Proteomic Analysis of a Detergent-resistant Membrane Skeleton from Neutrophil Plasma Membranes
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m205386200
ISSN1083-351X
AutoresThomas Nebl, Kersi Pestonjamasp, John Leszyk, Jessica L. Crowley, Sang Wook Oh, Elizabeth J. Luna,
Tópico(s)Blood properties and coagulation
ResumoPlasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H;d ∼1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, α-actinin 1, α-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Gαi-2). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Gαi-2 also resist extraction with 0.1 m octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 m sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Gαi staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains. Plasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H;d ∼1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, α-actinin 1, α-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Gαi-2). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Gαi-2 also resist extraction with 0.1 m octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 m sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Gαi staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains. detergent-resistant membrane fragments DRMs with higher buoyant densities (1.15–1.18 g/ml) DRMs with lower buoyant densities (1.09–1.13 g/ml) antibodies against the amino-terminal 340 amino acids of human supervillin glycophosphatidylinositol glutathione S-transferase the amino-terminal 340 amino acids of human supervillin horseradish peroxidase matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry membrane-type 6 matrix metalloproteinase post-source-decay 1,4-piperazinediethanesulfonic acid Plasma membranes in eukaryotic cells are organized into domains of specialized function. Many of these domains are relatively stable on the cell surface and involve a series of linkages between cytoplasmic cytoskeletal proteins, integral membrane proteins, and extracellular attachments to the substrate or to other cells. For example, intermediate filament anchorages to membranes at desmosomes and hemidesmosomes distribute mechanical stresses over epithelial surfaces and maintain the integrity of skin and other tissues (1Jonkman M.F. J. Dermatol. Sci. 1999; 20: 103-121Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 2McMillan J.R. Shimizu H. J. Dermatol. (Tokyo). 2001; 28: 291-298Crossref Scopus (52) Google Scholar). Plasma membrane domains anchored to actin filaments include spectrin-based meshworks, adsorptive cell surface microvilli, focal adhesions, and adherens junctions (3Bennett V. Baines A.J. Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (804) Google Scholar, 4Bloch R.J. Bezakova G. Ursitti J.A. Zhou D. Pumplin D.W. Soc. Gen. Physiol. 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Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar,36Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (106) Google Scholar). We also have used F-actin blot overlays and immunoblotting to show that neutrophil plasma membranes contain ezrin, moesin, and a 205-kDa F-actin-binding protein that we named supervillin. Molecular cloning of supervillin cDNA indicated that this protein contains a carboxyl terminus with predicted similarities to villin and a novel amino terminus (36Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (106) Google Scholar, 37Pope R.K. Pestonjamasp K.N. Smith K.P. Wulfkuhle J.D. Strassel C.P. Lawrence J.B. Luna E.J. Genomics. 1998; 52: 342-351Crossref PubMed Scopus (41) Google Scholar). The supervillin amino terminus contains functional nuclear targeting signals, sites for direct binding to F-actin, and regions that promote actin filament binding and bundling (38Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar). Supervillin is a tightly bound peripheral membrane protein that is concentrated at sites of epithelial cell-cell adhesion (36Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (106) Google Scholar). This protein also has been reported to be a minor constituent of lipid rafts from detergent-solubilized Jurkat T cells (39von Haller P.D. Donohoe S. Goodlett D.R. Aebersold R. Watts J.D. Proteomics. 