title: OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.
abstract: 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.
authors: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors: Jeffrey C
authors: JC
authors: Howard
authors: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors: Angelique
authors: A
authors: Florentinus-Mefailoski
authors: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors: Peter
authors: P
authors: Bowden
authors: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors: William
authors: W
authors: Trimble
authors: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors: Sergio
authors: S
authors: Grinstein
authors: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada. Electronic address: 4marshal@ryerson.ca.
authors: John G
authors: JG
authors: Marshall
mesh_terms: D002853:Chromatography, Liquid; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000737:chemistry*; D010587:Phagocytosis; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism; D051131:Receptors, Oxidized LDL / Q000737:chemistry*; D000072377:Syk Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000378:metabolism; D020298:U937 Cells
chemical_list: D007074:Immunoglobulin G; D008077:Lipoproteins, LDL; D011961:Receptors, Fc; D051131:Receptors, Oxidized LDL; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C099252:oxidized low density lipoprotein; C000606896:SYK protein, human; D000072377:Syk Kinase
keywords: IgG; MS; OxLDL; Phagocytosis; SQL; SYK