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Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 325 (2004) 175–184 www.elsevier.com/locate/yabio
Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins Peter S. Chang,a Afaf Absood,b Jennifer J. Linderman,a and Geneva M. Omannb,c,* a
c
Department of Chemical Engineering, University of Michigan Medical School, Ann Arbor, MI 48109, USA b Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA Department of Biological Chemistry, University of Michigan Medical School and Ann Arbor Veterans Administration Medical Center, Ann Arbor, MI 48105, USA Received 30 May 2003
Abstract A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of Gb2 in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 106 . These methods were coupled with immunodetection methods and an adenosine 50 -diphosphate-ribosylation assay to measure the amount of membrane-associated Gia proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Plasma membranes; G proteins; Magnetic beads; Neutrophils
To study the biochemistry that occurs at the cell plasma membrane, it has been essential to purify plasma membranes free from cytosol and other cytoplasmic membranes. Common isolation methods are differential centrifugation and density sedimentation of whole cell lysates. The first method utilizes sequential centrifugation steps at increasing speeds to selectively pellet membranes of the desired density [1]. The second method utilizes density gradients (e.g., sucrose, percoll), to separate membranes [2,3]. Both methods separate subcellular fractions based on differences in membrane densities. A limitation of these methods is that con* Corresponding author. Fax: 1-734-761-7693. E-mail address: [email protected] (G.M. Omann).
0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.10.039
taminating membranes may be of the same density as that of the plasma membrane. In addition, the recovered membranes are usually quantified as ‘‘membrane protein’’ using standard protein assays, which require amounts of membrane proteins isolated from relatively large numbers of cells (107 –108 ). In this paper, we present a method of isolating neutrophil plasma membranes from cell numbers as low as 106 , not by density, but by a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads. Recovered membranes were quantified by detection of the b subunit of the heterotrimeric guanine nucleotide binding protein after generating a Western blot of the membrane proteins. This membrane markerbased separation yielded highly pure membranes that
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specifically were shown to be free of secretory vesicles, primary granules, and secondary granules that may contaminate plasma membrane prepared by density centrifugation [4,5]. To show its applicability, this assay was used to measure the amount of membrane-associated Gia proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin, a toxin known to ADP-ribosylate Gi proteins, thereby preventing their coupling to receptors.
Materials and methods The overall scheme for plasma membrane isolation and G protein quantification is outlined in Fig. 1. Cells (controls or cells treated in some experimental manner) were disrupted by nitrogen cavitation to yield whole cell cavitate. Cell debris and nuclei were removed by centrifugation. The recovered cell lysate was then incubated with anti-CD15 antibody-conjugated superparamagnetic beads to bind the plasma membrane fraction; cytosol and other intracellular organelles were washed away. From this point on, the isolated membrane proteins were separated by subjecting aliquots of the membrane-bound beads to SDS–PAGE and electrotransfer to polyvinylidene fluoride (PVDF)1 membranes. Then, the PVDF membranes were assayed for plasma membrane marker Gb and Gia subunit. The recovery of Gia subunit was then quantified as a ratio of Gia subunit to Gb to correct for any potential differences in loading of the gel lanes. In cases where the extent of Gia subunit ADP-ribosylation was assessed, membrane-bound beads were treated with activated pertussis toxin (PT) in the presence of [32 P]nicotine adenine dinucleotide (NAD), and then radioactivity in the Gia subunit was quantified after SDS–PAGE. Several control experiments were performed to validate the concentration of beads used, the purity of the plasma membrane preparation, the plasma membrane marker used for membrane quantification, and the completeness of the ADP-ribosylation reaction. The amount of error involved in the quantification scheme was assessed.
Fig. 1. Schematic of plasma membrane isolation and G protein quantification protocol. Cells (controls or cells treated in some experimental manner) were disrupted by nitrogen cavitation to yield plasma membrane fragments. Cell debris and nuclei were removed by centrifugation. The recovered cell lysate was then incubated with antiCD15 antibody-conjugated superparamagnetic beads to bind the plasma membrane fraction; cytosol and other intracellular organelles were washed away. From this point on, membrane proteins were separated by subjecting aliquots of the membrane-bound beads to SDS–PAGE and electrotransfer to PVDF membranes that were assayed for plasma membrane marker and Gia subunit. The recovery of Gia subunit was then quantified as a ratio of Gia subunit to membrane marker to correct for any potential differences in loading of the gel lanes. In cases where the extent of Gia subunit ADP-riboyslation was assessed, membrane-bound beads were treated with activated pertussis toxin in the presence of [32 P]NADþ ; then radioactivity in the Gia subunit was quantified after SDS–PAGE. Several control experiments were performed (boxed information).
Materials Pertussis toxin was obtained from List Biologicals (Campbell, CA) and 50 protease inhibitor cocktail
1
Abbreviations used: PIC, protease inhibitor cocktail; MGB, modified GeyÕs buffer; CHO-MLF, N-formyl-methionyl-leucyl-phenylalanine; PT, pertussis toxin; FITC, fluorescein isothiocynate; NAD, nicotine adenine dinucleotide; PVDF, polyvinylidene fluoride; ECL+, enhanced chemiluminescence plus; DTT, D L -dithiothreitol; PM, plasma membrane; BCA, bicinchoninic acid; ELISA, enzyme-linked immunosorbent assay.
(PIC) was obtained from BD Biosciences (San Diego, CA). The superparamagnetic beads (Dynabeads M450) with or without anti-CD15 antibody were obtained from Dynal (Lake Success, NY). Antibodies used for flow cytometry were obtained as follows: mouse fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (clone 3G8) and anti-CD45 (clone BRA-55) antibodies were from Sigma Chemical Co. (St. Louis, MO); mouse phycoerythrin-conjugated anti-CD54 (clone HA58) and FITC-conjugated anti-CD58 (clone 1C3) and anti-CD44
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(clone L178) antibodies were from BD Biosciences. Antibodies utilized for Western blot analyses were obtained as follows: mouse anti-CD45 antibody (clone 69) was from BD Biosciences; rabbit antiserum for Gia1;2 (clone AS/7) was from Perkin–Elmer Life Sciences (Boston, MA) or from Calbiochem (San Diego, CA); rabbit antiserum for myeloperoxidase was from Rockland Inc. (Gilbertsville, PA); rabbit polyclonal antibody for tetranectin was from DAKO (Carpinteria, CA); rabbit polyclonal antibody for Gb was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Secondary antibodies against mouse and rabbit conjugated with horse radish peroxidase were from Jackson Immunological (West Grove, PA). The [32 P]NAD was obtained from Perkin–Elmer Life Sciences. The enhanced chemiluminescence plus (ECL+) was from Amersham (Oslo, Norway). The Quantikine MMP-8 Immunoassay kit was obtained from R&D Systems, Inc. (Minneapolis, MN). The Micro bicinchoninic acid (BCA) assay kit, the PAGEprep kit, and the Restore Western blot stripping buffer were from Pierce Biotechnology (Rockford, IL). All other chemicals were obtained from Sigma Chemical Co. Neutrophil isolation Neutrophils were isolated from whole blood by the method described in Tolley et al. [6]. Human neutrophils were partially purified from red cells by gelatin sedimentation and then further purified to >98% by counterflow elutriation. Purified neutrophils were resuspended in modified GeyÕs buffer (MGB) that consisted of 5 mM KCl, 147 mM NaCl, 1.9 mM KH2 PO4 , 0.22 mM Na2 HPO4 , 5.5 mM glucose, 0.3 mM MgSO4 , 1 mM MgCl2 , and 10 mM Hepes, pH 7.4. Identification of a suitable plasma membrane marker for plasma membrane quantification Normally, proteins quantified from bands on a Western blot are normalized by the total protein loaded in the lane. The protein quantification is typically done with a standard protein assay such as a BCA assay or a Lowry assay. Because in our protocol the protein assays would also quantify the antibody used to isolate the plasma membrane, accurate quantification of the plasma membrane sample would not be possible. To circumvent this limitation, we sought a plasma membrane marker that could also be quantified by Western immunoblotting; in this case, the expression of the plasma membrane marker must not change under different experimental conditions of interest. N-Formyl-methionylleucyl-phenylalanine (CHO-MLF) stimulation and PT treatment were two experimental conditions of interest, and thus a plasma membrane marker that did not change under these conditions was needed. The plasma
