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Blood, Vol. 114, Issue 9, 1919-1928, August 27, 2009
Miltenberger blood group antigen type III (Mi.III) enhances the expression of band 3
Blood Hsu et al.
114: 1919
Supplement materials for: Hsu et al
Antibodies
Anti-AE1 monoclonal antibodies used in this study include AE12-M (Alpha Diagnostic, TX, USA), BRIC170 and BRIC71 (BITS, UK). Anti-GPA and GPB monoclonal antibodies include E31 (Sigma, USA) and R1.32 (BITS). E3 recognizes a non-glycosylated, homologous region close to the transmembrane segment (in GPA: amino acids 61–64 or 64–67; in GPB: amino acids 32–35),1 whereas R1.3 recognizes the N-terminal non-sialylated protein sequence common to GPA and GPB.2 Other monoclonal antibodies include anti-GPA (clone E4 from Sigma), anti-AQP1 (clone 1/A5F6 from Serotec, UK), anti-CD240DCE (clone BRIC69 from Serotec), anti- 4.1R (clone 3D9 from Abnova, Taiwan), anti-α spectrin (clone 17C7 from Abcam, UK), and anti-β spectrin (clone 4C3 from Abcam). Polyclonal antibodies include anti-ANK1 (Abnova), anti-GLUT1 (Abcam), anti-actin (Sigma), anti-EPB42 (Novus Biologicals, USA), anti-stomatin (Novus Biologicals), and anti-RhAG (Abcam). Immunoprecipitation of the AE1-based complexes
Anti-AE1 (AE12-M; BRIC170) was DMP-crosslinked to magnet dynabeads according to the manufacturer’s protocol (Invitrogen). Erythrocyte ghosts were solubilized with an equal volume of the doubly-concentrated lysis buffer containing 2% CHAPS, 2% NP-40, 0.05% SDS, PBS, and Complete Protease Inhibitor Cocktails (CalBiochem). Equal quantities of the ghost lysates (usually 1 mg per sample) were applied for immunoprecipitation (IP) at 4°C for 12–16 hours. To remove nonspecific binding, antigen-bound beads were sequentially washed with increasing salt concentrations both beginning and ending with low salt (150 mM KCl, 0.1% CHAPS, 0.1% NP-40, and 50mM Tris-HCl pH8). The salt content was gradually increased to a maximum of 500 mM KCl3. Bound proteins were eluted in 100 µl of the elution buffer containing 1% SDS. One-tenth or one-twentieth of the immunoprecipitate (v∕v) per sample was loaded onto 4–12% SDS-PAGE. The immunoprecipitated proteins were identified by MS∕MS and subsequently validated by immunoblot. Reciprocal IP was also used for validation of protein-protein interaction. The starting materials for reciprocal IP were equal quantities of solubilized ghosts (usually 1 mg per sample). The antibodies previously used for immunoblot of the band 3 pulldown were now used for reciprocal IP, with the same protocol as described above. Quantitative mass spectrometry by iTRAQ
Sample preparation, iTRAQ labeling and strong cation exchange fractionation
To facilitate mass spectrometry-based protein identification, coimmunoprecipitated samples were deglycosylated in two sequential steps: (1) a 2-hour mild β-elimination followed by Michael addition with dithiothreitol (BEMAD);4 (2) a 3-day cocktail enzymatic deglycosylation (Enzymatic Deglycosylation kit, Glyco Inc., USA). BEMAD took place at 50°C in 10% triethylamine, 0.1% NaOH, and 10 mM DTT (pH 12–12.5),4 followed by dialysis for subsequent enzymatic deglycosylation. The deglycosylated samples were then pooled into 4 groups (control & Mi.III technical duplicates) to be digested with trypsin, labeled with iTRAQ reagents (Isobaric Tagging for Relative and Absolute Quantitation, Applied Biosystems), purified and fractionated with SCX for quantitative mass spectrometry. Specifically, the deglycosylated immunoprecipitates were dissolved in 20 µL of 0.1% SDS in 500 mM TEAB, reduced, alkylated and tryptic digested with a protein to enzyme ratio 20:1 at 37°C overnight. The iTRAQ reagent was dissolved in 70 µL of ethanol and added to the digest. The mixture was incubated at room temperature for 1h. Control samples were