Article: article from journal or magazin.
Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin.
Publication types: Journal Article ; Research Support, Non-U.S. Gov't Publication Status: ppublish
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.
Amino Acid Sequence, Antibodies/immunology, Antibodies/pharmacology, Antigens, CD/analysis, Cell Line, Cell Membrane/metabolism, Complement C3/immunology, Complement C3/metabolism, Complement Factor D/immunology, Complement Pathway, Alternative/immunology, Cytotoxicity, Immunologic, Diffusion, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Fluorescent Antibody Technique, Gene Expression, Humans, Membrane Proteins/chemistry, Membrane Proteins/genetics, Models, Immunological, Molecular Sequence Data, Properdin/chemistry, Properdin/genetics, Protein Binding/drug effects, Receptors, Platelet-Derived Growth Factor/chemistry, Receptors, Platelet-Derived Growth Factor/genetics, Recombinant Fusion Proteins/chemistry, Recombinant Fusion Proteins/genetics, Sequence Deletion, Solubility, Substrate Specificity, Transfection
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