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membrane proteins CD16, CD44, CD45, CD54, CD58, and Gb were tested for this purpose. First, intact neutrophils were stimulated with CHOMLF to assess the impact of stimulation on expression of plasma membrane markers. Neutrophils were incubated in MGB plus 1.5 mM Ca2þ at 2 106 cells/mL for 10 min at 37 °C. Then, CHO-MLF was added to a final concentration of 1 lM. After a 15-min incubation, cells were put on ice and the amount of plasma membrane marker was measured as described below. Second, intact neutrophils were treated with PT, which ADP-ribosylates the a subunit of Gi proteins, to determine the effect of such treatment on plasma membrane marker expression. Neutrophils were suspended in Krebs–Ringer buffer plus cytochrome c (6.3 mg/mL) at 5 107 cells/mL. PT was added at concentrations of 0, 0.5, 1.0, and 2.5 lg/mL, and samples were incubated at 37 °C for 120 min with gentle mixing. After incubation, cold MGB (4 °C) was added to stop PT action. The suspension was filtered through a nylon mesh to remove aggregates. Then, plasma membrane marker expression was quantified as described below. Two strategies for quantifying the amount of plasma membrane marker after CHO-MLF stimulation or PT treatment were used, one for membrane proteins with extracellular epitopes and one for intracellular membrane proteins. For the markers that have an extracellular epitope (CD16, CD44, CD45, CD54, CD58), flow cytometric assays were used to measure their expression. After either treatment condition, the cells 4 107 cells/ mL) were centrifuged and resuspended in MGB at 108 cells/mL. Antibody against the marker conjugated to FITC was added at a dilution of 1:5 in the cell suspension and incubated in the dark for 30 min at 4 °C. Cells were then washed and resuspended in MGB at a concentration of 4 106 cells/mL. Finally, cells were analyzed on a Becton–Dickinson FACScan flow cytometer, and fluorescent antibody binding was quantified as mean channel number of 5000 cells. Expression of CD16, CD44, CD45, CD54, and CD58 did not change with PT treatment. Expression of CD16 and CD44 changed with CHOMLF stimulation, but CD45, CD54, and CD58 remained unchanged, consistent with previous results [7]. Unfortunately, a search for suitable Western blotting antibodies for CD45, CD54, and CD58 yielded no antibodies that were sensitive enough to detect their presence in plasma membrane fractions from 106 cells. We note that although this approach did not suit our purpose, it is generally applicable to cell systems where there are sensitive antibodies available for flow cytometric assessment of changes (or lack thereof) in marker expression and quantification on Western blots. For our purposes, a second approach was necessary. To assess the feasibility of utilizing an intracellular plasma membrane marker (which could not easily be quantified by flow cytometry) such as Gb , large numbers
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of cells 108 ) were either stimulated with CHO-MLF or treated with PT. From these cells, plasma membrane was then isolated by differential centrifugation so that the total plasma membrane recovered could be measured with a standard protein assay. The plasma membrane marker was quantified by Western immunoblotting after separation of proteins by SDS–PAGE and normalized to total protein loaded per lane. This is a commonly utilized approach to quantifying changes in specific proteins in the membrane. However, it does assume that changes (if they occur) in the plasma membrane marker are highly specific and not coincident with a general change in total plasma membrane protein. For this protocol, a plasma membrane fraction was isolated by differential centrifugation following the method of Suchard and Mansfield [7]. Briefly, 3 108 neutrophils were suspended in SHE buffer (10 mM Hepes, pH 7.3, 0.1 M sucrose, 1 mM EGTA) with a 1/50 dilution of 50 PIC and disrupted by nitrogen cavitation [1]. The cavitate was then centrifuged for 10 min at 4 °C and 500g (1600 rpm in a Sorvall RT7 centrifuge) to remove intact cells and nuclei. The recovered supernatant was centrifuged in the medium-speed J2-MI Beckman centrifuge for 15 min at 4 °C and 8000g (8130 rpm in JA20 rotor) to remove granules. Finally, the plasma membrane fractions were isolated by centrifuging the supernatant from the previous step at 200,000g in an ultracentrifuge (34,200 rpm in 40 Ti rotor) for 30 min at 4 °C. The final supernatant was discarded and the resulting pellet was homogenized in 1 mL of SHE buffer and stored at –70 °C. A micro BCA protein assay was used to quantify protein, and Western blots were assayed to quantify the corresponding Gb in a given sample. Plasma membrane fraction isolation using CD15 antibody-conjugated beads Disruption of cells and generation of cell cavitate were performed using nitrogen cavitiation as described by Tolley et al. [6]. This method was chosen because it gives efficient lysis of the plasma membrane without disruption of intracellular organelle membranes [1,3], a point to which we will return in the discussion. Neutrophils were suspended (107 cells/mL) in homogenization buffer (0.34 M sucrose, 10 mM Hepes, 1 mM EDTA (disodium salt), and 2.5 mM MgCl2 ) with the addition of 1:50 dilution of 50 PIC. Neutrophils were then placed in a cell disruption bomb (Parr Instruments) and pressurized at 450 psi for 15 min. The pressure was slowly released, and the slurry of cellular debris and undisrupted whole cells (cell cavitate) was collected. The resulting cavitate was centrifuged at 600g for 10 min to remove unlysed cells and nuclei. Finally, the supernatant (cell lysate) was recovered.
Superparamagnetic Dynabeads with anti-CD15 antibodies were used to isolate plasma membrane fractions from the cell lysates. CD15 was chosen as the capture antigen because it is a cell surface marker that is found on neutrophils. It is a branched pentasacchaaride present on a subset of the LFA-1/HMAC-1/gp 150,95 glycoprotein family, CR1, CR3, and NCA-160 [8,9]. In addition to being on the plasma membrane, CD15 has been detected primarily in secondary/specific granules [10,11]. The concentration of beads added to aliquots of the cell lysate (107 cell equivalents/mL) was varied to determine the optimal ratio of beads to whole cell equivalents for efficient recovery of plasma membrane. Varying amounts of beads were added to microcentrifuge tubes containing 107 cell equivalents of cell lysate. The samples were incubated with agitation at 4 °C for 30 min and then placed in a holder with a magnet (Dynal) positioned near the bottom of the microcentrifuge tube. The magnet aggregated the beads near the bottom of the tubes such that the supernatants were easily decanted. Samples were removed from the magnet and the beads were resuspended in phosphate-buffered saline with bovine serum albumin (1 mg/mL). This washing step was performed twice to wash the beads, washing out the unattached proteins and organelles and leaving behind plasma membrane vesicles attached to the beads. In some experiments, the supernatant after the first wash step was incubated with beads again to determine whether more membrane could be recovered. As an additional control, nitrogen cavitate was incubated with beads without the anti-CD15 antibody. Neutrophil collagenase ELISA Lack of contamination by secondary/specific granules in the plasma membrane fractions was demonstrated by ELISA of neutrophil collagenase (MMP-8), a marker for secondary/specific granules [12], using the Quantikine MMP-8 immunoassay kit according to the manufacturerÕs directions. Briefly, samples (106 cell equivalents) and standards were aliquoted and incubated in 96-well plates for 2 h with agitation at room temperature. Wells were washed five times with the wash buffer. Detection antibody was added and the plates were incubated with agitation at room temperature. The wells were washed again and substrate was added before measuring absorbance on a plate reader. Protein blotting and phosphor screen detection For quantification of membrane recovery, quantification of Gia subunits, and verification of membrane purity, aliquots (106 cell equivalents) of the beadbound membrane suspension were boiled in Laemmli sample buffer [13] to denature proteins and dissociate
