OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL

Analytical Biochemistry 513 (2016) 7e20

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OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL Jeffrey C. Howard a, Angelique Florentinus-Mefailoski a, Peter Bowden a, William Trimble b, Sergio Grinstein b, John G. Marshall a, * a b

Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 December 2015 Received in revised form 21 June 2016 Accepted 19 July 2016 Available online 7 August 2016

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (~9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads. © 2016 Elsevier Inc. All rights reserved.

Keywords: OxLDL IgG Phagocytosis SYK MS SQL

1. Introduction Microscopic aggregates of oxLDL bind to receptors on the cell surface of macrophages in the intima of the arteries triggering the inflammatory phagocytic activation that may be a crucial step towards atherosclerosis leading to heart attack and stroke [1e4]. Macrophage scavenger receptors recognize pathogen molecules on the surface of micro-organisms and engulf foreign particles via

Abbreviations: ITAM, immunoreceptor tyrosine-based activation motifs; CBBR, Coomassie brilliant blue; ITIM, immunoreceptor tyrosine-based inhibitory motifs; SYK, spleen tyrosine kinase; pSYK525,526, phosphorylated SYK on amino acid residues Tyr-525 and Tyr-526; SFK, Src family kinases; oxLDL, oxidized LDL; LARC, Ligand affinity receptor chromatography; mgf, Mascot generic format; SQL, structured query language; ESI, electrospray ionization; STYP, serine threonine tyrosine phosphorylation; IBPC, internalized micro beads per cell; TBPC, total micro beads per cell; DIC, differential interference contrast; IF, immunofluorescence; TukeyHSD, Tukey honest significant difference; sem, standard error of the mean; SFM, serum free media; GM, growth media. * Corresponding author. Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, ON, Canada. E-mail address: [email protected] (J.G. Marshall). http://dx.doi.org/10.1016/j.ab.2016.07.021 0003-2697/© 2016 Elsevier Inc. All rights reserved.

phagocytosis [5]. Phagocytic activation in response to aggregates of pathological molecules like oxLDL [6,7] may trigger a chronic inflammatory process in macrophages that leads to cardiovascular disease [2,4,8]. The Toll Like Receptor 4 expressed on the plasma membrane is a scavenger receptor for oxidized low density lipoprotein (oxLDL) that plays an important role in atherosclerosis [9e13], and may signal in part via spleen tyrosine kinase (SYK) via the adaptor TRAF [14,15] [16]. The human monocyte cell line U937 [17] cultured in PMA may differentiate into macrophage-like cells that rapidly engulf large amounts of cytotoxic oxLDL [18e20]. The phagocytosis of oxLDL by macrophages may be an important aspect of atherosclerosis [2,4,7,21] but there is little information available regarding the mechanism that engulfs oxLDL aggregates [6,7]. Scavenger receptors that recognize many ligands, such as CD36, may interact with Fc receptors for the internalization of oxLDL particles [22]. Microbeads coated with bulk human immunoglobulin IgG are presumably recognized by the Fc receptors FcgRs [23,24]. Phagocytic ligands like the Fc region of IgG can be presented on microchromatography supports and subsequently used to both activate and capture receptor complexes from the surfaces of live cells

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[25,26]. Previously, ligand affinity receptor chromatography of the IgG-Fc receptor complex revealed that RHOG, CRKL and P115RHOGEF regulate IgG phagocytosis [25] in agreement with independent studies [27e29]. Ligand binding triggers FcgR clustering and activation through the phosphorylation of key tyrosine residues located within immunoreceptor tyrosine-based activation motifs (ITAM) that are part of either the receptor itself (FcgRIIA), or transmembrane adaptor proteins that non-covalently bind to FcgRI [30,31]. Receptor activation via ITAM signaling is modulated by inhibitory receptors that contain immunoreceptor tyrosine-based inhibitory motifs, or ITIMs [32]. The protein kinase SYK binds with high affinity to ITAMs phosphorylated on both tyrosine residues while protein phosphatases SHP1 and INPP5D are primarily recruited by phosphorylated ITIMs [26,33]. Fatal autoimmune diseases were observed in a number of FcgRIIB (ITIM containing inhibitory receptor) deficient strains of mice [34,35]. A pharmacological screen recently showed that SYK is required for the inflammatory phagocytic engulfment of aggregated oxLDL [1]. SYK is a proximal cell signaling protein associated with FcgRs [36,37] and FcεRI [38]. SYK functions as an adaptor molecule for SH2 containing proteins, such as PLC [39], due to the phosphorylation of one or more of its tyrosine residues [40]. SYK may be required for the effective expression of a functional Fc receptor activity since macrophages from SYK
/
mice do not bind IgG coated beads or ingest IgG coated erythrocytes [41,42]. SYK has also been shown to be activated by TLR4 that participates in inflammatory engulfment of oxLDL [11,13,14,43e45]. Two siRNA specific to the SYK(L) isoform have been described, but their effect on the phagocytosis of oxLDL has not been determined [46]. The recruitment of SYK to both oxLDL and IgG coated microbeads was examined by ligand affinity receptor chromatography and liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) indicating that SYK is enriched in the phagocytic receptor complex of aggregated oxLDL. In contrast to experiments with SYK
/
knock out mice that apparently do not develop or express effective Fc receptor complexes [41,42], or dominant genetic mutations that might have pleiotropic effects [47,48], this study shows SYK siRNAs had little effect on the phagocytosis of IgG coated microbeads but was required for oxLDL receptor mediated phagocytosis. Herein the isolation, protein purification and identification of SYK by Ligand Affinity Receptor Chromatography revealed the primary structure of the specific isoform of SYK that participates in the oxLDL receptor complex. Thus novel ligand affinity receptor chromatography, together with tandem mass spectrometry, specifically isolated and identified the long isoform of SYK (SYK-L), and most of the SYK interaction network, as specific to the oxLDL ligand that was confirmed by pharmacological inhibitors, plus independent SYK and SYK-L siRNA. 2. Methods 2.1. Materials U937 human leukemia cell line was obtained from the American Type Culture Collection (Manassas, VA). RPMI 1640 and heat inactivated Fetal Bovine Serum were obtained from Mediatech Inc. (Corning CELLGRO). Bulk human IgG 1 and the SDS-PAGE molecular mass standards were obtained from SIGMA-ALDRICH (St Louis Mo, USA). Microbeads (2 mm diameter, polystyrene beads) were obtained from Bangs Laboratories. Affinity reagents included, rabbitanti-human phospho-tyrosine-SYK (pSykY525,526) antibody (EMD Millipore) or a rabbit-anti-human SYK antibody (Cell Signaling Technologies) and included a monoclonal SYK-01 (Genway Biotech Inc.), rabbit anti-phospho-Syk (pTyr525,526) (Calbiochem, EMD Millipore). Fluorescent and HRP-labeled secondary

