Engineering of recombinant soluble CD46: an inhibitor of complement activation.
Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies. Following a single-step immunoaffinity purification, the protein resolved as a single band of approximately 56,000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rsCD46 (51 micrograms/ml) protected Chinese hamster ovary (CHO) cells from lysis initiated by a high titre rabbit anti-CHO antibody and complement from rabbit or human. The protection was specifically mediated by rsCD46 because the monoclonal antibody M177, which blocks interaction between CD46 and C3b/C4b, abrogated the protection. The results demonstrate that rsCD46 is effective as a fluid-phase regulator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Administration of a bolus injection of rsCD46 was effective at delaying hyperacute graft rejection. These data suggest that rsCD46 may have a role as a therapeutic agent.
Christiansen
D
D
Department of Surgery, University of Melbourne, Parkville, Australia.
Milland
J
J
Thorley
B R
BR
McKenzie
I F
IF
Mottram
P L
PL
Purcell
L J
LJ
Loveland
B E
BE
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D016466:CHO Cells; D003167:Complement Activation / Q000276:immunology*; D003165:Complement System Proteins / Q000276:immunology*; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D006084:Graft Rejection / Q000276:immunology / Q000628:therapy; D006801:Humans; D007167:Immunotherapy; D008297:Male; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D015703:Antigens, CD; C497857:CD46 protein, human; C497860:Cd46 protein, rat; D017931:DNA Primers; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D003165:Complement System Proteins