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membranes and proteins from the beads. The beads were removed by centrifugation and a measured aliquot of the supernatant was subjected to SDS–PAGE, based on the procedures developed by Laemmli [13], and transferred to PVDF membranes [15]. Membranes were probed with anti-Gia , anti-Gb , anti-tetranectin, or antimyeloperoxidase antibodies at dilutions of 1:1000, 1:1000, 1:100, or 1:5000, respectively. The membranes were incubated with either anti-mouse or anti-rabbit antibodies (depending on the host of the antibody as noted under Materials) conjugated with horseradish peroxidase. Then, the membranes were exposed with ECL+ as per manufacturerÕs instructions and the resulting fluorescent bands were quantified with a Molecular Dynamics Storm 860 imager. After probing for Gia , membranes were stripped by incubating the membranes in Restore Western blotting stripping buffer for 30 min at 37 °C. Then, the membranes were reprobed with antibody to Gb . Thus, potential differences in loading of samples in the gel lanes was accounted for by normalizing the Gia subunit signal to the Gb signal in the same lane. [32 P]ADP-ribosylation membranes
of
Gia
proteins
in
plasma
To determine the level of ADP-ribosylation of Gia proteins in the purified plasma membrane fragments, as a result of either endogenous enzymes or prior PT treatment of intact cells, purified membranes were treated with activated PT and [32 P]NAD. In cases where ADPribosylation occurred before cell disruption, only Gia proteins not previously ADP-ribosylated would become radiolabeled in the second ADP-ribosylation reaction. Control cells without prior ADP-ribosylation provided a measure of maximal obtainable radiolabeling. Magnetic beads with attached plasma membrane fractions were suspended in 200 lL of reaction mixture (110.6 lL PT cocktail, 60 lL activated PT (100 lg/mL), 20 lL 100 mM D L -dithiothreitol (DTT), 7 lL 5% (w%) Luberol, 4 lL 50 PIC, 0.06 lCi/lL [32 P]NAD), and incubated at 37 °C for 30 min [16,17]. The PT cocktail was made by mixing 2 mL of L -a-phosphatidylcholine, dimyristoyl (2.76 mg/mL in deionized water, sonicated for 20 min) and 7 mL of a solution of 180 mM Tris, 18 mM thymidine, 4.5 mM MgCl2 -6H2 O, 1.8 mM NaEDTA, 18 mM L -arginine, 180 lM GTP, and 1.8 mM ATP [16,17]. Activated PT (100 lg/mL) was prepared by a 1:1 dilution of a PT solution (200 lg/mL in sterile 0.1 M NaPO4 and 0.5 M NaCl) with an aqueous solution of 50 mM Tris–HCl and 100 mM DTT and incubating this mixture for 30 min at 37 °C [17,18]. Then, the protein in the mixture was concentrated using PAGEprep kit. bMercaptoethanol was added to a concentration of 5%, and the protein solution was boiled for 5 min at 100 °C before conducting SDS–PAGE and electrotransfer to
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PVDF membranes as described above. When appropriate, membranes were probed with anti-Gia and anti-Gb antibodies as described above. Furthermore, blots with 32 P-labeled proteins were exposed to a phosphor screen overnight and quantified with a Molecular Dynamics Storm 860 imager. Effect of neutrophil treatment conditions on Gia subunit membrane expression and ADP-ribosylation Two treatment conditions were evaluated for their effects on Gia ADP-ribosylation, activation with N-formyl peptide and pretreatment with pertussis toxin. Intact neutrophils were treated for 15 min with CHO-MLF or 2 h with PT as described above for identification of suitable membrane markers. After each treatment, cells were subjected to the membrane isolation and G protein quantification protocol as outlined in Fig. 1. Statistical analyses Statistical comparison of data was performed using Instat software from GraphPad Software (San Diego, CA) with the Student t test. A p value less than 0.05 was considered significant.
Results Validation of membrane markers for quantifying membrane recovery Gb was found to be a marker suitable for quantification of plasma membrane recovery. Sensitive antibodies were readily available for quantification of Gb on Western blots. Pretreatment of neutrophils for 15 min with CHO-MLF or 2 h with PT (Fig. 2) did not change (p > 0:05) the expression level of Gb in plasma membrane fractions isolated by differential centrifugation and quantified as total membrane protein. Thus Gb was utilized as a plasma membrane marker for quantification of membranes recovered by the superparamagnetic bead protocol. Optimization of the magnetic bead separation step To optimize the concentration of beads needed to maximize plasma membrane extraction, the concentration of beads was varied while maintaining the cell equivalent concentration at 107 /mL, and the amount of recovered G protein was measured. Saturation of plasma membrane G protein recovery was achieved at 5 107 beads/mL (Fig. 3A), which represented a bead to cell equivalent ratio of 50:1. The surface area of a bead (based on a diameter of 4.5lm, given by the manufacturer) is approximately 60 lm2 , whereas the
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Fig. 3. Efficiency of plasma membrane recovery by magnetic beads. (A) Cell lysate was incubated with a range of bead concentrations and recovered membrane proteins were separated by SDS–PAGE as described under Materials and methods. Membrane Gia was quantified after Western blot analysis and plotted as a percentage of the total Gia in the whole cell lysate. Each data point represents the average of three experiments. The error bars represent the standard error of the mean. (B) Cell lysate was incubated with two successive aliquots of magnetic beads. The beads were boiled in sample buffer and the samples subjected to SDS–PAGE and immunoblot analysis. The plasma membrane (detected as Gia in Western blots) was recovered from beads in the first incubation (lane 1) with undetectable levels of plasma membrane on the beads from the second incubation (lane 2).
Fig. 2. Validation of Gb as a plasma membrane marker. Cells were treated with or without PT or with and without CHO-MLF as described under Materials and methods. Plasma membranes were isolated by differential centrifugation and quantified as membrane protein using the BCA assay. Samples were subjected to SDS–PAGE and Gb was measured by immunoblotting as described under Materials and methods. Amount of Gb was normalized to the amount of protein loaded per lane. Data presented are the mean and standard error of the mean for three experiments. Control and treated samples did not differ by StudentÕs t test (p > 0:05).
surface area of a neutrophil is approximately 500 lm2 [19]. Thus the surface area of the beads must exceed the surface area of the cell equivalents by a factor of 8. This may be related to the surface density of the antibody on the beads, however, that information was not available from the manufacturer. The maximum membrane G protein recovered was approximately 55% of the total cellular G protein. This is consistent with the measurements of Bokoch et al. [20] and Keil et al. [21], who reported that 67–75% and 25%, respectively, of the total neutrophil Gia protein is at the plasma membrane.
To further confirm that the bead concentration was optimized at a ratio of 50 beads per cell equivalent, the supernatant recovered from a first bead incubation was treated a second time with beads under analogous conditions to see whether any more G proteins could be extracted. The Western blots comparing G protein bands from the first and second incubation of sample with beads showed that a negligible amount of G protein was recovered by beads in the second incubation (Fig. 3B), thus 50 beads per cell equivalent was sufficient to recover the plasma membrane fragments. To confirm specificity of plasma membrane binding to beads, nitrogen cavitate was incubated with beads with and without anti-CD15 antibody. Western blot analysis showed negligible amounts of G protein recovered by the naked beads compared to the beads with anti-CD15 antibody (Fig. 4), confirming that there was little nonspecific binding of proteins to beads. Validation of membrane purity The purity of the isolated plasma membrane fragments was determined by comparing the expression of tetranectin, myeloperoxidase, and neutrophil collagenase,
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Fig. 4. Specificity of magnetic bead isolation of plasma membrane fractions. Nitrogen cavitate was incubated with beads without antiCD15 antibody (NS) or with anti-CD15 antibody (SP). The beads were then boiled in sample buffer and the samples subjected to SDS–PAGE and immunoblot analysis. Recovered membrane was quantified as Gia . Plasma membrane was recovered only from beads coated with antiCD15 antibody.