divided into two groups and labeled with 114 and 115; Mi.III+ samples in two groups similarly labeled with 116 and 117. After labeling, the samples were admixed and dried down to a volume of 50 µL. The combined peptide mixture was fractionated by SCX chromatography on an 1100 HPLC system (Agilent) using a PolySulfoethyl A column (2.1 × 100 mm, 5µm, 300Å, PolyLC, Columbia, U.S.A.). Sample was dissolved in 4 mL of SCX loading buffer (25% v∕v acetonitrile, 10 mM KH2PO4, pH 2.8), pH was adjusted to 2.8 by adding 1 N phosphoric acid. The whole sample was loaded and washed isocratically for 30 min at 250 µL/min. Peptides were eluted with a gradient of 0–350 mM KCl (25% v∕v acetonitrile, 10 mM KH2PO4, pH 2.8) over 40 minutes at a flow rate of 250 µL/min. The absorbance at 214 nm was monitored and 10 SCX fractions were collected along the gradient. LC-MS∕MS analysis
Each SCX fraction was dried down and dissolved in 40 µL of 0.1% formic acid. The resulting fractions were analyzed on Qstar Pulsar™ (Applied Biosystems-MDS Sciex) interfaced with an Eksigent nano-LC system. Peptides were separated on a reverse-phase column packed with 10 cm of C18 beads (360 × 75 µm, 5 µm, 120Å, YMC ODS-AQ, waters, Milford, MA) with an emitter tip (New Objective, Woburn, MA ) attached. The HPLC gradient was 5–40% B for 60min (A, 0.1% formic acid; B, 90% acetonitrile in 0.1% formic acid) and the flow rate was 300 nL/min. Survey scans were acquired from m∕z 350–1200 with up to three precursors selected for MS∕MS using a dynamic exclusion of 45 sec. A rolling collision energy was used to promote fragmentation and the collision energy range was ~20% higher than that used for unlabeled peptides due to iTRAQ tags. Data analysis
The MS∕MS spectra were extracted and searched against SwissProt database using ProteinPilot™ software (Applied Biosystems) with Paragon™ method and the following parameters: all species, trypsin as enzyme (one missed cleavage allowed), cysteine static modification with methylmethanethiosulfate, and iTRAQ (peptide labeled at N-terminal and Lysine) as sample type. Mass tolerance was set to 0.2 atomic mass units for precursor and 0.15 atomic mass units for fragment ions. The raw peptide identification results from the Paragon™ Algorithm (Applied Biosystems) searches were further processed by the Pro Group™ Algorithm (Applied Biosystems) within the ProteinPilot™ software before they were displayed. The Pro Group™ Algorithm uses the peptide identification results to determine the minimal set of proteins that can be reported for a given protein confidence threshold. The peptide confidence threshold cutoff for this study was at least 90% confidence. The proteins that were identified with one peptide with confidence more than 90% (‘one hit wonders’) were inspected manually. For the purposes of formatting of the data for the Online Supplemental Tables and for archiving, data were reanalyzed with Scaffold 2.1.03 (Proteome Software). Briefly, the MS∕MS spectra were also searched against the SwissProt database (version 20070320; subset Homo sapiens; 165040 entries) using Protein Pilot in Mascot (Matrix Sciences) mode. The resulting .dat file was loaded into Scaffold v. 2.1.03 for subsequent searching with X! Tandem (a href="http://www.thegpm.org/" target="_blank">www.thegpm.org; version 2007.01.01.1) and protein validation. X! Tandem was set up to search the uniprot_sprot.fasta1 database (as of 10/22/08; 399749 entries). The fragment ion mass tolerance was 0.20 Da and the parent ion tolerance of 0.30 Da for all searches. Methyl methanethiosulfonate of cysteine and applied Biosystems iTRAQ™ multiplexed quantitation chemistry of lysine and the n-terminus were specified in Mascot and X! Tandem as fixed modifications. Oxidation of methionine and DTT_ST of serine and threonine were specified in Mascot and X! Tandem as variable modifications. Scaffold (version Scaffold_2.1.03, Proteome Software Inc., Portland, OR) was used to validate MS∕MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm (Keller, A et al. Anal. Chem. 2002; 74(20):5383–92). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, AI Anal Chem. 2003 Sep 1; 75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS∕MS analysis alone were grouped to satisfy the principles of parsimony. Measurement of HCO3−/Cl− transport capacities