2001; 1: 1010-1021Crossref PubMed Google Scholar) and to co-purify with P2X7 ATP receptors from embryonic kidney cells (40Kim M. Jiang L.H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (339) Google Scholar). Supervillin message levels are elevated in neutrophils stimulated with lipopolysaccharide from gingivitis-causing bacteria (41Morozumi T. Kubota T. Sugita N. Ohsawa Y. Yamazaki K. Yoshie H. J. Periodontal Res. 2001; 36: 160-168Crossref PubMed Scopus (15) Google Scholar), implying a role during physiological activation of neutrophils. The protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride, leupeptin, and bestatin, were purchased from Calbiochem-Novabiochem. Density gradient centrifugation media, PercollTM and OptiprepTM, were from Amersham Biosciences and Nycomed Pharma AS (Oslo, NOR), respectively. Tosyl-activated or protein A-conjugated Dynabeads M-280 were from Dynal Inc. (Lake Success, NY). Nonspecific control rabbit IgG and mouse monoclonal primary antibodies against α-actinin, β-actin, and vimentin were purchased from Sigma. Monoclonal anti-Lyn and anti-Gαi-2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-flotillin 2 was from BD Biosciences; monoclonal anti-α-fodrin was from ICN Biomedicals Inc. (Aurora, OH), and polyclonal rabbit anti-non-muscle myosin-II IgG was from Biomedical Technologies Inc. (Stoughton, MA). Myosin-IG was stained by a monoclonal antibody raised against chicken brush-border myosin (myosin-IA) that recognizes the head domain of multiple myosin-I isoforms (42Carboni J.M. Howe C.L. West A.B. Barwick K.W. Mooseker M.S. Morrow J.S. Am. J. Pathol. 1987; 129: 589-600PubMed Google Scholar, 43Peterson M.D. Mooseker M.S. J. Cell Sci. 1992; 102: 581-600Crossref PubMed Google Scholar) and also by affinity-purified rabbit IgG against a consensus myosin-IA peptide (G-371) (44Ruppert C. Godel J. Muller R.T. Kroschewski R. Reinhard J. Bähler M. J. Cell Sci. 1995; 108: 3775-3786Crossref PubMed Google Scholar). These antibodies were the kind gifts of Dr. Mark Mooseker (Yale University, New Haven, CT) and Dr. Martin Bähler (Westfälische Wilhelms-Universität, Münster, Germany), respectively. Horseradish peroxidase (HRP)-conjugated sheep IgG against bovine IgM was from Serotech Ltd. (Oxford, UK). HRP-conjugated, as well as alkaline phosphatase-conjugated, goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Jackson ImmunoResearch. AlexaFluor 488 goat F(ab′)2 anti-rabbit IgG and AlexaFluor 568 goat F(ab′)2 anti-mouse IgG were purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma. The HSV41 clone (37Pope R.K. Pestonjamasp K.N. Smith K.P. Wulfkuhle J.D. Strassel C.P. Lawrence J.B. Luna E.J. Genomics. 1998; 52: 342-351Crossref PubMed Scopus (41) Google Scholar) containing human supervillin cDNA sequences (GenBankTM accession number AF051851) was digested with EcoRI and NotI and ligated into the pGEMEX-1 vector (Promega Corp., Madison, WI). A chimeric fusion protein consisting of 260 amino acids of T7 gene 10 and linker sequence plus the first 340 amino acids of human supervillin (H340) was expressed in BL21(DE3) bacteria after induction with isopropyl β-d-thiogalactopyranoside. The fusion protein was purified as inclusion bodies and used as an immunogen for the production of rabbit polyclonal antisera (Research Genetics, Huntsville, AL). Antibodies against the amino-terminal 340 amino acids of human supervillin (αH340) were affinity-purified against a chimeric fusion protein of H340 and glutathioneS-transferase (GST). The GST-H340 protein was expressed in bacteria from sequences encoded by the pGEX-6P-1 vector and HSV41 cDNA and was purified on glutathione-SepharoseTM, as described by the manufacturer (Amersham Biosciences). All manipulations were carried out at 4 °C, unless stated otherwise. Purified GST-H340 (∼12 mg) was dialyzed against 20 mm sodium phosphate buffer (pH 7.6) and coupled to ∼1.5 ml of CNBr-activated Sepharose 6MB (Sigma) for 24 h. The column was blocked with 0.2m glycine ethyl ester (pH 8.0) for 24 h, washed with 40 ml of sodium phosphate buffer (pH 7.6), 2 ml of 4.5 mMgCl2, 10 ml TBS (25 mm Tris-HCl, 1 mm EDTA (pH 7.4)), and stored in TBS containing 0.1% thimerosal. The GST-H340 Sepharose beads were rotated overnight with 10-ml aliquots of specific rabbit antisera and washed sequentially at room temperature with the following: 1) ∼150 ml TBS; 2) 6 ml of 1m NaCl in TBS; 3) 4 ml of 2 m urea in TBS; and 4) 2 ml of 10 mm Tris-HCl (pH 7.5). High affinity antibodies were eluted with 