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endogeneous enzymes or by prior treatment of cells with pertussis toxin, isolated plasma membrane fractions adherent to magnetic beads were incubated with activated PT and [32 P]NAD following procedures described under Materials and methods. The time for incubation of this reaction was varied to determine the incubation time required to label all of the available G proteins. The PM fractions were incubated for 30 or 60 min to bracket the range of times typically utilized for this reaction [16–18]. Proteins of these samples were separated by SDS– PAGE, and the radioactivity in the Gi protein bands was quantified. Membranes incubated for 30 and 60 min exhibited the same levels of ADP-ribosylation of Gia (data not shown). Therefore, a 30 min incubation was deemed sufficient for the reaction to reach completion. Calculation of errors in quantifying total Gia and Gia available for signaling
Fig. 5. Lack of contamination of plasma membranes with secretory vesicles (tetranectin) and primary granules (myeloperoxidase). Equal cell equivalents of whole cell lysate (A), nitrogen cavitate (B1, B2), and plasma membrane isolated using magnetic beads (C1, C2) were assayed for the presence of tetranectin and myeloperoxidase by immunoblot analysis. Whereas whole cell lysate and nitrogen cavitate contained myeloperoxidase and tetranectin, the plasma membrane contained insignificant levels of each.
markers for secretory vesicles, azurophil/primary granules, and specific/secondary granules, respectively [12]. Secretory vesicles have been implicated as contaminants in plasma membrane isolates obtained by density sedimentation methods [4,5]. Azurophil and specific granules are reported to contain CD15 [10,11]. Although the epitopes for antibody binding were expected to be on the inside of these granules, it was important to confirm that these granules did not bind to the beads. Strong signals were seen in the bands for tetranectin and myeloperoxidase in the cell lysate and N2 cavitate, but unquantifiable levels were found in the purified plasma membrane fragments, given equal number of cell equivalents loaded in each lane of the gel (Fig. 5). Quantitative ELISAs of whole cell lysate, N2 cavitate, and plasma membrane fractions showed that only 3.8% of the neutrophil collegenase in N2 cavitate was in the plasma membrane fractions. Therefore, magnetic bead isolation yielded highly purified plasma membranes excluding both intracellular membrane vesicles of density similar to that of the plasma membrane and intracellular vesicles containing CD15 on the internal side of the granule membrane. Optimization of protocols for quantifying G proteins available for signaling To quantify Gi proteins available for signaling, i.e., Gi proteins that have not been ADP-ribosylated either by
Plasma membranes were isolated and incubated with PT and radiolabeled NAD, and proteins were separated by SDS–PAGE as outlined in Fig. 1. Each sample was run in triplicate gel lanes, and the optical densities of Gia , Gb , and [32 P]Gia were measured. Then, the measurements for Gia and [32 P]Gia bands were divided by the Gb measurements. The standard error of the mean for these ratios was calculated to be 20%. Application of the plasma membrane isolation and Gi protein quantification protocols The assay was used to investigate aspects of signal transduction that may be important in influencing neutrophil responses. Preincubation of neutrophils with PT is known to inhibit nearly all responses induced by NFormyl peptides by ADP-ribosylating Gi proteins, making them unavailable for coupling to receptors [22]. As a positive control for our protocols, neutrophils were pretreated with increasing concentrations of PT, and then plasma membranes isolated and total Gia and Gia available for signaling (i.e., Gia not ADP-ribosylated in the first incubation with PT) were quantified according to the scheme in Fig. 1. As expected, as the concentration of PT in the pretreatment was increased, the amount of G proteins available for signaling decreased in a statistically significant manner although the total Gia associated with the plasma membrane did not change (Fig. 6). Thus, increased PT treatment increased the ADP-ribosylation of Gi protein on the plasma membrane without altering the membrane association of the Gi protein. To measure the influence of chemoattractant-induced activation of neutrophils on plasma membrane-associated total Gia and Gia available for signaling, neutrophils were incubated with and without 1 lM CHO-MLF for 15 min as described under Materials and methods. Plasma membranes were isolated and total Gia and Gia
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Fig. 6. Effect of PT treatment on neutrophil plasma membrane Gia . Neutrophils were treated with a range of PT concentrations as indicated. Plasma membrane fractions were isolated and Gia available for signaling (A) and total Gia (B) were quantified and normalized to plasma membrane (detected as Gb ) loaded per lane according to the protocol in Fig. 1. Data are averaged for three experiments with error bars showing the standard error of the mean. Asterisks indicate values statistically different from the value for PT ¼ 0.
available for signaling were quantified by the procedures outlined in Fig. 1. Comparison of stimulated and control cells showed that stimulation decreased both the total Gia and the Gia available for signaling by approximately 50% (Fig. 7). Thus activation appears to cause release of the Gia subunit from the plasma membrane without the occurrence of endogenous ADPribosylation.
Discussion A method that allows quick isolation of plasma membranes from neutrophils yielding highly pure plas-
Fig. 7. Effect of CHO-MLF treatment on neutrophil plasma membrane Gia . Neutrophils were incubated with or without 1 lM CHOMLF. Plasma membrane fractions were isolated and Gia available for signaling (A) and total Gia (B) were quantified and normalized to plasma membrane (detected as Gb ) loaded per lane according to the protocol in Fig. 1. Data are averaged for three experiments with error bars showing the standard error of the mean. Asterisks indicate values statistically different from the value for the control.
ma membrane fractions from small numbers of cells has been devised. The key to this high purity is the specific isolation of the plasma membrane fragments by antibody-conjugated superparamagnetic beads. Because nitrogen cavitation lyses cell plasma membranes without bursting intracellular organelles, nitrogen cavitation has been used quite frequently to lyse neutrophils [1,23]. For the purposes of this beadisolation protocol, it is of importance that the intracellular organelles are not lysed, since normal processing of surface receptors (such as CD15 [9]) involves processing through intracellular vesicular compartments. In the intact vesicle the CD15 epitope is on the inside of the vesicle and therefore not accessible for binding to the magnetic beads, whereas the plasma membrane fraction
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produced by nitrogen cavitation is characterized by small closed vesicles with the right side (CD15 epitope) out [1]. This allowed for the recovery of purified plasma membranes without contaminating intracellular organelles. The ability of antibody-conjugated beads to recover all of the plasma membrane is dependent upon the epitope being relatively evenly distributed on the plasma membrane. This appeared to be the case for CD15, since experimental data indicated that the bulk of the plasma membrane was recovered. That is, the work of Bokoch et al [20] indicated that 50% of the total Gia2 protein is on the plasma membrane and the anti-CD15-conjugated beads recovered approximately 50% of the total cellular Gia2 protein (Fig. 3). However, it is well established that membrane proteins and lipids can be heterogeneously distributed in the plasma membrane [24–29]. The utility of the antibody-conjugated bead approach for isolating all the plasma membrane then becomes dependent upon the relative sizes of the plasma membrane vesicles (generated by nitrogen cavitation), the subdomains in which the recovery epitope resides, and the distribution of the subdomains. If the subdomains are relatively small compared to the plasma membrane vesicles and the subdomains are relatively randomly distributed, then all of the plasma membrane could be recovered by the beads. At the other extreme, where the subdomains are large or comparable in size to the plasma membrane vesicle surface area, the plasma membrane vesicles may segregate into distinct populations of vesicles with epitope and without. In this case, the antibody-bead isolation technique may be useful for obtaining plasma membrane fractions enriched in a particular subdomain. One other important aspect of this assay is the use of a membrane marker to quantify plasma membrane recovery. Commonly, membrane recovery is detected as the total membrane protein using a protein assay such as the bicinchononic acid assay. However, this assay cannot be used with our method because of the abundance of antiCD15 antibody conjugated onto the beads. An attempt to wash the membranes off of the beads would add an additional cumbersome step. As an alternative approach, we utilized a plasma membrane marker whose expression did not change under the experimental conditions used and for which antibodies for Western blot analysis were available. Any suitable plasma membrane marker must not change with relevant experimental situations. This is an important point since several cell surface antigens have been shown to vary with some experimental treatments [30,31]. For this study, Gb expression on intact cells did not change with changes in experimental conditions of interest, and thus relative membrane recovery was measured by immunodetection of Gb after separating membrane proteins by SDS–PAGE. Application of the newly developed protocol confirms, as expected, that incubation of intact neutrophils
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with increasing concentrations of PT causes increasing levels of ADP-ribosylated Gia (with a corresponding decrease in the number of Gia available for signaling). However, PT treatment does not change the level of total Gia associated with the plasma membrane. Stimulation of neutrophils with N-formyl peptides causes both the total Gia and the Gia available for signaling to drop by approximately 50%. These data suggest the hypothesis that desensitization of neutrophil responses could be related to a decrease in the amount of total Gia proteins at the plasma membrane. Although nitric oxide-induced endogeneous ADP-ribosylation of G proteins has been observed in a few mammalian cell types [14,32], our data provide no evidence for CHO-MLFinduced endogenous ADP-ribosylation in neutrophils. In summary, we have designed a protocol to isolate neutrophil plasma membrane fractions utilizing a capture antibody to a cell surface antigen. This general approach is applicable to other cell types for which an appropriate cell surface capture antigen is characterized. Additionally, other intracellular organelles could be isolated by this approach if an antibody is available to an antigen on the surface of the organelle (i.e., on its cytoplasmic side). The protocol is significantly faster than standard density sedimentation procedures.
Acknowledgments The authors thank Michael L. Keil for technical assistance. This work was supported by National Science Foundation Grant BES-9713856 and the Office of Research and Development, Medical Research Service, Department of Veteran Affairs.