antibodies were obtained from Jackson Immuno-Research Labs. Cy3 labeled Universal Negative Control #1 siRNA and SYK siRNAs (1s:Cy3-GAUGUACGAUCUCAUG AAUTT, 1as:AUUCAUGAGAUCGUACAUCTT; 2s:Cy3-GUAUCCAGAGGAAUUGAUUTT, 2as:A AUCAAUUCCUCUGGAUACTT) were prepared by Sigma-Aldrich (St. Louis MO, USA). DyLight547-labeled SYK (L) specific siRNAs (L1s 50 -GUUCCCAUCCUGCGACUUGTT, L1as 50 -CA AGUCGCAGGAUGGGAACTT; L2s 50 -GGUCA GCGGGUGGAAUAAUTT and L2as 50 -AUUAU UCCACCCGCUGACCTT) [46] were obtained from Thermo Fisher Scientific Biosciences. EM grade paraformaldehyde (16%) was obtained from Electron Microscopy Sciences. Pierce Slide-A-Lyzer Dialysis Cassettes (10K MWCO) were obtained from Thermo Scientific. All analytical grade solvents were obtained from Caledon Laboratories (Georgetown ON, Canada). SYK inhibitor BAY 613606 was obtained from EMD Millipore. The SYK inhibitors Piceatannol and the kinase inhibitor PP1 [48] were obtained from Sigma-Aldrich. All other buffers, salts and reagents were obtained from Sigma-Aldrich (St. Louis MO, USA). 2.2. Ligand microbeads Polystyrene microbeads (~100 ml, 2 mm diameter, 10% v/v stock slurry suspension, ~2.185  1010 microbeads/ml) were briefly pelleted (~12,000 rcf, 30s) and washed in PBS (3x) prior to being opsonized with appropriate affinity ligands as previously described [49]. A stock solution of human IgG (10 mg/ml) was prepared in PBS [25]. Oxidized low density lipoprotein was prepared as previously described and the final volume of oxLDL was extensively dialyzed and stored in the dark at 4  C for up to 4 weeks [50]. Micro-bead suspensions containing 100 ml of either 10 mg/ml oxLDL, antiCD36 IgA, anti-CD36 IgG, or mixed human IgG were gently incubated for 1 h at room temperature (RT) using a Ferris Wheel rotator. The IgG-, IgA, or oxLDL-coated microbeads were then washed (3x) (~12,000 rcf, 30 s) with PBS followed by (5 min, RT, rotator, pelleted and then re-suspended in ~100 ml of PBS. 2.3. Affinity chromatography treatments The Fc domain of IgG affixed to microbeads presumably interacts with the Fc receptor. The variable Fab domain of the divalent or trivalent IgA anti CD36 presumably binds the CD36 receptor on the surface of the live macrophage cells. Specific affinity chromatography treatments and control groups included: Live cells þ antiCD36 IgA coated microbeads (Live CD36 IgA); Crude cell extract þ anti-CD36 IgA coated microbeads (Crude CD36 IgA); Live cells þ anti-CD36 IgG coated microbeads (Live CD36 IgG); Crude cell extract þ anti-CD36 IgG coated microbeads (Crude CD36 IgG); oxLDL cell media þ uncoated microbeads (Media oxLDL None); Live cells þ human IgG coated microbeads (Live hIgG); Crude cell extract þ human IgG coated microbeads (Crude hIgG); IgG cell media þ uncoated microbeads (IgG Media None); Live cells þ oxLDL coated microbeads (Live oxLDL); Crude cell extract þ oxLDL coated microbeads (Crude oxLDL). The controls to demonstrate the potential for non-specific binding was Crude cell extract þ uncoated microbeads (Crude None) and Live cells þ uncoated microbeads with no ligand (Live None). 2.4. U937 cell cultures The human monocyte cell line U937 was expanded and maintained in RPMI 1640 media supplemented with 10% heat inactivated fetal bovine serum (humidified, 5% CO2, 37  C). The U937 cells were treated with PMA (100 nM) for 3 days in order to induce monocyte differentiation into adherent macrophages [18]. A model system of adherent human macrophages

J.C. Howard et al. / Analytical Biochemistry 513 (2016) 7e20

binding to oxLDL, anti CD36 IgA, anti CD36 IgG or human bulk IgG was created with the PMA treated U937 cells in culture. Ligand affinity chromatography experiments with oxLDL, anti CD36 IgA and IgG as well as bulk human IgG (hIgG) were performed in 75 cm2 flasks. Pharmaceutical inhibitor and siRNA experiments cells were cultured in 6 well dishes on 25 mm glass cover slips.

2.5. Ligand Affinity Receptor Chromatography Ligand-receptor complexes were isolated from the surface of live human cells or crude homogenates as previously described [25,49,51]. Microbeads with or without ligands were added to the 75 cm2 flasks of adherent U937 cells on ice cells for 30 min and excess beads washed away. The cells were warmed to 37  C and sampled immediately or at 30 min, 1 h, 2 h and 4 h and combined for isolation of the receptor complex. For crude extract treatments the cells were scraped on ice in PBS and the microbeads added immediately prior to disruption in a French Press. The microbeadligand-receptor complex was collected by centrifugation (14,000 rcf, 5 min, 4  C), re-suspended in PBS and then re-collected to wash the beads 3x. The ligand-receptor microbeads were purified over sucrose gradients at 100,000 RCF as previously described. Proteins bound to the microbeads were eluted using a NaCl step-gradient (200 ml PBS with 50e1000 mM of added NaCl), followed by a final 50% acetonitrile extraction step. Eluted NaCl and 50% acetonitrile fractions were digested with trypsin made to final concentration of 200 mM with urea in pH 8.8 tris and 5% acetonitrile. The residual proteins on the beads digested with a decreasing step gradient of organic solvents were made to final concentration of 200 mM with urea in pH 8.8 tris and 5% acetonitrile. The entire experiment with all affinity treatments was replicated three times on independent occasions, using fresh cells and reagents a few months apart.

2.6. LC-ESI-MS/MS analysis Replicate blocks of the fractionated sub-samples from each treatment were randomly and independently sampled. A new (150 mm x 15 cm) analytical C18 column was struck and quality control tested for each ligand affinity receptor isolation that was eluted with increasing salt into 17 step gradient fractions of intact proteins prior to digestion of the fractions, and the beads for LC-ESMS/MS, and then the column was discarded [49,51]. The LC-ESI-MS/ MS system was calibrated with the manufactures standard mixture, tuned with GluFib and Angiotensin before sensitivity was tested using an infusion of increasing GluFib and Angiotensin concentrations. A fresh C18 column was struck, conditioned and quality controlled for each individual sample fraction-set using a digest of cytochrome C, alcohol dehydrogenase and glycogen phosphorylase B as previously described [52]. Eluted fractions from microbeadreceptor complexes were digested with sequencing grade trypsin and collected over C18 micro chromatography columns [53]. Bound peptides were eluted using 2 ml of a 65% acetonitrile þ5% formic acid mixture and the eluent was diluted to 20 ml using 5% formic acid prior to manual injection via a 20 ml loop and resolved by C18 reverse phase liquid chromatography (LC) over a 90 min gradient from 12% to 65% acetonitrile at a flow rate of ~0.2 ml per min with an Agilent 1100 series capillary pump. A random and unbiased sampling of the five most intense spectral peaks eluting at any time was permitted up to 200 milli seconds to fill the ion trap and the MS/MS scan was automatically repeated up to four times for each analyte to achieve a high signal to noise ratio [49,52,54,55].

