title: Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.
abstract: BACKGROUND
  Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.
  
  
  RESULTS
  Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.
  
  
  CONCLUSIONS
  Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.
authors.affiliation: The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, Texas, United States of America.
authors.forename: Felipe
authors.initials: F
authors.lastname: Gazos-Lopes
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Mauricio M
authors.initials: MM
authors.lastname: Oliveira
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Lucas V B
authors.initials: LV
authors.lastname: Hoelz
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Danielle P
authors.initials: DP
authors.lastname: Vieira
authors.affiliation: The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, Texas, United States of America; Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Parasitologia, Pampulha, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Alexandre F
authors.initials: AF
authors.lastname: Marques
authors.affiliation: The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, Texas, United States of America.
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil; Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco H, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marta T
authors.initials: MT
authors.lastname: Gomes
authors.affiliation: The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, Texas, United States of America.
authors.forename: Nasim G
authors.initials: NG
authors.lastname: Salloum
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Pedro G
authors.initials: PG
authors.lastname: Pascutti
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Thaïs
authors.initials: T
authors.lastname: Souto-Padrón
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco H, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Cidade Universitária, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Angela H
authors.initials: AH
authors.lastname: Lopes
authors.affiliation: The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, Texas, United States of America.
authors.forename: Igor C
authors.initials: IC
authors.lastname: Almeida
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D008244:Lysophosphatidylcholines / Q000737:chemistry* / Q000494:pharmacology; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010972:Platelet Activating Factor / Q000737:chemistry*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014230:Triazoles / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000737:chemistry*
chemical_list: D001381:Azepines; D008244:Lysophosphatidylcholines; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086
references.citation: Annu Rev Biophys Biomol Struct. 2000;29:291-325
references.pmid: 10940251
references.citation: Infect Immun. 1999 Jun;67(6):2810-4
references.pmid: 10338485
references.citation: J Exp Med. 2007 Apr 16;204(4):929-40
references.pmid: 17420269
references.citation: Annu Rev Biochem. 2000;69:419-45
references.pmid: 10966465
references.citation: Parasitology. 2013 Feb;140(2):147-59
references.pmid: 23010131
references.citation: J Chromatogr A. 2008 Oct 31;1209(1-2):88-94
references.pmid: 18823632
references.citation: Toxicon. 1997 May;35(5):649-57
references.pmid: 9203289
references.citation: Proteins. 2007;69 Suppl 8:108-17
references.pmid: 17894355
references.citation: Antioxid Redox Signal. 2002 Oct;4(5):791-8
references.pmid: 12470507
references.citation: J Lipid Res. 2010 Oct;51(10):2957-66
references.pmid: 20610733
references.citation: J Mass Spectrom. 2003 Jul;38(7):752-63
references.pmid: 12898655