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Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins Peter S. Chang,a Afaf Absood,b Jennifer J. Linderman,a and Geneva M. Omannb,c,* a
c
Department of Chemical Engineering, University of Michigan Medical School, Ann Arbor, MI 48109, USA b Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA Department of Biological Chemistry, University of Michigan Medical School and Ann Arbor Veterans Administration Medical Center, Ann Arbor, MI 48105, USA Received 30 May 2003
Abstract A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of Gb2 in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 106 . These methods were coupled with immunodetection methods and an adenosine 50 -diphosphate-ribosylation assay to measure the amount of membrane-associated Gia proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Plasma membranes; G proteins; Magnetic beads; Neutrophils
To study the biochemistry that occurs at the cell plasma membrane, it has been essential to purify plasma membranes free from cytosol and other cytoplasmic membranes. Common isolation methods are differential centrifugation and density sedimentation of whole cell lysates. The first method utilizes sequential centrifugation steps at increasing speeds to selectively pellet membranes of the desired density [1]. The second method utilizes density gradients (e.g., sucrose, percoll), to separate membranes [2,3]. Both methods separate subcellular fractions based on differences in membrane densities. A limitation of these methods is that con* Corresponding author. Fax: 1-734-761-7693. E-mail address: [email protected] (G.M. Omann).
0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.10.039
taminating membranes may be of the same density as that of the plasma membrane. In addition, the recovered membranes are usually quantified as ‘‘membrane protein’’ using standard protein assays, which require amounts of membrane proteins isolated from relatively large numbers of cells (107 –108 ). In this paper, we present a method of isolating neutrophil plasma membranes from cell numbers as low as 106 , not by density, but by a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads. Recovered membranes were quantified by detection of the b subunit of the heterotrimeric guanine nucleotide binding protein after generating a Western blot of the membrane proteins. This membrane markerbased separation yielded highly pure membranes that
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specifically were shown to be free of secretory vesicles, primary granules, and secondary granules that may contaminate plasma membrane prepared by density centrifugation [4,5]. To show its applicability, this assay was used to measure the amount of membrane-associated Gia proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin, a toxin known to ADP-ribosylate Gi proteins, thereby preventing their coupling to receptors.
Materials and methods The overall scheme for plasma membrane isolation and G protein quantification is outlined in Fig. 1. Cells (controls or cells treated in some experimental manner) were disrupted by nitrogen cavitation to yield whole cell cavitate. Cell debris and nuclei were removed by centrifugation. The recovered cell lysate was then incubated with anti-CD15 antibody-conjugated superparamagnetic beads to bind the plasma membrane fraction; cytosol and other intracellular organelles were washed away. From this point on, the isolated membrane proteins were separated by subjecting aliquots of the membrane-bound beads to SDS–PAGE and electrotransfer to polyvinylidene fluoride (PVDF)1 membranes. Then, the PVDF membranes were assayed for plasma membrane marker Gb and Gia subunit. The recovery of Gia subunit was then quantified as a ratio of Gia subunit to Gb to correct for any potential differences in loading of the gel lanes. In cases where the extent of Gia subunit ADP-ribosylation was assessed, membrane-bound beads were treated with activated pertussis toxin (PT) in the presence of [32 P]nicotine adenine dinucleotide (NAD), and then radioactivity in the Gia subunit was quantified after SDS–PAGE. Several control experiments were performed to validate the concentration of beads used, the purity of the plasma membrane preparation, the plasma membrane marker used for membrane quantification, and the completeness of the ADP-ribosylation reaction. The amount of error involved in the quantification scheme was assessed.
Fig. 1. Schematic of plasma membrane isolation and G protein quantification protocol. Cells (controls or cells treated in some experimental manner) were disrupted by nitrogen cavitation to yield plasma membrane fragments. Cell debris and nuclei were removed by centrifugation. The recovered cell lysate was then incubated with antiCD15 antibody-conjugated superparamagnetic beads to bind the plasma membrane fraction; cytosol and other intracellular organelles were washed away. From this point on, membrane proteins were separated by subjecting aliquots of the membrane-bound beads to SDS–PAGE and electrotransfer to PVDF membranes that were assayed for plasma membrane marker and Gia subunit. The recovery of Gia subunit was then quantified as a ratio of Gia subunit to membrane marker to correct for any potential differences in loading of the gel lanes. In cases where the extent of Gia subunit ADP-riboyslation was assessed, membrane-bound beads were treated with activated pertussis toxin in the presence of [32 P]NADþ ; then radioactivity in the Gia subunit was quantified after SDS–PAGE. Several control experiments were performed (boxed information).
Materials Pertussis toxin was obtained from List Biologicals (Campbell, CA) and 50 protease inhibitor cocktail
1
Abbreviations used: PIC, protease inhibitor cocktail; MGB, modified GeyÕs buffer; CHO-MLF, N-formyl-methionyl-leucyl-phenylalanine; PT, pertussis toxin; FITC, fluorescein isothiocynate; NAD, nicotine adenine dinucleotide; PVDF, polyvinylidene fluoride; ECL+, enhanced chemiluminescence plus; DTT, D L -dithiothreitol; PM, plasma membrane; BCA, bicinchoninic acid; ELISA, enzyme-linked immunosorbent assay.
(PIC) was obtained from BD Biosciences (San Diego, CA). The superparamagnetic beads (Dynabeads M450) with or without anti-CD15 antibody were obtained from Dynal (Lake Success, NY). Antibodies used for flow cytometry were obtained as follows: mouse fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (clone 3G8) and anti-CD45 (clone BRA-55) antibodies were from Sigma Chemical Co. (St. Louis, MO); mouse phycoerythrin-conjugated anti-CD54 (clone HA58) and FITC-conjugated anti-CD58 (clone 1C3) and anti-CD44
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(clone L178) antibodies were from BD Biosciences. Antibodies utilized for Western blot analyses were obtained as follows: mouse anti-CD45 antibody (clone 69) was from BD Biosciences; rabbit antiserum for Gia1;2 (clone AS/7) was from Perkin–Elmer Life Sciences (Boston, MA) or from Calbiochem (San Diego, CA); rabbit antiserum for myeloperoxidase was from Rockland Inc. (Gilbertsville, PA); rabbit polyclonal antibody for tetranectin was from DAKO (Carpinteria, CA); rabbit polyclonal antibody for Gb was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Secondary antibodies against mouse and rabbit conjugated with horse radish peroxidase were from Jackson Immunological (West Grove, PA). The [32 P]NAD was obtained from Perkin–Elmer Life Sciences. The enhanced chemiluminescence plus (ECL+) was from Amersham (Oslo, Norway). The Quantikine MMP-8 Immunoassay kit was obtained from R&D Systems, Inc. (Minneapolis, MN). The Micro bicinchoninic acid (BCA) assay kit, the PAGEprep kit, and the Restore Western blot stripping buffer were from Pierce Biotechnology (Rockford, IL). All other chemicals were obtained from Sigma Chemical Co. Neutrophil isolation Neutrophils were isolated from whole blood by the method described in Tolley et al. [6]. Human neutrophils were partially purified from red cells by gelatin sedimentation and then further purified to >98% by counterflow elutriation. Purified neutrophils were resuspended in modified GeyÕs buffer (MGB) that consisted of 5 mM KCl, 147 mM NaCl, 1.9 mM KH2 PO4 , 0.22 mM Na2 HPO4 , 5.5 mM glucose, 0.3 mM MgSO4 , 1 mM MgCl2 , and 10 mM Hepes, pH 7.4. Identification of a suitable plasma membrane marker for plasma membrane quantification Normally, proteins quantified from bands on a Western blot are normalized by the total protein loaded in the lane. The protein quantification is typically done with a standard protein assay such as a BCA assay or a Lowry assay. Because in our protocol the protein assays would also quantify the antibody used to isolate the plasma membrane, accurate quantification of the plasma membrane sample would not be possible. To circumvent this limitation, we sought a plasma membrane marker that could also be quantified by Western immunoblotting; in this case, the expression of the plasma membrane marker must not change under different experimental conditions of interest. N-Formyl-methionylleucyl-phenylalanine (CHO-MLF) stimulation and PT treatment were two experimental conditions of interest, and thus a plasma membrane marker that did not change under these conditions was needed. The plasma
