TLR2 is one of the endothelial receptors for beta 2-glycoprotein I.
During the antiphospholipid syndrome, beta2-gpI interacts with phospholipids on endothelial cell (EC) surface to allow the binding of autoantibodies. However, induced-pathogenic intracellular signals suggest that beta2-gpI associates also with a receptor that is still not clearly identified. TLR2 and TLR4 have long been suspected, yet interactions between TLRs and beta2-gpI have never been unequivocally proven. The aim of the study was to identify the TLR directly involved in the binding of beta2-gpI on EC surface. beta2-gpI was not synthesized and secreted by ECs in vitro, but rather taken up from FCS. This uptake occurred through association with TLR2 and TLR4 which partitioned together in the lipid rafts of ECs. After coimmunoprecipitation, mass-spectrometry identification of peptides demonstrated that TLR2, but not TLR4, was implicated in the beta2-gpI retention. These results were further confirmed by plasmon resonance-based studies. Finally, siRNA were used to obtain TLR2-deficient ECs that lost their ability to bind biotinylated beta2-gpI and to trigger downstream phosphorylation of kinases and activation of NFkappaB. TLR4 may upregulate TLR2 expression, thereby contributing to beta2-gpI uptake. However, our data demonstrate that direct binding of beta2-gpI on EC surface occurs through direct interaction with TLR2. Furthermore, signaling for anti-beta2-gpI may be envisioned as a multiprotein complex concentrated in lipid rafts on the EC membrane.
Alard
Jean-Eric
JE
Equipe d'Accueil 2216, Université de Brest, France.
Gaillard
Fanny
F
Daridon
Capucine
C
Shoenfeld
Yehuda
Y
Jamin
Christophe
C
Youinou
Pierre
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004730:Endothelium, Vascular / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020298:U937 Cells; D053482:beta 2-Glycoprotein I / Q000096:biosynthesis / Q000378:metabolism*
D010455:Peptides; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D053482:beta 2-Glycoprotein I