references.citation: Immunol Lett. 2004 Jan 30;91(1):17-21
references.pmid: 14757365
references.citation: Biochem Biophys Res Commun. 2002 May 24;293(5):1358-63
references.pmid: 12054663
references.citation: J Cell Sci. 2000 Apr;113 ( Pt 7):1299-307
references.pmid: 10704380
references.citation: Nature. 1991 Jan 24;349(6307):342-6
references.pmid: 1846231
references.citation: Fed Proc. 1987 Jan;46(1):133-8
references.pmid: 2948837
references.citation: Exp Parasitol. 2009 Jan;121(1):22-8
references.pmid: 18851965
references.citation: Infect Immun. 2002 Aug;70(8):4247-53
references.pmid: 12117933
references.citation: FEBS J. 2005 Dec;272(24):6354-64
references.pmid: 16336272
references.citation: Stroke. 2005 May;36(5):965-70
references.pmid: 15845889
references.citation: Am J Trop Med Hyg. 1990 Sep;43(3):274-81
references.pmid: 2121055
references.citation: J Biol Chem. 1997 Mar 21;272(12):7846-54
references.pmid: 9065450
references.citation: J Chromatogr A. 2006 Dec 29;1137(2):127-37
references.pmid: 17097094
references.citation: J Immunol. 2001 Mar 1;166(5):3423-31
references.pmid: 11207300
references.citation: Biochim Biophys Acta. 2007 Nov;1771(11):1364-71
references.pmid: 17950662
references.citation: PLoS One. 2013 Sep 30;8(9):e76233
references.pmid: 24312681
references.citation: Microbes Infect. 2003 Jul;5(9):789-96
references.pmid: 12850205
references.citation: Rapid Commun Mass Spectrom. 2010 Jul 30;24(14):2074-82
references.pmid: 20552712
references.citation: Am J Physiol. 1997 Jan;272(1 Pt 2):H17-24
references.pmid: 9038918
references.citation: Parasitology. 1994 Apr;108 ( Pt 3):323-34
references.pmid: 8022658
references.citation: Prostaglandins Other Lipid Mediat. 2009 Sep;89(3-4):73-81
references.pmid: 19383550
references.citation: Clin Exp Immunol. 1999 May;116(2):326-31
references.pmid: 10337026
references.citation: Nat Protoc. 2010 Apr;5(4):725-38
references.pmid: 20360767
references.citation: Parasitology. 2007 Apr;134(Pt 4):491-502
references.pmid: 17121684
references.citation: J Leukoc Biol. 2007 Dec;82(6):1501-9
references.pmid: 17884992
references.citation: J Infect Dis. 2010 Nov 1;202(9):1369-79
references.pmid: 20863227
references.citation: Biochem Soc Trans. 2013 Feb 1;41(1):205-12
references.pmid: 23356284
references.citation: PLoS One. 2011 Feb 15;6(2):e16959
references.pmid: 21347238
references.citation: Biochem J. 2005 May 1;387(Pt 3):617-26
references.pmid: 15588229
references.citation: Parasitol Res. 2011 Jun;108(6):1473-8
references.pmid: 21161276
references.citation: Protein Eng Des Sel. 2006 Nov;19(11):511-6
references.pmid: 17032692
references.citation: J Biochem. 2002 Jun;131(6):773-9
references.pmid: 12038971
references.citation: J Biol Chem. 1957 May;226(1):497-509
references.pmid: 13428781
references.citation: J Mol Cell Cardiol. 2003 Nov;35(11):1375-84
references.pmid: 14596794
references.citation: J Biol Chem. 2003 Jul 25;278(30):27766-71
references.pmid: 12740385
references.citation: Mol Microbiol. 2007 Feb;63(4):1078-95
references.pmid: 17238918
references.citation: J Exp Biol. 2010 Oct 1;213(Pt 19):3305-10
references.pmid: 20833923
references.citation: J Comput Aided Mol Des. 2012 Mar;26(3):301-9
references.pmid: 22395902
references.citation: Biol Pharm Bull. 2011;34(8):1159-62
references.pmid: 21804199
references.citation: J Biol Chem. 1995 Nov 10;270(45):27244-53
references.pmid: 7592983
references.citation: Curr Protoc Protein Sci. 2007 Nov;Chapter 2:Unit 2.9
references.pmid: 18429317
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3784-8
references.pmid: 12824418
references.citation: Enzyme Res. 2011;2011:392082
references.pmid: 21603263
references.citation: J Am Soc Mass Spectrom. 2010 Apr;21(4):657-69
