Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin.
Properdin (P) is a serum glycoprotein that stabilizes the labile C3 convertase (C3bBb) of the alternative pathway of the complement system (AP). Thanks to its oligomeric nature, P specifically upregulates AP on surfaces without activating AP in the fluid-phase. We investigated whether human cells, displaying P at their membrane, could activate autologous AP. The cDNAs encoding human P and the transmembrane domain of human platelet derived growth factor receptor were fused together and expressed in human embryo kidney cells (HEK-293). Selected cells displayed P at their surface as shown by FACS. In contact with human serum at 37 degrees C, they triggered AP-mediated C3 deposition. SDS-PAGE analysis showed C3 covalently bound to various membrane proteins, but not to P itself. However, displayed P affinity could bind to serum or purified C3i at 4 degrees C. C3 binding was restricted to the cells displaying P, was inhibited by an anti-P mAb, and did not require serum P. Bound C3 allowed further C5, C7 and C9 deposition as well as cell lysis after blocking CD59 function. In contrast, wild-type cells, cells displaying factor D or truncated P (deleted from its 6th thrombospondin-like repeat) did not activate AP. We hypothesize that displayed P activates AP by stabilizing bystander C3b and/or by capturing serum C3iBb convertase. Finally, we suggest that P could be used for retargeting autologous complement to AP-resistant pathogens and tumor cells.
Vuagnat
B B
BB
Biochemistry Institute, University of Lausanne, CH-1066, Epalinges, Switzerland.
Mach
J
J
Le Doussal
J M
JM
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003176:Complement C3 / Q000276:immunology / Q000378:metabolism; D011416:Complement Factor D / Q000276:immunology; D003170:Complement Pathway, Alternative / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018448:Models, Immunological; D008969:Molecular Sequence Data; D011414:Properdin / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D013379:Substrate Specificity; D014162:Transfection
D000906:Antibodies; D015703:Antigens, CD; D003176:Complement C3; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011414:Properdin; D017479:Receptors, Platelet-Derived Growth Factor; C493488:CFD protein, human; D011416:Complement Factor D