Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.
BACKGROUND
It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement.


METHODS
cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis.


RESULTS
Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration.


CONCLUSIONS
These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.
Miyagawa
Shuji
S
Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. miyagawa@orgtrp.med.osaka-u.ac.jp
Fukuta
Daisuke
D
Kitano
Etsuko
E
Kobayashi
Chizuko
C
Fumimoto
Yuichi
Y
Shirasu
Akio
A
Hattori
Hiroyuki
H
Shirakura
Ryota
R
Fukuzawa
Masahiro
M
D000818:Animals; D018958:CD55 Antigens / Q000235:genetics / Q000494:pharmacology*; D016466:CHO Cells; D003167:Complement Activation / Q000187:drug effects*; D016384:Consensus Sequence; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D018076:DNA, Complementary; D005434:Flow Cytometry; D012091:Repetitive Sequences, Nucleic Acid; D013552:Swine; D014162:Transfection; D014183:Transplantation, Heterologous / Q000276:immunology*
D018958:CD55 Antigens; D018076:DNA, Complementary