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membrane proteins CD16, CD44, CD45, CD54, CD58, and Gb were tested for this purpose. First, intact neutrophils were stimulated with CHOMLF to assess the impact of stimulation on expression of plasma membrane markers. Neutrophils were incubated in MGB plus 1.5 mM Ca2þ at 2 106 cells/mL for 10 min at 37 °C. Then, CHO-MLF was added to a final concentration of 1 lM. After a 15-min incubation, cells were put on ice and the amount of plasma membrane marker was measured as described below. Second, intact neutrophils were treated with PT, which ADP-ribosylates the a subunit of Gi proteins, to determine the effect of such treatment on plasma membrane marker expression. Neutrophils were suspended in Krebs–Ringer buffer plus cytochrome c (6.3 mg/mL) at 5 107 cells/mL. PT was added at concentrations of 0, 0.5, 1.0, and 2.5 lg/mL, and samples were incubated at 37 °C for 120 min with gentle mixing. After incubation, cold MGB (4 °C) was added to stop PT action. The suspension was filtered through a nylon mesh to remove aggregates. Then, plasma membrane marker expression was quantified as described below. Two strategies for quantifying the amount of plasma membrane marker after CHO-MLF stimulation or PT treatment were used, one for membrane proteins with extracellular epitopes and one for intracellular membrane proteins. For the markers that have an extracellular epitope (CD16, CD44, CD45, CD54, CD58), flow cytometric assays were used to measure their expression. After either treatment condition, the cells 4 107 cells/ mL) were centrifuged and resuspended in MGB at 108 cells/mL. Antibody against the marker conjugated to FITC was added at a dilution of 1:5 in the cell suspension and incubated in the dark for 30 min at 4 °C. Cells were then washed and resuspended in MGB at a concentration of 4 106 cells/mL. Finally, cells were analyzed on a Becton–Dickinson FACScan flow cytometer, and fluorescent antibody binding was quantified as mean channel number of 5000 cells. Expression of CD16, CD44, CD45, CD54, and CD58 did not change with PT treatment. Expression of CD16 and CD44 changed with CHOMLF stimulation, but CD45, CD54, and CD58 remained unchanged, consistent with previous results [7]. Unfortunately, a search for suitable Western blotting antibodies for CD45, CD54, and CD58 yielded no antibodies that were sensitive enough to detect their presence in plasma membrane fractions from 106 cells. We note that although this approach did not suit our purpose, it is generally applicable to cell systems where there are sensitive antibodies available for flow cytometric assessment of changes (or lack thereof) in marker expression and quantification on Western blots. For our purposes, a second approach was necessary. To assess the feasibility of utilizing an intracellular plasma membrane marker (which could not easily be quantified by flow cytometry) such as Gb , large numbers
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of cells 108 ) were either stimulated with CHO-MLF or treated with PT. From these cells, plasma membrane was then isolated by differential centrifugation so that the total plasma membrane recovered could be measured with a standard protein assay. The plasma membrane marker was quantified by Western immunoblotting after separation of proteins by SDS–PAGE and normalized to total protein loaded per lane. This is a commonly utilized approach to quantifying changes in specific proteins in the membrane. However, it does assume that changes (if they occur) in the plasma membrane marker are highly specific and not coincident with a general change in total plasma membrane protein. For this protocol, a plasma membrane fraction was isolated by differential centrifugation following the method of Suchard and Mansfield [7]. Briefly, 3 108 neutrophils were suspended in SHE buffer (10 mM Hepes, pH 7.3, 0.1 M sucrose, 1 mM EGTA) with a 1/50 dilution of 50 PIC and disrupted by nitrogen cavitation [1]. The cavitate was then centrifuged for 10 min at 4 °C and 500g (1600 rpm in a Sorvall RT7 centrifuge) to remove intact cells and nuclei. The recovered supernatant was centrifuged in the medium-speed J2-MI Beckman centrifuge for 15 min at 4 °C and 8000g (8130 rpm in JA20 rotor) to remove granules. Finally, the plasma membrane fractions were isolated by centrifuging the supernatant from the previous step at 200,000g in an ultracentrifuge (34,200 rpm in 40 Ti rotor) for 30 min at 4 °C. The final supernatant was discarded and the resulting pellet was homogenized in 1 mL of SHE buffer and stored at –70 °C. A micro BCA protein assay was used to quantify protein, and Western blots were assayed to quantify the corresponding Gb in a given sample. Plasma membrane fraction isolation using CD15 antibody-conjugated beads Disruption of cells and generation of cell cavitate were performed using nitrogen cavitiation as described by Tolley et al. [6]. This method was chosen because it gives efficient lysis of the plasma membrane without disruption of intracellular organelle membranes [1,3], a point to which we will return in the discussion. Neutrophils were suspended (107 cells/mL) in homogenization buffer (0.34 M sucrose, 10 mM Hepes, 1 mM EDTA (disodium salt), and 2.5 mM MgCl2 ) with the addition of 1:50 dilution of 50 PIC. Neutrophils were then placed in a cell disruption bomb (Parr Instruments) and pressurized at 450 psi for 15 min. The pressure was slowly released, and the slurry of cellular debris and undisrupted whole cells (cell cavitate) was collected. The resulting cavitate was centrifuged at 600g for 10 min to remove unlysed cells and nuclei. Finally, the supernatant (cell lysate) was recovered.
Superparamagnetic Dynabeads with anti-CD15 antibodies were used to isolate plasma membrane fractions from the cell lysates. CD15 was chosen as the capture antigen because it is a cell surface marker that is found on neutrophils. It is a branched pentasacchaaride present on a subset of the LFA-1/HMAC-1/gp 150,95 glycoprotein family, CR1, CR3, and NCA-160 [8,9]. In addition to being on the plasma membrane, CD15 has been detected primarily in secondary/specific granules [10,11]. The concentration of beads added to aliquots of the cell lysate (107 cell equivalents/mL) was varied to determine the optimal ratio of beads to whole cell equivalents for efficient recovery of plasma membrane. Varying amounts of beads were added to microcentrifuge tubes containing 107 cell equivalents of cell lysate. The samples were incubated with agitation at 4 °C for 30 min and then placed in a holder with a magnet (Dynal) positioned near the bottom of the microcentrifuge tube. The magnet aggregated the beads near the bottom of the tubes such that the supernatants were easily decanted. Samples were removed from the magnet and the beads were resuspended in phosphate-buffered saline with bovine serum albumin (1 mg/mL). This washing step was performed twice to wash the beads, washing out the unattached proteins and organelles and leaving behind plasma membrane vesicles attached to the beads. In some experiments, the supernatant after the first wash step was incubated with beads again to determine whether more membrane could be recovered. As an additional control, nitrogen cavitate was incubated with beads without the anti-CD15 antibody. Neutrophil collagenase ELISA Lack of contamination by secondary/specific granules in the plasma membrane fractions was demonstrated by ELISA of neutrophil collagenase (MMP-8), a marker for secondary/specific granules [12], using the Quantikine MMP-8 immunoassay kit according to the manufacturerÕs directions. Briefly, samples (106 cell equivalents) and standards were aliquoted and incubated in 96-well plates for 2 h with agitation at room temperature. Wells were washed five times with the wash buffer. Detection antibody was added and the plates were incubated with agitation at room temperature. The wells were washed again and substrate was added before measuring absorbance on a plate reader. Protein blotting and phosphor screen detection For quantification of membrane recovery, quantification of Gia subunits, and verification of membrane purity, aliquots (106 cell equivalents) of the beadbound membrane suspension were boiled in Laemmli sample buffer [13] to denature proteins and dissociate
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membranes and proteins from the beads. The beads were removed by centrifugation and a measured aliquot of the supernatant was subjected to SDS–PAGE, based on the procedures developed by Laemmli [13], and transferred to PVDF membranes [15]. Membranes were probed with anti-Gia , anti-Gb , anti-tetranectin, or antimyeloperoxidase antibodies at dilutions of 1:1000, 1:1000, 1:100, or 1:5000, respectively. The membranes were incubated with either anti-mouse or anti-rabbit antibodies (depending on the host of the antibody as noted under Materials) conjugated with horseradish peroxidase. Then, the membranes were exposed with ECL+ as per manufacturerÕs instructions and the resulting fluorescent bands were quantified with a Molecular Dynamics Storm 860 imager. After probing for Gia , membranes were stripped by incubating the membranes in Restore Western blotting stripping buffer for 30 min at 37 °C. Then, the membranes were reprobed with antibody to Gb . Thus, potential differences in loading of samples in the gel lanes was accounted for by normalizing the Gia subunit signal to the Gb signal in the same lane. [32 P]ADP-ribosylation membranes
