Expression of rat CD59: functional analysis confirms lack of species selectivity and reveals that glycosylation is not required for function.
This study reports the expression and functional characterization of the rat analogue of the human complement regulatory molecule CD59. We here describe the expression in chinese hamster ovary (CHO) cells of rat CD59 and a modified rat CD59 in which an N-glycosylation site at Asn-16 has been deleted by point mutation. The complement-inhibiting capacity of these two forms of rat CD59 has been analysed and compared. Expressed rat CD59 efficiently inhibited complement lysis of CHO cells when rat serum was used as a source of complement and also inhibited lysis by complement from all other species tested, confirming that rat CD59 displayed little or no species restriction of activity. Blocking of expressed rat CD59 with a monoclonal antibody abrogated the inhibition of lysis for all sources of complement, confirming that the expressed molecule was responsible for the protection. The glycosylation mutant had a much reduced molecular weight on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12,000 MW as compared with 20,000-28,000 MW for unmutated), confirming that it was unglycosylated. However, the glycosylation mutant had complement-inhibitory activity which was at least as potent as that of the unmutated molecule, demonstrating that the large, N-linked carbohydrate moiety was not required for function.
Rushmere
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NK
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Tomlinson
S
S
Morgan
B P
BP
D000818:Animals; D015153:Blotting, Western; D018957:CD59 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D003602:Cytotoxicity, Immunologic; D006031:Glycosylation; D016296:Mutagenesis; D017354:Point Mutation; D051381:Rats; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014162:Transfection
D018957:CD59 Antigens