9

2.7. MS/MS spectra correlation MS/MS spectra from parent ions with at least 1000 arbitrary intensity counts (to avoid type I error) were exported as Mascot Generic Files (.mgf) for correlation and statistical analysis [49,51,52,54,55]. The MS/MS spectra were correlated to fully tryptic peptides of a federated human protein library using the rigorous OMSSA and X!TANDEM algorithms, plus the sensitive SEQUEST algorithm, that automatically considered the loss of water, ammonia and the addition of phosphate PO3 to the precursor peptides and/or resulting fragments [56e58]. Several constraints were used during the fully tryptic searches, including: (i) precursor values were considered with 0 to ± 3 m/z units; (ii) daughter fragment ion mass tolerance was within 0.5 Da; (iii) only peptide charged states of þ2, þ3 were considered; and (iv) up to three missed cleavages were allowed [49,52,54]. The data from SEQUEST [57] provided via the manufacturer's algorithm was further limited to the default setting of 0.05 maximum delta correlation of parent ions with maximum peptide mass set to 5000 Da and peptide length set to  6 amino acids. The correlated peptide sequences were parsed into an SQL relational database [52,59,60]. The results of the correlation algorithm with no modification (Filter 0) was filtered using the unique hash tag to ensure that only a single top scoring charge state (filter 1) and peptide sequence (filter 2) for each MS/MS spectra was accepted (Filter 2). As shown, the large majority of precursor peptide delta values are within ±1 m/z unit. 2.8. Protein-protein interaction networks using cytoscape The Michigan molecular interaction (MiMI v3.1) Plugin feature of Cytoscape (v2.8.3) [61] was used to build a protein-protein interaction (PPI) network based on human SYK. All known SYK binding proteins were retrieved using the following settings: (i) MiMI query: SYK; (ii) species: homo sapiens; (iii) molecule type: protein; (iv) data source: all; and (v) retrieval type: Query protein þ nearest neighbors. In total 92 proteins were identified from all of the available databases (BIND, CCSB, DIP, GRID, HPRD, IntAct, KEGG, MDC, MINT, PubMed, reactome). Precursor peptide counts for all detected SYK interacting protein with their affinity chromatography treatments and control groups were retrieved from the U-937 SQL Server database, imported into Cytoscape and mapped onto the network nodes using the visual mapping browser of Cytoscape. 2.9. Western Blot analysis Ligand affinity receptor samples were washed with PBS, retreated with protease inhibitors, and isolated by sucrose density gradient ultracentrifugation, washed with PBS, and immediately re-suspended in 2x Laemmli buffer and boiled. Protein samples (40 mg total protein) were subjected to Tricine gel (7%) electrophoresis, transferred to PVDF membrane, stained with CBBR in 50% MEOH to confirm equal loading, de-stained in 50% MEOH and probed with a Rabbit-anti-human phospho-tyrosine-SYK (pSykY525,526) antibody or a mouse anti-human SYK antibody. ECL solutions were prepared as previously described [62] and the resulting protein-specific band intensities digitally captured using a Bio-Rad ChemiDoc™ XRS þ System. The mass of the ~72 kDa SYK band was calculated relative to molecular mass standards. 2.10. Pharmacological inhibitor treatments The U937 cells were seeded onto 25 mm diameter cover slips in 6 well plates and differentiated with PMA for 72 h, the wells were washed 3x to remove remaining serum factors from the growth

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media. A stock solution at 1000 x the final concentration required for cellular assays was prepared in water for BAY 613606 (5 mM) while piceatannol (15 mg/ml) and PP1 (15 mM) were prepared in DMSO. The stock inhibitor solutions were diluted 2 ml in 2 ml (0.1% DMSO final) of experimental media that was then applied to the cells for 1 h prior to phagocytosis assays.

washes and one PBS wash (5 min, shaker, RT, dark). Glass coverslips were then mounted onto glass slides with DAKO aqueous fluorescent mounting media for laser confocal microscopy. 2.14. Laser confocal microscopy

Differentiated U937 cells in 6 well plates on glass cover slips were maintained for 72 h prior to experiments (~70% confluent) in growth media. Differentiated U937 cells were transfected with siRNAs using Extreme-Gene (XGene, Roche) transfection reagent according to the manufacture's established protocols. Briefly, 200 pMoles of either Cy3-or DyLight547-labeled control siRNAs or SYK siRNAs were used per well. Growth media was changed 2 days post-transfection prior to performing phagocytosis assays (day 3).

Images were acquired using a Zeiss LSM510 META laser confocal microscope equipped with an AxioCam high resolution digital camera and Plan-Neofluor 40/1.3 Oil DIC or Plan-Apochromat 100/1.4 Oil DIC (Differential interference) objective lens. Image acquisition settings were optimized to avoid pixel saturation and treatments were recorded under identical laser, amplifier, background and filter conditions. For CY3 the 543 nm laser with the NFT 545 mirror and the band pass 565 to 615 emission filter were used. For CY5 the 633 nm laser with NFT 545 and the band pass 565e615 was employed. The photo multiplier tube used was a Hamamatsu R6357.

2.12. Phagocytosis assay

2.15. Statistical analysis

IgG or oxLDL coated microbeads as prepared above in 100 ml of PBS were used for each 6 well plate. Equal volumes (~15 ml) of IgGcoated microbeads were then added to each well of the 6-well plate. Cells were then incubated with the prepared microbeads for 2 h at 37  C, gently washed (3x) with cold PBS to halt phagocytosis and then immediately fixed with PBS containing 4% paraformaldehyde (EM grade, 20 min, shaker, RT). The assays were performed either in growth media with serum refreshed 1 h prior to assay, immediately after transfer to serum free media, or after the cells were serum starved for 1 h prior to assays. After incubation with the ligand beads for up to 2 h as indicated the excess beads were gently washed away and the samples were fixed in 0.1% paraformaldehyde for 20 min at room temperature, the fixation was stopped by washing with ice cold PBS 3x, quenched with 5% glycine (5 min, shaker, RT), washed 3x with PBS (5 min, shaker, RT). The external IgG beads were stained (30 min, shaker, RT) with a 1:10,000 dilution of a DyLight649-labeled Donkey-anti-human IgG. Cells were washed (3x) with PBS (5 min, shaker, RT) and then mounted onto glass slides using DAKO aqueous fluorescent mounting media. The external oxLDL-coated microbeads were stained with a ~1:800 dilution of a Rabbit anti-Cuþ2 oxLDL polyclonal antibody (1 h, shaker, RT). Cells were then washed (3x) with PBS (5 min, shaker, RT) and then incubated with a 1:10,000 dilution of DyLight labeled secondary antibodies. Cells were then washed 3x with PBS (5 min, shaker, RT) and the coverslips mounted onto glass slides using DAKO aqueous fluorescent mounting media. Phagocytosis measurements were then quantified by counting the total versus external ligand beads by analyzing laser confocal images.

The protein and peptide frequency and intensity data stored in the SQL Server database. The p-values for SYK were computed from the protein of SYK peptide probabilities provided by X!TANDEM [56]. The frequency data was analyzed using a 64-bit open source R Studio program Chi Square (c2) and log transformation followed by ANOVA [63].

2.11. siRNA treatment

2.13. Confirmation of anti-SYK siRNA knockdown by immunofluorescence U937 cells treated with control, or anti SYK siRNA, were fixed and then blocked with PBS containing 5% skim milk and 1% donkey normal serum (30 min, shaker, RT). Cells were then washed with PBS before Immunoflourescent (IF) cell staining that was carried out on permeabilized cells previously fixed and blocked. Briefly, cells were blocked (PBS þ 0.1% Triton-X100þ 5% skim milk þ 1% goat serum) for 30 min (shaker, RT) and then washed 3x with PBS (5 min, RT, shaker). Cells were incubated for 1 h, at room temperature with a 1:25 dilution of a monoclonal anti-SYK antibody (SYK01, Genway Biotech Inc.) and then washed 3x with PBS (5 min, shaker, RT). Cells were then incubated with a 1:10,000 dilution of DyLight649 labeled Donkey-anti-mouse secondary antibody (30 min, shaker, RT, dark) followed by three PBS/0.01% triton