The HCO3−/Cl− transport capacity across RBC membrane was monitored in terms of the changes of intracellular Cl− concentrations (Cl−in) with respect to changes of extracellular Cl− concentrations (Cl−out). Whole blood withdrawn within 1–2 days in Acid/Citrate/Dextrose tubes was washed with saline and fractionated for erythrocytes. Red cells (1% hematocrit) were incubated with 5 mM Cl−-sensitive dye SPQ (Invitrogen) in 1:1 H2O : Hank’s Balanced Saline Buffer (HBSS containing 137.9 mM NaCl, 5.33 mM KCl, 0.441 mM KH2PO4, 4.17 mM NaHCO3, 0.338 mM Na2HPO4, 5.56 mM glucose, and 20 mM HEPES (pH 7.5)) for 15 minutes (hypotonic shock), followed by recovery in isotonic HBSS for another 15 minutes, as previously described.5 SPQ-loaded erythrocytes were then washed with a balanced buffer free of Cl− and HCO3−, and then individually challenged with different concentrations of extracellular Cl− in a FluoroNunc 96-well plate (Nunc, USA). Cl−out variations were made from mixing a high NaCl buffer (100 mM Cl−) and a high NaNO3 buffer (0 Cl−). The high NaCl buffer contained (in mM) NaCl, 99.6; Na+-gluconate, 43.7; KH2PO4, 0.441; Na2HPO4, 0.338; glucose, 5.56; and HEPES, 20 (pH 7.5). The high NaNO3 buffer (in mM) contained NaNO3, 99.6; Na+-gluconate, 43.7; KH2PO4, 0.441; Na2HPO4, 0.338; glucose, 5.56; and HEPES, 20 (pH 7.5). Bicarbonate was supplemented by addition of 1 M NaHCO3. The Cl−-free and HCO3−-free buffer contained (in mM) K+-gluconate, 5.33; Na+-gluconate, 138; KH2PO4, 0.441; Na2HPO4, 0.338; glucose, 5.56; and HEPES, 20 (pH 7.5). Most of the buffer (i.e. 170 µL out of 175 µL per well) was removed by 400xg centrifugation prior to fluorescence measurement, in order to minimize the fluorescence interference from solution. The SPQ fluorescence from wet erythrocytes was excited at 350 nm, and its emission collected at 430 nm by SpectraMAX Gemini XS. For SPQ calibration, Cl− permeability was clamped by the double ionophore method.5 Briefly, SPQ-loaded RBCs were incubated in high-K+ calibration solutions containing 5 µM of nigericin (K+/H+ antiporter) and 10 µM of tributyltin (Cl−/OH− antiporter). Cl−out variations in the high K+ calibration solutions were made from mixing a high KCl buffer (100 mM Cl−) and a high KNO3 buffer (0 Cl−). The high KCl buffer contained (in mM) KCl, 99.6; K+-gluconate, 43.7; KH2PO4, 0.441; Na2HPO4, 0.338; glucose, 5.56; and HEPES, 20 (pH 7.5). The high KNO3 solution contained (in mM) KNO3, 99.6; K+-gluconate, 43.7; KH2PO4, 0.441; Na2HPO4, 0.338; glucose, 5.56; and HEPES, 20 (pH 7.5). Since SPQ fluorescence is quenched upon binding to Cl−, the relative level of intracellular Cl− is expressed as Fo/F where F is the SPQ fluorescence intensity and Fo is the maximal intensity in the absence of the quencher Cl−. Intracellular chloride concentrations were calculated from Fo/F based on individual calibration equations.5 Measurement of intracellular pH by flow cytometry