4.5 m MgCl2, 72.5 mm Tris (pH 7.0) and collected in 1-ml fractions with equivalent volumes of 10 mm Tris-HCl (pH 7.5). Fractions containing affinity-purified IgG were pooled, repeatedly diluted with 10 mm Tris-HCl (pH 7.5), and re-concentrated to ∼0.3 ml by centrifugation at 2000 × g using Ultra-Free-10TM ultrafiltrate concentrators (Millipore Corp., Bedford, MA). Concentrated antibody was clarified by centrifugation at 13,000 × g for 5 min and stored in aliquots at −20 °C with ∼1 mg/ml bovine serum albumin and 50% (v/v) sterile glycerol. The specificity of αH340 antibody was confirmed by immunoblots of whole neutrophil extracts (Fig.1 A), and the suitability for staining of paraformaldehyde-fixed samples was demonstrated using immunolabeling experiments with COS-7 cells expressing a GFP-tagged bovine supervillin (not shown). H340 antigen was purified by cleavage from GST-H340 and chromatography on glutathioneTM-Sepharose and DEAE-Sepharose, as recommended by the manufacturer (AmershamBiosciences). Polymorphonuclear leukocytes were routinely isolated from 8–10 liters of bovine blood provided by Research 87 Inc. (Marlborough, MA). Peripheral blood was collected from a single animal, immediately mixed 9:1 (v/v) with anti-coagulant solution (3.8% (w/v) sodium citrate, 2.5 mm EDTA, 50,000 units of heparin), and stored on ice overnight. Neutrophils (∼95% pure) were obtained by hypo-osmotic lysis of erythrocytes, followed by fractionation on preformed gradients of isotonic PercollTM(27Mottola C. Gennaro R. Marzullo A. Romeo D. Eur. J. Biochem. 1980; 111: 341-346Crossref PubMed Scopus (26) Google Scholar, 28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar, 45Del Buono B.J. Luscinskas F.W. Simons E.R. J. Cell. Physiol. 1989; 141: 636-644Crossref PubMed Scopus (22) Google Scholar). Cells were washed twice and resuspended in Hanks' deficient saline solution buffered with 10 mm HEPES (pH 7.4) (HBSS). All manipulations were carried out at 4 °C. Neutrophils were treated with 5 mm diisopropylphosphofluoridate for 15 min, washed, and resuspended in relaxation buffer (28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar) (10 mmPIPES, 100 mm KCl, 3 mm NaCl, 3.5 mm MgCl2, 1 mm ATP, 1 mm EGTA, pH 7.3) containing a mix of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 0.4 μmaprotinin, 10 μm E-64, 20 μm leupeptin, 10 μm bestatin, 5 μm N-acetyl-Leu-Leu-Met-al, 2 μm pepstatin), and disrupted by nitrogen cavitation. All manipulations were carried out at 0–4 °C. Cavitates were centrifuged at 1000 × g for 10 min to remove nuclei, and 10-ml aliquots of postnuclear supernatant were loaded onto 28 ml of 1.06 g/ml PercollTM suspension in relaxation buffer. Initially, plasma membranes were separated from secretory vesicles, granules, and cytosol using PercollTMstep gradients, as described previously (28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar, 36Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (106) Google Scholar). As a faster alternative to manual pipetting, PercollTM density gradients were generated by centrifugation. Density gradients were generated by centrifugation at 50,000 × g for 30 min in a Beckman VTi50 vertical rotor. Neutrophil plasma membranes corresponded to the "γ fraction" (∼1.04 g/ml) from these gradients. This fraction was enriched ∼10-fold in cell surface (28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar) and plasma membrane (27Mottola C. Gennaro R. Marzullo A. Romeo D. Eur. J. Biochem. 1980; 111: 341-346Crossref PubMed Scopus (26) Google Scholar, 45Del Buono B.J. Luscinskas F.W. Simons E.R. J. Cell. Physiol. 1989; 141: 636-644Crossref PubMed Scopus (22) Google Scholar) markers. After centrifugation at 140,000 × g for 2 h to remove residual PercollTM particles, plasma membranes were resuspended in relaxation buffer containing protease inhibitors, disrupted with 10–20 strokes of a tight-fitting Teflon Dounce homogenizer, and stored in aliquots at −80 °C. Neutrophils suspended in HBSS were seeded onto coverslips that were coated with poly-l-lysine or zymosan-activated plasma for 15 min at 22 °C (28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar). Zymosan-activated plasma was prepared by incubating bovine plasma (from the same batch of peripheral blood used to isolate the neutrophils) with 5 mg/ml zymosan at 37 °C for 60 min and clarified by centrifugation at 