3. Results 3.1. SYK protein interaction complex The protein tyrosine kinase SYK has been previously associated with the phagocytosis of IgG and the engulfment of oxLDL [1,22,26,33] and was observed to be detected most frequently in oxLDL and anti CD36 IgA affinity chromatography (Supplemental Data and Tables). Many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), as well as RNA binding and metabolism proteins were also observed with oxLDL ligands. SYK itself and many of the known interacting proteins including BLNK, BTK, CBL, CBLB, CD19, CD22, CD3E, CD79A, CD79B, COL1A1, COL1A2, CRKL, CSF2RB, CSF3R, CTTN, DBN, DTYMK, DUSP3, EGFR, EPOR, ERBB2, ERBB3, ERBB4, EZR, FGR, FYN, GAB2, GP6, GRB2, HCLS1, HMGCS2, HNRNPU, IL15RA, IL2RB, ITGB2, JAK1, JUN, LAT, LAX1, LCK, LCP2, LYN, MAP4K1, MAPK3, MAX, MS4A2, MSN, MYC, NFAM1, NFKBIB, PAG1, PIK3AP1, PIK3R1, PIK3R2, PLCG1, PLCG2, POU2AF1, PRKCA, PRKD1, PTK2, PTK2B, PTPN6, PXN, RASA1, RPS6KA1, RPS6KA2, SELPLG, SH2B2, SH3BP2, SHC1, SIT1, SLA, SLC4A1, SNCA, SRC, STAT1, STAT3, STAT5A, TAF1, TLR4, TRAF6, TUBA1A, TUBA4A, TYROBP, UBB, UCP2, VAV1, and VAV2 were summarized together as peptide-to-protein counts across treatments (Table 1). The peptide to protein counts for each gene symbol were mapped onto the SYK protein-protein interaction (PPI) network using Cytoscape [61]. The peptide-to-protein counts of the putative SYK binding proteins were compared between ligands and controls across the ligand affinity conditions and some including HNRNPU and MSN showed high levels of enrichment with the oxLDL ligand (Table 1). Statistical analysis of the SYK PPI network for each LARC group revealed that the oxLDL-ligand presented to live cells showed significant (p < 0.012) enrichment for SYK binding proteins when compared to all six control groups. Anti-CD36/IgA isolated receptor complex was also significantly (p < 0.012) enriched for SYK and its binding partners, but only when compared to media (p < 0.01) and live cell uncoated microbead (p < 0.013) control groups. The peptide per protein count data indicates that SYK and SYK-interacting proteins were frequently

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Table 1 The mean log10 precursor (peptide) intensity and total peptide counts for previously established protein interaction network of SYK compared across different affinity ligands presented to live cells versus incubation with crude extracts. The correlation of MS/MS spectra to peptides was performed for fully tryptic peptides or tryptic peptides plus phosphorylation at serine, threonine of tyrosine (STYP). The summary provided is the aggregate results for the gene symbols BLNK, BTK, CBL, CBLB, CD19, CD22, CD3E, CD79A, CD79B, COL1A1, COL1A2, CRKL, CSF2RB, CSF3R, CTTN, DBN, DTYMK, DUSP3, EGFR, EPOR, ERBB2, ERBB3, ERBB4, EZR, FCER1G, FCGR1A, FCGR2A, FGR, FYN, GAB2, GP6, GRB2, HCLS1, HMGCS2, HNRNPU, IL15RA, IL2RB, ITGB2, JAK1, JUN, LAT, LAX1, LCK, LCP2, LYN, MAP4K1, MAPK3, MAX, MS4A2, MSN, MYC, NFAM1, NFKBIB, PAG1, PIK3AP1, PIK3R1, PIK3R2, PLCG1, PLCG2, POU2AF1, PRKCA, PRKD1, PTK2, PTK2B, PTPN6, PXN, RASA1, RPS6KA1, RPS6KA2, SELPLG, SH2B2, SH3BP2, SHC1, SIT1, SLA, SLC4A1, SNCA, SRC, STAT1, STAT3, STAT5A, SYK, TAF1, TLR4, TRAF6, TUBA1A, TUBA4A, TYROBP, UBB, UCP2, VAV1, VAV2 with Filter 2 (best charge state and peptide sequence for each MS/MS). The mean precursor intensity for the whole set of gene symbols with standard deviation and the number of observations are shown over the ligand affinity and control treatments. The probability that the precursor intensity means are the same across treatments by ANOVA is  E-16 (see Supplemental Fig. 1). Treatment/Affinity resin

log intensity

STND_DEV

Peptide count

Tryptic

Log intensity

STND_DEV

Peptide count

Tryptic STYP

Live antiCD36_IgA Crude antiCD36_IgA

4.99 4.92

0.49 0.44

34,904 18,193

4.95 4.85

0.51 0.45

19,487 11,130

Live antiCD36_IgG Crude antiCD36_IgGS

5.05 4.75

0.49 0.42

10,085 5166

5.01 4.69

0.52 0.44

5670 3273

Live oxLDL Crude oxLDL Media_oxLDL Beads

5.02 4.71 4.43

0.54 0.48 0.53

46,126 29,364 235

4.97 4.65 4.12

0.55 0.49 0.46

27,015 17,791 140

Live IgG Crude IgG Media IgG Beads

4.72 4.83 4.39

0.54 0.49 0.59

10,205 9751 473

4.65 4.77 4.20

0.55 0.51 0.65

6140 5898 323

Live Uncoated Beads Crude Uncoated Beads

4.61 5.03

0.61 0.50

567 14,343

4.73 4.97

0.60 0.52

694 9098

detected with the oxLDL or anti CD36 IgA ligands from live cells compared to IgG or controls (Fig. 1). The potential SYK substrates PIK3AP1, PIK3R1, PIK3R2, PLCG1, PLCG2 and VAV1&2 were observed with the oxLDL ligand-receptor complex.

3.2. Tryptic peptides and phosphopeptides from human SYK The oxLDL- and IgG-ligand receptor complexes were isolated alongside controls from adherent U937 cells leading to the identification of seven unique accession numbers for SYK that differ by at least one amino acid but showed the greatest number of correlations to the long isoform of spleen tyrosine kinase SYK (L) NP_003168.2 that migrates at ~72 kDa. The probability that SYK was mis-identified from a total of 1216 peptides correlations from precursor ions 1000 counts was calculated by the OMSSA (E
78.3) and X!TANDEM (E
207.6) algorithms while SEQUEST showed a large sum X-Corr value (359.2) where 2.5 to 3.5 is generally considered by be significant (Supplemental Table 1). High-intensity peptides that ranged from about 9*E3 to 2*E5 counts were correlated to SYK and especially SYK (L) NP_003168.2 or similar forms of SYK that contain the sequence TWSAGGIISRIKSYSFPPGHRKK but the peptide data does not rule out other isoforms. Filtering the results using an SQL database indicated that 243 MS/MS cannot be better explained by peptides at a different charge state, and 211 of which cannot be better explained by considering an alternative amino acid sequence (Supplemental Table 2). The oxLDL ligand efficiently captured SYK from the surface of live cells compared to IgG control, media controls or uncoated beads (Supplemental Table 2) in agreement with previous results [20,43,46]. Overlapping and nested amino acid sequences, and multiple correlations to the same polypeptides on the SYK protein were observed (Fig. 2A). Multiple serine, threonine or tyrosine (STY) phosphorylated peptides were also identified that were tightly clustered in the SH2 domains, the ATP binding site, catalytic domain and the activation loop from the live oxLDL and anti CD36 IgA affinity treatments (Fig. 2B). The phosphorylation

sites S379 & T384 that were previously annotated in the SYK (L) NP_003168 General Peptide Report were observed by LC-ESI-MS/ MS. The preferred cleavage sites of trypsin were consistently located at a local hydrophilic maximum. Many distinct, but overlapping or nested peptides and/or phosphopeptides from SYK(L) were identified from the MS/MS spectra that essentially rule out any possibility of false positive identification [64] (see Supplemental Table 3). 3.3. Western blot of SYK High levels of SYK(L) phospho Y525/Y526 at ~72 kDa was associated with oxLDL and to a lesser degree with IgG-activated receptor complexes in U937 cells (Fig. 3). The oxLDL and human bulk IgG also showed strong ~72 kDa SYK(L) bands (Fig. 3). The Western blot data agreed with the trends to greater levels of SYK phosphorylation and greater levels of SYK recruitment to the oxLDL phagocytic receptor complex observed by mass spectrometry. The sensitive LC-ESI-MS/MS detected SYK less frequently and/or at lower intensity levels in the controls in agreement with the Western Blot that shows SYK from crude extracts may adsorb to the surface of polystyrene beads in the absence of detergents and blocking agents. 3.4. Pharmacological inhibitors of SYK The function of SYK during oxLDL- and IgG-coated microbead phagocytosis was examined using pharmacological inhibitors that target the catalytic site of SYK or closely related kinases. The mean number Internalized microBeads Per Cell (IBPC) and Total microBeads Per Cell (TBPC) were quantified by differential immunofluorescent (IF) staining and confocal microscopy. In agreement with previous results [1], the SYK inhibitor BAY 613,606 (p < 0.05) impaired oxLDL-coated microbead phagocytosis in both the presence and absence of serum, but had no significant effect on the phagocytosis of IgG-coated microbeads (Fig. 4). In addition, the