of
Gia
proteins
in
plasma
To determine the level of ADP-ribosylation of Gia proteins in the purified plasma membrane fragments, as a result of either endogenous enzymes or prior PT treatment of intact cells, purified membranes were treated with activated PT and [32 P]NAD. In cases where ADPribosylation occurred before cell disruption, only Gia proteins not previously ADP-ribosylated would become radiolabeled in the second ADP-ribosylation reaction. Control cells without prior ADP-ribosylation provided a measure of maximal obtainable radiolabeling. Magnetic beads with attached plasma membrane fractions were suspended in 200 lL of reaction mixture (110.6 lL PT cocktail, 60 lL activated PT (100 lg/mL), 20 lL 100 mM D L -dithiothreitol (DTT), 7 lL 5% (w%) Luberol, 4 lL 50 PIC, 0.06 lCi/lL [32 P]NAD), and incubated at 37 °C for 30 min [16,17]. The PT cocktail was made by mixing 2 mL of L -a-phosphatidylcholine, dimyristoyl (2.76 mg/mL in deionized water, sonicated for 20 min) and 7 mL of a solution of 180 mM Tris, 18 mM thymidine, 4.5 mM MgCl2 -6H2 O, 1.8 mM NaEDTA, 18 mM L -arginine, 180 lM GTP, and 1.8 mM ATP [16,17]. Activated PT (100 lg/mL) was prepared by a 1:1 dilution of a PT solution (200 lg/mL in sterile 0.1 M NaPO4 and 0.5 M NaCl) with an aqueous solution of 50 mM Tris–HCl and 100 mM DTT and incubating this mixture for 30 min at 37 °C [17,18]. Then, the protein in the mixture was concentrated using PAGEprep kit. bMercaptoethanol was added to a concentration of 5%, and the protein solution was boiled for 5 min at 100 °C before conducting SDS–PAGE and electrotransfer to
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PVDF membranes as described above. When appropriate, membranes were probed with anti-Gia and anti-Gb antibodies as described above. Furthermore, blots with 32 P-labeled proteins were exposed to a phosphor screen overnight and quantified with a Molecular Dynamics Storm 860 imager. Effect of neutrophil treatment conditions on Gia subunit membrane expression and ADP-ribosylation Two treatment conditions were evaluated for their effects on Gia ADP-ribosylation, activation with N-formyl peptide and pretreatment with pertussis toxin. Intact neutrophils were treated for 15 min with CHO-MLF or 2 h with PT as described above for identification of suitable membrane markers. After each treatment, cells were subjected to the membrane isolation and G protein quantification protocol as outlined in Fig. 1. Statistical analyses Statistical comparison of data was performed using Instat software from GraphPad Software (San Diego, CA) with the Student t test. A p value less than 0.05 was considered significant.
Results Validation of membrane markers for quantifying membrane recovery Gb was found to be a marker suitable for quantification of plasma membrane recovery. Sensitive antibodies were readily available for quantification of Gb on Western blots. Pretreatment of neutrophils for 15 min with CHO-MLF or 2 h with PT (Fig. 2) did not change (p > 0:05) the expression level of Gb in plasma membrane fractions isolated by differential centrifugation and quantified as total membrane protein. Thus Gb was utilized as a plasma membrane marker for quantification of membranes recovered by the superparamagnetic bead protocol. Optimization of the magnetic bead separation step To optimize the concentration of beads needed to maximize plasma membrane extraction, the concentration of beads was varied while maintaining the cell equivalent concentration at 107 /mL, and the amount of recovered G protein was measured. Saturation of plasma membrane G protein recovery was achieved at 5 107 beads/mL (Fig. 3A), which represented a bead to cell equivalent ratio of 50:1. The surface area of a bead (based on a diameter of 4.5lm, given by the manufacturer) is approximately 60 lm2 , whereas the
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Fig. 3. Efficiency of plasma membrane recovery by magnetic beads. (A) Cell lysate was incubated with a range of bead concentrations and recovered membrane proteins were separated by SDS–PAGE as described under Materials and methods. Membrane Gia was quantified after Western blot analysis and plotted as a percentage of the total Gia in the whole cell lysate. Each data point represents the average of three experiments. The error bars represent the standard error of the mean. (B) Cell lysate was incubated with two successive aliquots of magnetic beads. The beads were boiled in sample buffer and the samples subjected to SDS–PAGE and immunoblot analysis. The plasma membrane (detected as Gia in Western blots) was recovered from beads in the first incubation (lane 1) with undetectable levels of plasma membrane on the beads from the second incubation (lane 2).
Fig. 2. Validation of Gb as a plasma membrane marker. Cells were treated with or without PT or with and without CHO-MLF as described under Materials and methods. Plasma membranes were isolated by differential centrifugation and quantified as membrane protein using the BCA assay. Samples were subjected to SDS–PAGE and Gb was measured by immunoblotting as described under Materials and methods. Amount of Gb was normalized to the amount of protein loaded per lane. Data presented are the mean and standard error of the mean for three experiments. Control and treated samples did not differ by StudentÕs t test (p > 0:05).
surface area of a neutrophil is approximately 500 lm2 [19]. Thus the surface area of the beads must exceed the surface area of the cell equivalents by a factor of 8. This may be related to the surface density of the antibody on the beads, however, that information was not available from the manufacturer. The maximum membrane G protein recovered was approximately 55% of the total cellular G protein. This is consistent with the measurements of Bokoch et al. [20] and Keil et al. [21], who reported that 67–75% and 25%, respectively, of the total neutrophil Gia protein is at the plasma membrane.
To further confirm that the bead concentration was optimized at a ratio of 50 beads per cell equivalent, the supernatant recovered from a first bead incubation was treated a second time with beads under analogous conditions to see whether any more G proteins could be extracted. The Western blots comparing G protein bands from the first and second incubation of sample with beads showed that a negligible amount of G protein was recovered by beads in the second incubation (Fig. 3B), thus 50 beads per cell equivalent was sufficient to recover the plasma membrane fragments. To confirm specificity of plasma membrane binding to beads, nitrogen cavitate was incubated with beads with and without anti-CD15 antibody. Western blot analysis showed negligible amounts of G protein recovered by the naked beads compared to the beads with anti-CD15 antibody (Fig. 4), confirming that there was little nonspecific binding of proteins to beads. Validation of membrane purity The purity of the isolated plasma membrane fragments was determined by comparing the expression of tetranectin, myeloperoxidase, and neutrophil collagenase,
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Fig. 4. Specificity of magnetic bead isolation of plasma membrane fractions. Nitrogen cavitate was incubated with beads without antiCD15 antibody (NS) or with anti-CD15 antibody (SP). The beads were then boiled in sample buffer and the samples subjected to SDS–PAGE and immunoblot analysis. Recovered membrane was quantified as Gia . Plasma membrane was recovered only from beads coated with antiCD15 antibody.
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endogeneous enzymes or by prior treatment of cells with pertussis toxin, isolated plasma membrane fractions adherent to magnetic beads were incubated with activated PT and [32 P]NAD following procedures described under Materials and methods. The time for incubation of this reaction was varied to determine the incubation time required to label all of the available G proteins. The PM fractions were incubated for 30 or 60 min to bracket the range of times typically utilized for this reaction [16–18]. Proteins of these samples were separated by SDS– PAGE, and the radioactivity in the Gi protein bands was quantified. Membranes incubated for 30 and 60 min exhibited the same levels of ADP-ribosylation of Gia (data not shown). Therefore, a 30 min incubation was deemed sufficient for the reaction to reach completion. Calculation of errors in quantifying total Gia and Gia available for signaling
Fig. 5. Lack of contamination of plasma membranes with secretory vesicles (tetranectin) and primary granules (myeloperoxidase). Equal cell equivalents of whole cell lysate (A), nitrogen cavitate (B1, B2), and plasma membrane isolated using magnetic beads (C1, C2) were assayed for the presence of tetranectin and myeloperoxidase by immunoblot analysis. Whereas whole cell lysate and nitrogen cavitate contained myeloperoxidase and tetranectin, the plasma membrane contained insignificant levels of each.