references.pmid: 20171120
references.citation: J Parasitol Res. 2012;2012:625838
references.pmid: 22132309
references.citation: J Proteome Res. 2013 Feb 1;12(2):883-97
references.pmid: 23214914
references.citation: J Biol Chem. 2001 Nov 2;276(44):41325-35
references.pmid: 11535583
references.citation: Recent Pat Inflamm Allergy Drug Discov. 2008 Jan;2(1):72-6
references.pmid: 19075994
references.citation: J Biol Chem. 2003 Apr 18;278(16):14379-86
references.pmid: 12586833
references.citation: Infect Immun. 2008 Dec;76(12):5543-52
references.pmid: 18794282
references.citation: Int J Parasitol. 2006 Feb;36(2):165-73
references.pmid: 16337632
references.citation: Parasitology. 2010 Aug;137(9):1357-92
references.pmid: 20602846
references.citation: J Chromatogr A. 1998 May 22;807(2):165-84
references.pmid: 9646494
references.citation: Mem Inst Oswaldo Cruz. 1983 Jul-Sep;78(3):317-33
references.pmid: 6361445
references.citation: Nature. 2000 Jan 13;403(6766):199-203
references.pmid: 10646605
references.citation: PLoS One. 2013 May 31;8(5):e65155
references.pmid: 23741477
references.citation: Cell Signal. 2012 Nov;24(11):2035-42
references.pmid: 22759791
references.citation: Biochem J. 1996 Sep 1;318 ( Pt 2):575-81
references.pmid: 8809049
references.citation: Proteins. 2009;77 Suppl 9:100-13
references.pmid: 19768687
references.citation: Acta Trop. 2010 Jul-Aug;115(1-2):14-21
references.pmid: 19932071
references.citation: Biochim Biophys Acta. 2007 Apr;1768(4):923-40
references.pmid: 17078925
references.citation: Bioinformatics. 2010 Dec 15;26(24):3067-74
references.pmid: 20947525
references.citation: J Protozool. 1982 May;29(2):264-9
references.pmid: 7047731
references.citation: Circulation. 2001 Jul 10;104(2):181-6
references.pmid: 11447083
references.citation: Rapid Commun Mass Spectrom. 2008 Nov;22(22):3579-87
references.pmid: 18937225
references.citation: Biochem Pharmacol. 2013 May 1;85(9):1317-29
references.pmid: 23500543
references.citation: Mol Pharmacol. 2003 Jun;63(6):1256-72
references.pmid: 12761335
references.citation: Biochim Biophys Acta. 2007 Apr;1768(4):794-807
references.pmid: 17188232
references.citation: Mem Inst Oswaldo Cruz. 2000 Jan-Feb;95(1):97-102
references.pmid: 10656713
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 2005 Aug;372(2):131-8
references.pmid: 16200394
references.citation: J Lipids. 2011;2011:532145
references.pmid: 21912747
references.citation: Clin Microbiol Rev. 1992 Oct;5(4):400-19
references.pmid: 1423218
references.citation: Rev Inst Med Trop Sao Paulo. 1964 May-Jun;6:93-100
references.pmid: 14177814
references.citation: Protein Sci. 1993 Sep;2(9):1511-9
references.pmid: 8401235
references.citation: Mol Biochem Parasitol. 2013 Feb;187(2):77-86
references.pmid: 23275096
references.citation: Exp Cell Res. 1964 Jun;35:9-13
references.pmid: 14190667
references.citation: Semin Immunopathol. 2012 Nov;34(6):753-70
references.pmid: 23076807
references.citation: Am J Physiol Heart Circ Physiol. 2003 Oct;285(4):H1786-9
references.pmid: 12805023
references.citation: Int J Parasitol. 2001 May 1;31(5-6):472-81
references.pmid: 11334932
references.citation: Curr Protoc Bioinformatics. 2006 Oct;Chapter 5:Unit-5.6
references.pmid: 18428767
references.citation: PLoS Negl Trop Dis. 2008 Sep 24;2(9):e300
references.pmid: 18820747
references.citation: Biochem J. 1993 Jun 15;292 ( Pt 3):617-29
references.pmid: 8391253
references.citation: Adv Physiol Educ. 2007 Mar;31(1):5-16
references.pmid: 17327576
references.citation: J Med Chem. 2006 Jun 1;49(11):3315-21
references.pmid: 16722650
references.citation: J Am Soc Mass Spectrom. 2007 Apr;18(4):783-90
references.pmid: 17303435