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Fig. 2. The primary structural summary of the affinity mass spectrometry results for the long isoform of Spleen Tyrosine Kinase SYK(L) NP_003168. Panels: A, The location of peptide detected at least 5 times in the FASTA sequence of SYK with phosphorylations sites shown in red and the location of the 23 amino acid linker region is shown “¼TWSAGGIISRIKSYSFPPGHRKK¼”. Symbols; B, the clustering of phosphorylation sites in discrete locations in SYK that map to the N terminus, the SH2 domains, the ATP binding site and the catalytic domain including a cluster in both substrate binding site and the activation loop where agreement with the phosphorylation sites of NP_003168 SYK(L) are shown in red. A peptide from the SYK activation region shared by some SYK short and long protein sequences “kfdtlwqlvehysyk” was detected. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

independent kinase inhibitors piceatannol and PP1 also significantly reduced the receptor mediated phagocytosis of oxLDL compared to controls.

3.5. Anti SYK siRNAs Experiments with the pooled set of SYK siRNAs that targets both the long (L) and short (S) isoforms SYK inhibited the phagocytosis of oxLDL but that had no effect on the engulfment of IgG (Fig. 5). Cells transfected with SYK siRNAs in serum or serum free experimental media showed significantly lower phagocytosis of oxLDL aggregates compared to controls siRNA transfected cells (Fig. 5). In

contrast, serum-starved cells transfected with SYK siRNA showed significantly higher mean internalization of oxLDL microbeads compared to controls. SYK siRNAs did not significantly alter the number of engulfed IgG-coated microbeads compared to controls in either the presence or absence of serum (Fig. 5). The results indicate that SYK differentially regulated the engulfment of oxLDL in the context of yet unknown serum factors.

3.6. Immuno confirmation of SYK knockdown by siRNA The knockdown of SYK expression using siRNAs was confirmed by immuno fluorescent staining using SYK-specific antibodies and

Fig. 1. Quantitative analysis of the SYK protein interaction complex between oxLDL versus IgG ligands presented to live cells on 2 mm polystyrene microbeads. The Gene Symbols of the known interacting proteins of SYK were obtained from the Michigan Molecular Interaction (MiMI v3.1) Panels: A, the oxLDL ligand; B, the IgG ligand. Plugin feature of Cytoscape and matched to the peptide to protein counts (Supplemental Data 1). The peptide to protein counts of many of the known SYK binding proteins, BLNK, BTK, CBL, CBLB, CD19, CD22, CD3E, CD79A, CD79B, COL1A1, COL1A2, CRKL, CSF2RB, CSF3R, CTTN, DBN, DTYMK, DUSP3, EGFR, EPOR, ERBB2, ERBB3, ERBB4, EZR, FCER1G, FCGR1A, FCGR2A, FGR, FYN, GAB2, GP6, GRB2, HCLS1, HMGCS2, HNRNPU, IL15RA, IL2RB, ITGB2, JAK1, JUN, LAT, LAX1, LCK, LCP2, LYN, MAP4K1, MAPK3, MAX, MS4A2, MSN, MYC, NFAM1, NFKBIB, PAG1, PIK3AP1, PIK3R1, PIK3R2, PLCG1, PLCG2, POU2AF1, PRKCA, PRKD1, PTK2, PTK2B, PTPN6, PXN, RASA1, RPS6KA1, RPS6KA2, SELPLG, SH2B2, SH3BP2, SHC1, SIT1, SLA, SLC4A1, SNCA, SRC, STAT1, STAT3, STAT5A, SYK, TAF1, TLR4, TRAF6, TUBA1A, TUBA4A, TYROBP, UBB, UCP2, VAV1, and VAV2 are shown.

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5. Discussion 5.1. Ligand affinity receptor chromatography The results show that U937 monocytes cultured in PMA apparently expressed functional phagocytic receptor complexes for aggregated IgG and oxLDL [1]. The ligand microbead system isolated the CD19, TLR4 and several Fc receptors that may function in the recognition and engulfment of pathogen associated molecules by macrophages such an oxLDL [16,19,22,65,66]. The ligand microbeads apparently provide a model of monocytes that bind and adhere onto the aggregated inflammatory ligands such as oxLDL in the fatty streaks of the artery wall where they differentiate into a macrophage phenotype [1,67,68]. Human U937 cells challenged with oxLDL versus IgG microbeads may serve as a useful comparative model of authentic human macrophages binding aggregated oxLDL [1] to perform the isolation and primary identification of the factors required for innate immune functions. In addition to the Fc and Toll receptors and receptor kinases such as EGFRs, many of the molecules are known to interact with SYK by independent genetic or biochemical methods [61], including TLR4 and TRAF6 [16] were observed in the complex. A previous pharmacological screen indicated that SYK may play a key role in the phagocytosis of oxLDL. Herein the importance of SYK-L for the phagocytosis of oxLDL was confirmed by independent pharmacological, biochemical and genetic methods. 5.2. Specificity of ligand affinity receptor chromatography with LCESI-MS/MS

Fig. 3. Western blot using anti SYK and anti phospho SYK antibodies against phagocytic receptor complexes isolated from live U937 cells using oxLDL versus IgG ligands alongside crude controls with no ligand. Equal loading between treatments was confirmed by CBBR staining of the PVDF blot prior to stripping and probing. Panels: A, PVDF membranes and probed with a rabbit anti-human phospho-SYK (pSykY525,526) specific antibody; B, PVDF membrane probed with mouse SYK-01 anti-human SYK antibody. Lanes: OxLDL, the oxLDL ligand presented on 2 mm beads to live cells; IgG, the IgG control ligand presented on 2mm beads to live cells; beads with no ligand mixed with crude extract.

laser confocal microscopy (Fig. 6). Expressing SYK siRNA had a notable effect on SYK expression that was statistically significant. The expression of SYK was lower in siRNA transfected cells that engulfed fewer oxLDL coated microbeads. The loss of SYK expression as measured by immunofluorescence was quantified against the levels of fluorescent siRNA within the cells that showed a statistically significant relationship.

4. Anti SYK(L) siRNA Experiments with two distinct siRNAs that target only the long isoform SYK (L) both inhibited the phagocytosis of oxLDL. Cells transfected with either SYK-L1 or SYK-L2 siRNAs showed significantly lower phagocytosis of oxLDL microbeads in the presence of serum compared to control siRNA transfected cells (Fig. 7). Thus SYK(L) was strongly associated with the oxLDL receptor complex by ligand affinity receptor chromatography with LC-ESI-MS/MS or Western blot and the same isoform was found to be required for the phagocytic engulfment of oxLDL.

