigh LE, Ghebrehiwet B, Perera TP, Bird IN, Strong P, Kishore U, Reid KBM, Eggleton P (1998) C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms. Biochem J 330:247–254 22. Vegh Z, Kew R, Gruber B, Ghebrehiwet B (2005) Chemotaxis of human monocyte derived dendritic cells toward C1q is mediated by gC1qR and cC1qR. Mol Immunol 43:1402–1407 23. Dedio J, Jahnen-Dechent W, Bachmann M, Müller-Esterl W (1998) The multi-ligandbinding protein gC1q-R, putative C1q receptor, is a mitochondrial protein. J Immunol 160: 3534–35542 24. Ghebrehiwet B, Hosszu KK, Valentino A, Peerschke EI (2012) The C1q family of proteins: insights into the emerging non-traditional functions. Front Immunol 3; doi:pii:52