Fresh red cells were loaded with 1 µM fluorescent pH indicator carboxy SNARF-1 (Invitrogen) for 10 minutes, followed by HBSS wash. For intracellular pH (pHi) calibration, SNARF-1–loaded cells were incubated with high K+-balanced solutions containing 10 µM nigericin. The composition of the high K+-balanced solution is a modification of HBSS containing 100 mM K+ instead of Na+. The similar K+ concentrations inside and outside the cells were expected to drive pH equilibration inside and outside in the presence of K+/H+ antiporter nigericin. For Cl−-free stimulation, Cl− was substituted with gluconate− in the HBSS-based composition. For bicarbonate-free stimulation, 4.17 mM NaHCO3 was eliminated from the HBSS composition. The SNARF-1–labeled RBCs were divided and incubated in different conditioned solutions (e.g. calibration buffers of different pH’s, Cl−-free or HCO3−-free solutions). Flow cytometry measured the pHi-elicited SNARF-1 fluorescence from cells under different conditions (usually 10000 cells for each challenge). SNARF-1 was excited with a 488 nm excitation laser, and its emission at yellow and red fluorescence channels (FL2 and FL3, respectively in FACSCalibur) was collected. Because SNARF-1 exhibits a pH-dependent spectral shift, pHi was calculated from the ratios of fluorescence intensities (in geometric means) from FL2 and FL3, as described in the manufacturer’s protocol (Invitrogen).6 REFERENCES 1. Telen MJ, Scearce RM, Haynes BF. Human erythrocyte antigens. III. Characterization of a panel of murine monoclonal antibodies that react with human erythrocyte and erythroid precursor membranes. Vox Sang. 1987;52:236–243.
2. King MJ, Poole J, Anstee DJ. An application of immunoblotting in the classification of the Miltenberger series of blood group antigens. Transfusion. 1989;29:106–112.
3. Hsu K, Seharaseyon J, Dong P, Bour S, Marban E. Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel. Mol Cell. 2004;14:259–267.
4. Wells L, Vosseller K, Cole RN, Cronshaw JM, Matunis MJ, Hart GW. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics. 2002;1:791–804.
5. Pilas B, Durack G. A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). Cytometry. 1997;28:316–322.
6. van Erp PE, Jansen MJ, de Jongh GJ, Boezeman JB, Schalkwijk J. Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry. Cytometry. 1991;12:127–132.
Files in this Data Supplement:
- Table S1. Scaffold 2.1.3 peptide report (XLS, 116 KB)
- Table S2. Protein parameters (XLS, 44 KB)
- Figure S1. AE1 pulldowns were deglycosylated by a combination of BEMAD and enzymatic cocktails, prior to iTRAQ analysis (JPG, 107 KB)
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Anti-AE1 antibody (clone AE12-M) was chemically conjugated to magnetic beads coated with protein A, and the conjugated beads were used to pull down AE1-bound components from equal quantities of ghost lysates (m∕m). “Bead”, the negative-control immunoprecipitation lacking a reactive antibody. (A) Coomassie blue and silver stains for the immunoprecipitate of a control sample. (B) The efficacy of this combined deglycosylation approach was verified by glyco-silver stain. Only glycosylated proteins, and not unglycosylated or completely-deglycosylated ones, were shown on a glyco-silver stained gel.
- Figure S2. AE1 immunoblot of the deglycosylated ghost lysates (JPG, 214 KB)
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Freshly-isolated red cell ghosts were deglycosylated, solubilized, and loaded at equal protein quantities (2 mg/sample) for immunoblot comparison. Homozygous Mi.III samples are marked +∕+.
- Figure S3. Mi.III+ and the control red cells exhibited similar cytoskeleton organization (JPG, 193 KB)
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Extracted RBC cytoskeletons were negatively stained and visualized by transmission electron microscopy (JOEL JEM-1200EXII). The star symbol indicates a cell membrane glob not removed by Triton X-100 solubilization. JC, junctional complexes; Sp, spectrin filaments; Ank, ankyrin. Spectrin lengths and cytoskeleton organization were not significantly different between Mi.III+ and control red cells. Scale bar = 100 nm.
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