SYK was more frequently observed with the activated IgG receptor complex in murine RAW 264.7 by mass spectrometry and expression of GFP constructs [25,26]. The affinity purified oxLDLreceptor complexes showed dozens of correlations to SYK that averaged up to ~220,000 arbitrary counts (compared to ~9000 counts in the media controls) and many of the known SYK binding proteins with more than 10 independent best-fit peptides consistent with successful MS/MS fragmentation and identification at high signal to noise. The statistical distributions of the peptides in terms of peptide-to-protein counts, expectation values and intensity essentially preclude false positive identification of SYK with the phagocytic oxLDL receptor complex [54,69]. Moreover, the probability of observing nested and overlapping sets of tryptic peptides that map to the same location on the kinase is remote and also rules out false positive identification by LC-ESI-MS/MS [60,64]. Furthermore, the multiply observed peptides that mapped to the most hydrophilic portions of SYK that would presumably be readily accessed by the protease were again consistent with the veracity of the LC-ESI-MS/MS experiment. The tryptic and phospho tryptic peptides from SYK were frequently observed with the oxLDL ligand or anti CD36 IgA compared to the controls. Thus the Chi Square value of E
13 for SYK distribution across treatments clearly indicates SYK(L) was specifically associated with the oxLDL receptor complex. Effective affinity chromatography of the ligand receptor complex provided much greater frequency and/or intensity of the SYK protein interaction network compared to the crude extract on uncoated beads and other controls that unambiguously and clearly confirmed that SYK was specifically captured by the oxLDL affinity ligand. 5.3. Phosphorylation of SYK The apparent phosphorylation of SYK at Y525/526 in the catalytic domain in response to shear stress is equated with activation of the kinase in platelets [70]. Phosphorylation of SYK was detected

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Fig. 4. The effect of SYK enzyme inhibitors on the phagocytic activity of the oxLDL versus IgG receptor complexes in cultured U937 macrophages. PMA differentiated U937 cells attached to 25 mm diameter cover glasses were pre-incubated (1 h) with the indicated drugs 5 mM BAY 61 3606, 15 mM PP1, 15 mg/ml Piceatannol (Pic), or control vehicle (H2O, DMSO) and assayed for phagocytosis. Panels: A, Phagocytosis of oxLDL coated microbeads in growth media (n ¼ 1044); B, Phagocytosis of oxLDL coated microbeads in serum free media (n ¼ 1066); C, illustration of oxLDL phagocytosis assays where external beads are shown as blue; D, phagocytosis of IgG coated microbeads in growth media (n ¼ 959); E, phagocytosis of IgG coated microbeads in serum free media (n ¼ 777); F, illustration of IgG phagocytosis assays where external beads are shown as blue. The internal beads per cell (IBPC) and the total beads per cell (TBPC) are shown with mean and Standard Error. The p-values from ANOVA followed by the Tukey Kramer HSD test are shown. Immunofluorescent images of drug- or control vehicle (H2O, 0.1% DMSO) treated U937 cells engulfing oxLDL-coated beads are shown along with the corresponding DIC images. The inset scale bar is 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. The effect of anti SYK siRNA on the activity of the oxLDL versus IgG phagocytic receptor complex. PMA differentiated U937 cells attached to 25 mm diameter cover glasses were transfected with either SYK siRNAs, control siRNA, or no siRNAs (Negative Control) and assayed for either oxLDL- or IgG-coated microbead phagocytosis. The internal beads per cell (IBPC) and the total beads per cell (TBPC) are shown with mean and Standard Error (SEM). Panels: A, The mean number of IBPC (-) and TBPC (,) were scored by confocal microscopy for each type of ligand-coated microbeads. The total number of cells samples were oxLDL n ¼ 5,085, IgG n ¼ 2136 in GM, growth media (GM) or Serum Free Media (SFM). Statistical differences by ANOVA and Tukey Kramer HSD means test are indicated by the p values.

by Western blot and independently by ligand affinity chromatography together with tandem mass spectrometry of the oxLDLreceptor complex. Immunoblots of macrophages showed that SYK Y525/526 was most phosphorylated in receptor complexes isolated with the ligand oxLDL but there was detectable phosphorylation with IgG beads as well. Mass spectrometry demonstrated that the receptor chromatography ligands oxLDL, anti CD36 IgA/G, and bulk human IgG all result in the detection of SYK phosphorylation at multiple locations and seems to indicate that phosphorylated SYK binds the oxLDL or IgG receptor complexes with greater efficiency. The phosphorylation sites detected with oxLDL, anti CD36 and human IgG affinity chromatography showed agreement with the previously annotated phosphorylation sites of SYK at S379 and T384 consistent with the veracity of the affinity LC-ESI-MS/MS results. Most serine, threonine or tyrosine residues showed no modification. In contrast, dense islands of STYP sites were apparently fully phosphorylated, showed multiple phosphorylations in close proximity in the SH2, substrate binding site, catalytic domain and activation loop, indicating the phosphorylation of SYK from ligand-receptor binding may play a role in the function of SYK. The overlapping, fully tryptic phophopeptides detected essentially rule out false positive identification of the phosphorylation sites. 5.4. Pharmacological treatments The inhibition of oxLDL-mediated phagocytosis by BAY 61 3606, PP1, and Piceatannol is consistent with the notion that SYK or closely related kinases play a key role in the activation of receptor

mediated phagocytosis. Pharmacological inhibitors of SYK prevented the engulfment of oxLDL aggregates and seemed to indicate that inhibiting SYK will be effective to prevent the inflammatory phagocytic activation of macrophages by aggregated oxLDL. Inhibiting the inflammatory phagocytic engulfment of oxLDL by human macrophages may be the mechanism by which drugs targeting SYK effectively prevent atherogenesis [71]. 5.5. Anti SYK siRNA The results here clearly show that the phagocytosis of IgG opsonized particles by U937 macrophages did not require SYK catalytic activity and was independent of SYK expression. The use of siRNA with cultured macrophages was able to clearly demonstrate that SYK plays little role in IgG-mediated phagocytosis in human U937 macrophages. In contrast, macrophages from SYK
/
mice may fail to develop or express an effective Fc receptor activity [41,42] which in turn confounds measurements of SYK function's within the receptor complex pathway towards engulfment. Phagocytosis of aggregate IgG has been previously documented using Syk
/
and Hck
/
/Fgr
/
/Lyn
/
deficient primary macrophages that efficiently engulf various sized ligand coated particles (0.2e5.5 mm) [72] and Escherichia coli and heat killed zymosan [41,42]. Kiefer and co-workers also showed that Syk
/
deficient primary macrophages could engulf zymosan coated particles in the absence of Syk [41,42]. SYK may be modulated by both ITAM stimulatory and ITIM inhibitory immuno receptors that together may integrate innate immune responses involving integrins,

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Fig. 6. Confirmation and quantification of the knockdown of SYK expression by pan SYK siRNA. Panels: A, Representative images of SYK siRNACY3 transfected and non-transfected U937 cells stained with anti-SYK antibody are shown. External oxLDL-coated beads were stained using an anti-oxLDL antibody prior to co-staining for SYK. The inset scale bar is 20 mm. B, SYK knockdown using siRNAs was confirmed by immunofluorescence (IF) using an anti-SYK IgG antibody Confocal images were analyzed to determine the mean pixel intensity values per cell per fluorescent channel as described in Experimental Procedures. The confidence plot of the IF intensity levels for anti-SYK staining versus SYKCY3 siRNA fluorescent levels is shown (▬ median, A mean) using R. Means comparison by ANOVA followed by Tukey Kramer HSD test as indicated by the p values; C, anti-SYK Ab (DyLight649) staining of SYK siRNACY3 transfected cells are shown along with examples of the siRNA visual scoring or each cell in the panel (arrows þ numbered levels).

interleukins receptors, C-type Lectins, and scavenger receptors such as TLR4 [13,14,22,43,44,73e77]. Here specific SYK siRNAs were used to unambiguously confirm that SYK functions in the phagocytosis of oxLDL. 5.6. Anti-SYK(L) siRNA The results with siRNA specific to SYK(L) agree with the immunoblots, and affinity chromatography mass spectrometry, that the long isoform of Spleen Tyrosine Kinase, SYK(L), regulates the phagocytosis of aggregated oxLDL. The amino acid sequence of the peptide TWSAGGIISRIKSYSFPPGHRKK maps to the 23 amino acid linker region that is unique to SYK(L) and was observed 7 times and so unambiguously confirms the presence of the long isoform in the complex [78] but does not rule out the presence of other forms of SYK. Targeted inhibition of SYK(L) accounts for all of the observed inhibition of phagocytosis elicited by either pharmacological inhibitors or global (L þ S) SYK siRNAs. SYK (L) apparently plays a role in oxLDL phagocytosis by integrating the context of serum factors that makes an important addition to the molecular mechanism governing phagocytosis of pro-inflammatory aggregates. 6. Conclusion Ligand affinity chromatography together with direct LC-ESI-

