title: Human beta 2-glycoprotein I binds to endothelial cells through a cluster of lysine residues that are critical for anionic phospholipid binding and offers epitopes for anti-beta 2-glycoprotein I antibodies.
abstract: Beta 2-Glycoprotein I (beta 2GPI) is a phospholipid-binding protein recognized by serum autoantibodies from the anti-phospholipid syndrome both in cardiolipin- and beta 2GPI-coated plates. We found that: 1) recombinant wild-type beta 2GPI bound to HUVEC and was recognized by both human monoclonal IgM and affinity-purified polyclonal IgG anti-beta 2GPI anti-phospholipid syndrome Abs; and 2) a single amino acid change from Lys286 to Glu significantly reduced endothelial adhesion. Double and triple mutants (from Lys284,287 to Glu284,287, from Lys286,287 to Glu286,287, and from Lys284,286,287 to Glu284,286,287) completely abolished endothelial binding. A synthetic peptide (P1) spanning the sequence Glu274-Cys288 of the beta 2GPI fifth domain still displayed endothelial adhesion. Another peptide (P8), identical with P1 except that Cys281 and Cys288 were substituted with serine residues, did not bind to HUVEC. Anti-beta 2GPI Abs, once bound to P1 adhered to HUVEC, induced E-selectin expression and up-regulated IL-6 secretion. Control experiments conducted with irrelevant Abs as well as with the P8 peptide did not show any endothelial Ab binding nor E-selectin and IL-6 modulation. Our results suggest that: 1) beta 2GPI binds to endothelial cells through its fifth domain; 2) the major phospholipid-binding site that mediates the binding to anionic phospholipids is also involved in endothelial binding; 3) HUVEC provide a suitable surface for beta 2GPI binding comparable to that displayed by anionic phospholipids dried on microtiter wells; and 4) the formation of the complex between beta 2GPI and the specific Abs leads to endothelial activation in vitro.
authors.affiliation: Istituto di Medicina Interna, Malattie Infettive & Immunopatologia-Istituto Di Ricerca e Eura a Carattere Scientifica Policlinico, University of Milan, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Del Papa
authors.forename: Y H
authors.initials: YH
authors.lastname: Sheng
authors.forename: E
authors.initials: E
authors.lastname: Raschi
authors.forename: D A
authors.initials: DA
authors.lastname: Kandiah
authors.forename: A
authors.initials: A
authors.lastname: Tincani
authors.forename: M A
authors.initials: MA
authors.lastname: Khamashta
authors.forename: T
authors.initials: T
authors.lastname: Atsumi
authors.forename: G R
authors.initials: GR
authors.lastname: Hughes
authors.forename: K
authors.initials: K
authors.lastname: Ichikawa
authors.forename: T
authors.initials: T
authors.lastname: Koike
authors.forename: G
authors.initials: G
authors.lastname: Balestrieri
authors.forename: S A
authors.initials: SA
authors.lastname: Krilis
authors.forename: P L
authors.initials: PL
authors.lastname: Meroni
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001323:Autoantibodies / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D014471:Umbilical Veins; D015854:Up-Regulation / Q000276:immunology; D053482:beta 2-Glycoprotein I
chemical_list: D000838:Anions; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D019040:E-Selectin; D000939:Epitopes; D006023:Glycoproteins; D015850:Interleukin-6; D010446:Peptide Fragments; D010743:Phospholipids; D053482:beta 2-Glycoprotein I; D008239:Lysine