markers for secretory vesicles, azurophil/primary granules, and specific/secondary granules, respectively [12]. Secretory vesicles have been implicated as contaminants in plasma membrane isolates obtained by density sedimentation methods [4,5]. Azurophil and specific granules are reported to contain CD15 [10,11]. Although the epitopes for antibody binding were expected to be on the inside of these granules, it was important to confirm that these granules did not bind to the beads. Strong signals were seen in the bands for tetranectin and myeloperoxidase in the cell lysate and N2 cavitate, but unquantifiable levels were found in the purified plasma membrane fragments, given equal number of cell equivalents loaded in each lane of the gel (Fig. 5). Quantitative ELISAs of whole cell lysate, N2 cavitate, and plasma membrane fractions showed that only 3.8% of the neutrophil collegenase in N2 cavitate was in the plasma membrane fractions. Therefore, magnetic bead isolation yielded highly purified plasma membranes excluding both intracellular membrane vesicles of density similar to that of the plasma membrane and intracellular vesicles containing CD15 on the internal side of the granule membrane. Optimization of protocols for quantifying G proteins available for signaling To quantify Gi proteins available for signaling, i.e., Gi proteins that have not been ADP-ribosylated either by
Plasma membranes were isolated and incubated with PT and radiolabeled NAD, and proteins were separated by SDS–PAGE as outlined in Fig. 1. Each sample was run in triplicate gel lanes, and the optical densities of Gia , Gb , and [32 P]Gia were measured. Then, the measurements for Gia and [32 P]Gia bands were divided by the Gb measurements. The standard error of the mean for these ratios was calculated to be 20%. Application of the plasma membrane isolation and Gi protein quantification protocols The assay was used to investigate aspects of signal transduction that may be important in influencing neutrophil responses. Preincubation of neutrophils with PT is known to inhibit nearly all responses induced by NFormyl peptides by ADP-ribosylating Gi proteins, making them unavailable for coupling to receptors [22]. As a positive control for our protocols, neutrophils were pretreated with increasing concentrations of PT, and then plasma membranes isolated and total Gia and Gia available for signaling (i.e., Gia not ADP-ribosylated in the first incubation with PT) were quantified according to the scheme in Fig. 1. As expected, as the concentration of PT in the pretreatment was increased, the amount of G proteins available for signaling decreased in a statistically significant manner although the total Gia associated with the plasma membrane did not change (Fig. 6). Thus, increased PT treatment increased the ADP-ribosylation of Gi protein on the plasma membrane without altering the membrane association of the Gi protein. To measure the influence of chemoattractant-induced activation of neutrophils on plasma membrane-associated total Gia and Gia available for signaling, neutrophils were incubated with and without 1 lM CHO-MLF for 15 min as described under Materials and methods. Plasma membranes were isolated and total Gia and Gia
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Fig. 6. Effect of PT treatment on neutrophil plasma membrane Gia . Neutrophils were treated with a range of PT concentrations as indicated. Plasma membrane fractions were isolated and Gia available for signaling (A) and total Gia (B) were quantified and normalized to plasma membrane (detected as Gb ) loaded per lane according to the protocol in Fig. 1. Data are averaged for three experiments with error bars showing the standard error of the mean. Asterisks indicate values statistically different from the value for PT ¼ 0.
available for signaling were quantified by the procedures outlined in Fig. 1. Comparison of stimulated and control cells showed that stimulation decreased both the total Gia and the Gia available for signaling by approximately 50% (Fig. 7). Thus activation appears to cause release of the Gia subunit from the plasma membrane without the occurrence of endogenous ADPribosylation.
Discussion A method that allows quick isolation of plasma membranes from neutrophils yielding highly pure plas-
Fig. 7. Effect of CHO-MLF treatment on neutrophil plasma membrane Gia . Neutrophils were incubated with or without 1 lM CHOMLF. Plasma membrane fractions were isolated and Gia available for signaling (A) and total Gia (B) were quantified and normalized to plasma membrane (detected as Gb ) loaded per lane according to the protocol in Fig. 1. Data are averaged for three experiments with error bars showing the standard error of the mean. Asterisks indicate values statistically different from the value for the control.
ma membrane fractions from small numbers of cells has been devised. The key to this high purity is the specific isolation of the plasma membrane fragments by antibody-conjugated superparamagnetic beads. Because nitrogen cavitation lyses cell plasma membranes without bursting intracellular organelles, nitrogen cavitation has been used quite frequently to lyse neutrophils [1,23]. For the purposes of this beadisolation protocol, it is of importance that the intracellular organelles are not lysed, since normal processing of surface receptors (such as CD15 [9]) involves processing through intracellular vesicular compartments. In the intact vesicle the CD15 epitope is on the inside of the vesicle and therefore not accessible for binding to the magnetic beads, whereas the plasma membrane fraction
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produced by nitrogen cavitation is characterized by small closed vesicles with the right side (CD15 epitope) out [1]. This allowed for the recovery of purified plasma membranes without contaminating intracellular organelles. The ability of antibody-conjugated beads to recover all of the plasma membrane is dependent upon the epitope being relatively evenly distributed on the plasma membrane. This appeared to be the case for CD15, since experimental data indicated that the bulk of the plasma membrane was recovered. That is, the work of Bokoch et al [20] indicated that 50% of the total Gia2 protein is on the plasma membrane and the anti-CD15-conjugated beads recovered approximately 50% of the total cellular Gia2 protein (Fig. 3). However, it is well established that membrane proteins and lipids can be heterogeneously distributed in the plasma membrane [24–29]. The utility of the antibody-conjugated bead approach for isolating all the plasma membrane then becomes dependent upon the relative sizes of the plasma membrane vesicles (generated by nitrogen cavitation), the subdomains in which the recovery epitope resides, and the distribution of the subdomains. If the subdomains are relatively small compared to the plasma membrane vesicles and the subdomains are relatively randomly distributed, then all of the plasma membrane could be recovered by the beads. At the other extreme, where the subdomains are large or comparable in size to the plasma membrane vesicle surface area, the plasma membrane vesicles may segregate into distinct populations of vesicles with epitope and without. In this case, the antibody-bead isolation technique may be useful for obtaining plasma membrane fractions enriched in a particular subdomain. One other important aspect of this assay is the use of a membrane marker to quantify plasma membrane recovery. Commonly, membrane recovery is detected as the total membrane protein using a protein assay such as the bicinchononic acid assay. However, this assay cannot be used with our method because of the abundance of antiCD15 antibody conjugated onto the beads. An attempt to wash the membranes off of the beads would add an additional cumbersome step. As an alternative approach, we utilized a plasma membrane marker whose expression did not change under the experimental conditions used and for which antibodies for Western blot analysis were available. Any suitable plasma membrane marker must not change with relevant experimental situations. This is an important point since several cell surface antigens have been shown to vary with some experimental treatments [30,31]. For this study, Gb expression on intact cells did not change with changes in experimental conditions of interest, and thus relative membrane recovery was measured by immunodetection of Gb after separating membrane proteins by SDS–PAGE. Application of the newly developed protocol confirms, as expected, that incubation of intact neutrophils
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with increasing concentrations of PT causes increasing levels of ADP-ribosylated Gia (with a corresponding decrease in the number of Gia available for signaling). However, PT treatment does not change the level of total Gia associated with the plasma membrane. Stimulation of neutrophils with N-formyl peptides causes both the total Gia and the Gia available for signaling to drop by approximately 50%. These data suggest the hypothesis that desensitization of neutrophil responses could be related to a decrease in the amount of total Gia proteins at the plasma membrane. Although nitric oxide-induced endogeneous ADP-ribosylation of G proteins has been observed in a few mammalian cell types [14,32], our data provide no evidence for CHO-MLFinduced endogenous ADP-ribosylation in neutrophils. In summary, we have designed a protocol to isolate neutrophil plasma membrane fractions utilizing a capture antibody to a cell surface antigen. This general approach is applicable to other cell types for which an appropriate cell surface capture antigen is characterized. Additionally, other intracellular organelles could be isolated by this approach if an antibody is available to an antigen on the surface of the organelle (i.e., on its cytoplasmic side). The protocol is significantly faster than standard density sedimentation procedures.
Acknowledgments The authors thank Michael L. Keil for technical assistance. This work was supported by National Science Foundation Grant BES-9713856 and the Office of Research and Development, Medical Research Service, Department of Veteran Affairs.
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