MS/MS analysis of high-intensity peptides agreed with known protein-interaction data, plus independent genetic and biochemical methods including Western blot, confocal microscopy of immuno fluorescent antibodies, enzyme inhibitors and siRNA, that SYK(L) is functionally associated with the aggregated oxLDL receptor complex. All of the data in this study agreed that targeting the SYK(L) with drugs or siRNA should prevent the inflammatory phagocytic activation of macrophages by oxLDL aggregates that may be a crucial step towards atherosclerosis [2e4,7,21,79]. The important role that SYK(L) plays in regulating oxLDL phagocytosis as seen in this study seems to indicate that targeting SYK(L) may be an effective strategy in preventing oxLDL related chronic inflammatory activation in the circulatory system. The affinity ligand oxLDL was effective for isolating the receptor complex component SYK(L) that may be a therapeutic target in atherosclerosis and to reveal its phosphorylations. The simplest explanation of the data presented here and elsewhere is that macrophages are directly activated by aggregated lipoproteins such as oxLDL for phagocytic engulfment by scavenger receptors without any other opsonin [6] and that SYK plays the role of integrating information from the oxLDL receptor(s) with other phagocytic ligand receptors [73e77,80] that function together [22] to engulf pro-inflammatory aggregated oxLDL. It is apparently possible to activate and capture the receptor complex for oxLDL from the surface of live macrophages and to provide the

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Fig. 7. SYK(L) is the isoform that regulates the engulfment of oxLDL micro particles in circulation. Panels: A, Human U937 cells were also transfected with a DyLight547 labeled long (L) isoform specific siRNAs (L1 and L2), or a matching DyLight547 labeled universal control siRNA. Cells were then assayed for oxLDL-coated microbead phagocytosis. The mean ± sem of IBPC (-) and TBPC (,) were scored by confocal microscopy with a total of 1642 cells assays with at least 370  per group in the presence of serum growth media, GM. The results of ANOVA followed by Tukey HSD means are indicated by the p values; B, Representative images of control and SYK(L) siRNA transfected U937 cells are shown along with the accompanying DIC and overlay images. The inset scale bar is 20 mm.

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primary structural identity by LC-ESI-MS/MS as confirmed by drug and siRNA assays of receptor pathway function that might have profound implications for discovery of new therapeutic treatments. Conflict of interest

[21] [22] [23] [24]

The authors have no conflicts of interests in the publication of this study. Acknowledgements

[25]

[26]

This work was supported by an NSERC Discovery Grant to JGM and an NSERC CRD grant CRDPJ 386003 - 2009 to JGM with the collaborators SG and WST from The Natural Science and Engineering Research Council of Canada.

[27]

Appendix A. Supplementary data

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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ab.2016.07.021.

[28] [29]

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References [1] D.T. Vance, et al., A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages, Anal. Biochem. 500 (2016) 24e34. [2] M.O. Pentikainen, et al., Modified LDL - trigger of atherosclerosis and inflammation in the arterial intima, J. Intern Med. 247 (3) (2000) 359e370. [3] H. Maxeiner, et al., Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2, J. Exp. Med. 188 (12) (1998) 2257e2265. [4] R. Ross, Atherosclerosis is an inflammatory disease, Am. Heart J. 138 (5 Pt 2) (1999) S419eS420. [5] R.S. Flannagan, V. Jaumouille, S. Grinstein, The cell biology of phagocytosis, Annu. Rev. Pathol. 7 (2012) 61e98. [6] L. Liu, L. Zhou, Y. Li, W. Bai, N. Liu, W. Li, Y. Gao, Z. Liu, R. Han, High-density lipoprotein acts as an opsonin to enhance phagocytosis of group A streptococcus by U937 cells, Microbiol. Immunol. 59 (7) (2015 Jul) 419e425. [7] C.B. Guest, et al., Phagocytosis of cholesteryl ester is amplified in diabetic mouse macrophages and is largely mediated by CD36 and SR-A, PLoS One 2 (6) (2007) e511. [8] F.K. Swirski, M. Nahrendorf, Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure, Science 339 (6116) (2013) 161e166. [9] Y.S. Bae, et al., Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2, Circ. Res. 104 (2) (2009) 210e218, 21pp. following 218. [10] S.H. Choi, et al., Lipoprotein accumulation in macrophages via toll-like receptor-4-dependent fluid phase uptake, Circ. Res. 104 (12) (2009) 1355e1363. [11] Y.I. Miller, et al., Toll-like receptor-4 and lipoprotein accumulation in macrophages, Trends Cardiovasc. Med. 19 (2009) 227e232. [12] C.R. Stewart, et al., CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer, Nat. Immunol. 11 (2010) 155e161. [13] X.H. Xu, et al., Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL, Circulation 104 (2001) 3103e3108. [14] K. Yang, et al., Oxidized low-density lipoprotein promotes macrophage lipid accumulation via the toll-like receptor 4-Src pathway, Circ. J. 79 (11) (2015) 2509e2516. [15] Z. Lu, et al., TLR4 antagonist attenuates atherogenesis in LDL receptordeficient mice with diet-induced type 2 diabetes, Immunobiology 220 (11) (2015) 1246e1254. [16] Y.C. Lin, et al., The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3, Sci. Signal (289) (2013) 6 p. ra71. € m, Establishment and characterization of a human histiocytic [17] C. Sundstro lymphoma cell line (U-937), Int. J. Cancer 17 (1976) 565e577. [18] K. Nilsson, et al., Differentiation in vitro of human leukemia and lymphoma cell lines, in: International Symposium on New Trends in Human Immunology and Cancer Immunotherapy, 1980. [19] J. Frostegard, et al., Oxidized low density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 904e908. [20] I. Olsson, et al., Induction of differentiation of the human histiocytic

[33]

[34] [35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

[43] [44]

[45]

[46] [47] [48]

[49]

[50]

[51] [52]

