Annexin-II, DNA, and histones serve as factor H ligands on the surface of apoptotic cells.
Apoptotic cells are opsonized by complement components such as C1q and C3b, which increases their susceptibility to phagocytosis. Soluble complement inhibitors such as factor H (fH) also recognize apoptotic cells to minimize the pro-inflammatory effects of downstream complement activation. We used four radiolabeled protein constructs that span different regions of the 20 complement control protein (CCP) modules that make up fH and found that fragments comprising CCPs 6-8, CCPs 8-15, and CCPs 19-20 but not CCPs 1-4, bound to apoptotic Jurkat T cells. There are four possible ligand types on apoptotic cells that could recruit fH: proteins, carbohydrates, lipids, and DNA. We found that CCPs 6-8 of fH bind to annexin-II, a trypsin-insensitive protein that becomes exposed on surfaces of apoptotic cells. The second ligand of fH, which interacts with CCPs 6-8 and 19-20, is DNA. Confocal microscopy showed co-localization of fH with antibodies specific for DNA. fH also binds to histones devoid of DNA, and CCPs 1-4, 6-8, and 8-15 mediate this interaction. Treatment of apoptotic cells with neuraminidase, chondroitinase, heparitinase, and heparinase did not change fH binding. Treatment of apoptotic cells with phospholipase A(2) dramatically increased both binding of fH and cell-surface DNA. We also excluded the possibility that fH interacts with lysophospholipids using surface plasmon resonance and flow cytometry with lipid-coated beads. Identification of annexin-II as one of the fH ligands on apoptotic cells together with the fact that autoantibodies against annexin-II are found in systemic lupus erythematosus provides further insight into understanding the pathogenesis of this disease.
Leffler
Jonatan
J
From the Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden and.
Herbert
Andrew P
AP
the Edinburgh Biological NMR Unit, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Norström
Eva
E
From the Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden and.
Schmidt
Christoph Q
CQ
the Edinburgh Biological NMR Unit, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Barlow
Paul N
PN
the Edinburgh Biological NMR Unit, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Blom
Anna M
AM
From the Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden and. Electronic address: Anna.Blom@med.lu.se.
Martin
Myriam
M
From the Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden and.
D017306:Annexin A2 / Q000378:metabolism*; D017209:Apoptosis; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D050716:Complement C4b-Binding Protein / Q000378:metabolism; D017242:Complement Factor H / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism*; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008024:Ligands; D018613:Microscopy, Confocal; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009336:Necrosis; D054467:Phospholipases A2 / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020349:Surface Plasmon Resonance
D017306:Annexin A2; D050716:Complement C4b-Binding Protein; D004273:DNA, Neoplasm; D006025:Glycosaminoglycans; D006657:Histones; D008024:Ligands; D017242:Complement Factor H; D054467:Phospholipases A2; D019158:N-Acetylneuraminic Acid