100,000 × g for 60 min. Non-adherent cells were removed, and adherent unactivated (poly-l-lysine) or activated (zymosan-activated plasma) cells were incubated at 37 °C in 5% CO2 for 30–60 min in 10 mm potassium phosphate, 130 mm NaCl, 0.6 mm CaCl2, 1 mm MgCl2, 2.7 mm KCl, 0.18% glucose, pH 7.4. Neutrophils were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min, washed three times in PBS, and then permeabilized using 0.5% Triton X-100 for 10 min at 22 °C. Exposed cytoplasmic surfaces of neutrophil plasma membranes were obtained by disrupting cells with a focused jet of 60 mm PIPES, 25 mm HEPES, 10 mm EGTA, 2 mm MgCl2 (pH 6.9) (28Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (156) Google Scholar, 46Hartwig J.H. Chambers K.A. Stossel T.P. J. Cell Biol. 1989; 108: 467-479Crossref PubMed Scopus (121) Google Scholar), delivered from a syringe with a 26-gauge needle at an oblique angle across the surface of the coverslip. Sheared cells were immediately fixed in 2% paraformaldehyde in the same buffer for ∼15 min at 22 °C. Fixed samples were washed 3 times for 5 min in PBS and blocked at 4 °C overnight, or for up to 3 days, with 10% bovine serum, 0.02% sodium azide, 0.02% thimerosal in PBS (pH 7.4). Samples then were incubated 2–4 h with 3 μg/ml affinity-purified αH340 or nonspecific rabbit IgG alone or together with mouse monoclonal anti-Gαi-2antibody at 2 μg/ml in blocking solution. As an additional specificity control, aliquots of affinity-purified αH340 were incubated for 2 h with 10 μg/ml purified H340 antigen before addition to coverslips. After three washes in PBS, samples were incubated with blocking solution containing 2 μg/ml AlexaFluor 488-labeled F(ab′)2 fragment of goat anti-rabbit IgG and either 2 μg/ml AlexaFluor 568-labeled F(ab′)2 fragment of goat anti-mouse IgG or 50 nm rhodamine phalloidin. At these antibody concentrations, no significant background was observed in the absence of primary antibody (not shown). After final washes, samples were mounted with VectaShieldTM mounting medium (Vector Laboratories Inc., Burlingame, CA) and observed using an MC100 Axioskop epifluorescence microscope with filters for Texas Red and fluorescein (Carl Zeiss Inc., Thornwood, NY). Phase contrast and fluorescence micrographs were acquired with a model Retiga-1300 QImaging Camera and processed using Openlab imaging software (Improvision Inc., Lexington, MA). Confocal immunofluorescence images were recorded on a Bio-Rad MRC 1024 laser scanning confocal microscope equipped with a krypton/argon laser and LaserSharp version 3.2 software (Bio-Rad). No bleed-through fluorescence between different channels was detected in double-label experiments. Detergent-resistant membranes were prepared as shown (Fig. 2 A and Fig. 3 A). Pelleted neutrophil plasma membranes (Fig. 3 A, fraction 1) were resuspended to 1–2 mg of membrane protein per ml of Triton X-100 extraction buffer (TEB: 1% Triton X-100, 25 mm Tris, 250 mm NaCl, 2.5 mm MgCl2, 1 mm EGTA, 1 mm ATP) containing protease inhibitors and were incubated for 60 min on ice. Samples (6 × 2.7 ml) were underlayered with 0.3 ml of 48% (v/v) OptiprepTMin TEB and centrifuged at 100,000 × g for 60 min at 4 °C in a Beckman TLA 100.3 rotor. Supernatant was discarded. The sharp opalescent band at the top of the OptiprepTM cushion, which represented the Triton X-100 extracted plasma membranes (Fig.3 A, fraction 2), was collected and adjusted to 30% (v/v) with TEB and/or 60% OptiprepTM. Centrifugation of aliquots (8 × 500 μl) at 350,000 × g for 2 h at 4 °C in a Beckman TLA120.1 rotor generated gradients of OptiprepTM with detergent-resistant membrane fragments (DRMs) floating at low buoyant density (Fig. 2 A). Fractions (50 μl) were collected and analyzed for total protein concentration using BCA assay reagent (Pierce), for total cholesterol concentration using Infinity cholesterol assay reagent (Sigma), and for refractive indices using a model 334610 refractometer (Bausch & Lomb, Rochester, NY), according to manufacturer's instructions. Cholesterol-rich DRM fractions were then pooled (total DRMs; Fig. 3 A,fraction 4), mixed with an equal volume of TEB, and centrifuged into linear 20–45% (w/v) sucrose density gradients at 200,000 × g for 18 h at 4 °C in a Beckman SW50.1 rotor. Single gradients were fractionated (300 μ
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