19

lymphoma cell line U-937 by 1a, 25-dihydroxycholecalciferol, Cancer Res. 43 (1983) 5862e5867. A.C. Li, C.K. Glass, The macrophage foam cell as a target for therapeutic intervention, Nat. Med. 8 (11) (2002) 1235e1242. B. Heit, et al., Multimolecular signaling complexes enable Syk-mediated signaling of CD36 internalization, Dev. Cell 24 (2013) 372e383. M.B. Overdijk, et al., Crosstalk between human IgG isotypes and murine effector cells, J. Immunol. 189 (7) (2012) 3430e3438. J.V. Ravetch, S. Bolland, IgG Fc receptors, Annu. Rev. Immunol. 19 (2001) 275e290. A. Jankowski, P. Zhu, J.G. Marshall, Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography, Anal. Biochem. 380 (2008) 235e248. J.G. Marshall, et al., Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptormediated phagocytosis, J. Cell Biol. 153 (2001) 1369e1380. N. Dong, L. Liu, F. Shao, A bacterial effector targets host DH-PH domain RhoGEFs and antagonizes macrophage phagocytosis, EMBO J. 29 (8) (2010) 1363e1376. W.L. Lee, et al., Role of CrkII in Fcgamma receptor-mediated phagocytosis, J. Biol. Chem. 282 (15) (2007) 11135e11143. G. Tzircotis, V.M. Braga, E. Caron, RhoG is required for both FcgammaR- and CR3-mediated phagocytosis, J. Cell Sci. 124 (Pt 17) (2011) 2897e2902. J.A. Odin, et al., Regulation of phagocytosis and [Ca2þ]i flux by distinct regions of an Fc receptor, Sci. (New York, N. Y. 254 (1991) 1785e1788. S. Amigorena, et al., Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG, Nature 358 (1992) 337e341. T. Muta, et al., A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB modulates B-cell receptor signalling, Nature 368 (1994) 70e73. M. Ono, et al., Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc gamma RIIB, Nature 383 (1996) 263e266. T. Takai, et al., Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice, Nature 379 (1996) 346e349. S. Bolland, J.V. Ravetch, Spontaneous autoimmune disease in Fc(gamma)RIIBdeficient mice results from strain-specific epistasis, Immunity 13 (2000) 277e285. J.G. Park, A.D. Schreiber, Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7381e7385. D. Cox, et al., Syk tyrosine kinase is required for immunoreceptor tyrosine activation motif-dependent actin assembly, J. Biol. Chem. 271 (1996) 16597e16602. J.E. Hutchcroft, et al., FcεRI-Mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 9107e9111. C.L. Law, et al., Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk, Mol. Cell. Biol. 16 (1996) 1305e1315. csai, J. Ruland, V.L.J. Tybulewicz, The SYK tyrosine kinase: a crucial A. Mo player in diverse biological functions, Nat. Rev. Immunol. 10 (2010) 387e402. M.T. Crowley, et al., A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages, J. Exp. Med. 186 (1997) 1027e1039. F. Kiefer, et al., The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in macrophages and neutrophils, Mol. Cell. Biol. 18 (1998) 4209e4220. S.-H. Choi, et al., Lipoprotein accumulation in macrophages via toll-like receptor-4-dependent fluid phase uptake, Circ. Res. 104 (2009) 1355e1363. Y.-C. Lin, et al., Anti-inflammatory actions of Syk inhibitors in macrophages involve non-specific inhibition of toll-like receptors-mediated JNK signaling pathway, Mol. Immunol. 47 (2010) 1569e1578. A. Shiratsuchi, et al., Inhibitory effect of Toll-like receptor 4 on fusion between phagosomes and endosomes/lysosomes in macrophages, J. Immunol. 172 (4) (2004) 2039e2047. P. Prinos, et al., Alternative splicing of SYK regulates mitosis and cell survival, Nat. Struct. Mol. Biol. 18 (2011) 673e679. Y. Shi, Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis, Blood 107 (2006) 4554e4562. M. Majeed, et al., Role of Src kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome-lysosome fusion, J. Leukoc. Biol. 70 (5) (2001) 801e811. A.K. Florentinus, et al., Capture and qualitative analysis of the activated Fc receptor complex from live cells. Current protocols in protein science/editorial board, John E. Coligan. (2012). Chapter 19: p. Unit 19.22. L.-H. Shen, et al., Oxidized low-density lipoprotein induces differentiation of RAW264.7 murine macrophage cell line into dendritic-like cells, Atherosclerosis 199 (2008) 257e264. A.K. Florentinus, et al., The Fc receptor-cytoskeleton complex from human neutrophils, J. Proteom. 75 (2011) 450e468. P. Bowden, et al., Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry, J. Proteome Res. 11 (2012) 2032e2047.

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[53] J. Marshall, et al., Human serum proteins preseparated by electrophoresis or chromatography followed by tandem mass spectrometry, J. Proteome Res. 3 (3) (2004) 364e382. [54] P. Zhu, et al., Chi-square comparison of tryptic peptide-to-protein distributions of tandem mass spectrometry from blood with those of random expectation, Anal. Biochem. 409 (2) (2011) 189e194. [55] P. Zhu, et al., Peptide-to-protein distribution versus a competition for significance to estimate error rate in blood protein identification, Anal. Biochem. 411 (2011) 241e253. [56] R. Craig, R.C. Beavis, TANDEM: matching proteins with tandem mass spectra, Bioinformatics 20 (9) (2004) 1466e1467. [57] J.R. Yates 3rd, et al., Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database, Anal. Chem. 67 (8) (1995) 1426e1436. [58] L.Y. Geer, et al., Open mass spectrometry search algorithm, J. Proteome Res. 3 (5) (2004) 958e964. [59] P. Bowden, R. Beavis, J. Marshall, Tandem mass spectrometry of human tryptic blood peptides calculated by a statistical algorithm and captured by a relational database with exploration by a general statistical analysis system, J. Proteom. 73 (2009) 103e111. [60] P. Bowden, et al., Meta sequence analysis of human blood peptides and their parent proteins, J. Proteom. 73 (2010) 1163e1175. [61] P. Shannon, et al., Cytoscape: a software environment for integrated models of biomolecular interaction networks, Genome Res. 13 (11) (2003) 2498e2504. [62] C. Haan, I. Behrmann, A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background, J. Immunol. Methods 318 (2007) 11e19. [63] A.K. Florentinus, et al., Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance, J. Proteom. 75 (2012) 1303e1317. [64] M. Tucholska, et al., The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC-ESI-Paul ion trap and Qq-TOF, J. Proteomics 73 (6) (2010) 1254e1269. [65] O.C. Ferreira Jr., et al., Phorbol ester-induced differentiation of U937 cells enhances attachment to fibronectin and distinctly modulates the alpha 5 beta 1 and alpha 4 beta 1 fibronectin receptors, Exp. Cell Res. 193 (1) (1991) 20e26. [66] P. Harris, P. Ralph, Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines, J. Leukoc. Biol. 37 (1985) 407e422. [67] M. Janabi, et al., Oxidized LDL-induced NF-kappa B activation and subsequent

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76] [77]

[78] [79] [80]

expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients, Arterioscler. Thromb. Vasc. Biol. 20 (8) (2000) 1953e1960. S. Nozaki, et al., Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects, J. Clin. Investig. 96 (4) (1995) 1859e1865. B.J. Cargile, J.L. Bundy, J.L. Stephenson Jr., Potential for false positive identifications from large databases through tandem mass spectrometry, J. Proteome Res. 3 (5) (2004) 1082e1085. H.E. Speich, et al., Platelets undergo phosphorylation of Syk at Y525/526 and Y352 in response to pathophysiological shear stress, Am. J. Physiol. Cell Physiol. 295 (4) (2008) C1045eC1054. I. Hilgendorf, et al., The oral spleen tyrosine kinase inhibitor fostamatinib attenuates inflammation and atherogenesis in low-density lipoprotein receptor-deficient mice, Arteriosclerosis, Thrombosis, Vasc. Biol. 31 (2011) 1991e1999. C.J. Fitzer-Attas, et al., Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn, J. Exp. Med. 191 (2000) 669e682. X. Hu, et al., Crosstalk among Jak-STAT, Toll-like receptor, and ITAMdependent pathways in macrophage activation, J. Leukoc. Biol. 82 (2) (2007) 237e243. csai, et al., Integrin signaling in neutrophils and macrophages uses A. Mo adaptors containing immunoreceptor tyrosine-based activation motifs, Nat. Immunol. 7 (2006) 1326e1333. Y. Minami, et al., Protein tyrosine kinase Syk is associated with and activated by the IL-2 receptor: possible link with the c-myc induction pathway, Immunity 2 (1995) 89e100. A.M. Kerrigan, G.D. Brown, Syk-coupled C-type lectins in immunity, Trends Immunol. 32 (2011) 151e156. T. Ichinohe, et al., Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI, J. Biol. Chem. 272 (1997) 63e68. S. Latour, et al., A unique insert in the linker domain of Syk is necessary for its function in immunoreceptor signalling, EMBO J. 17 (9) (1998) 2584e2595. W. Li, et al., CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs, J. Clin. Investig. 120 (11) (2010) 3996e4006. A. Ravandi, et al., Relationship of IgG and IgM autoantibodies and immune complexes to oxidized LDL with markers of oxidation and inflammation and cardiovascular events: results from the EPIC-Norfolk Study, J. Lipid Res. 52 (10) (2011) 1829e1836.