Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.
Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.
Jokiranta
T Sakari
TS
Department of Bacteriology and Immunology, Haartman Institute and Huslab, FIN-00014, University of Helsinki, Helsinki, Finland. sakari.jokiranta@helsinki.fi
Cheng
Zhu-Zhu
ZZ
Seeberger
Harald
H
Jòzsi
Mihály
M
Heinen
Stefan
S
Noris
Marina
M
Remuzzi
Giuseppe
G
Ormsby
Rebecca
R
Gordon
David L
DL
Meri
Seppo
S
Hellwage
Jens
J
Zipfel
Peter F
PF
D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D015933:Complement C3d / Q000378:metabolism; D017242:Complement Factor H / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D000071228:Polyelectrolytes; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014471:Umbilical Veins / Q000166:cytology
D000911:Antibodies, Monoclonal; D015933:Complement C3d; D017242:Complement Factor H; D006025:Glycosaminoglycans; D006493:Heparin; D000071228:Polyelectrolytes; D011108:Polymers; D011994:Recombinant Proteins