Molecular cloning and functional characterization of the pig analogue of CD59: relevance to xenotransplantation.
In this work, we report the cloning of the cDNA for the porcine analogue of human CD59. Degenerate primers, derived from the N-terminal sequence of pig erythrocyte CD59, were used to obtain the corresponding cDNA sequence. From this sequence, gene-specific primers were designed and used to amplify the 3' and 5' ends of the cDNA using the rapid amplification of cDNA ends (RACE) method. The complete 768-bp cDNA so obtained consisted of a 84-bp 5' untranslated region, a 26-amino-acid NH2-signal peptide, a 98-amino-acid coding region, including putative N-glycosylation sites and a glycosylphosphatidylinositol-anchoring signal, and a 312-bp 3' untranslated region. The mature protein sequence was 48% identical to human CD59 at the amino acid level. Northern blot analysis revealed several distinct CD59 transcripts, and a variability in expression levels of the different transcripts in the panel of tissues screened. Stable expression of pig CD59 in a CD59-negative human cell line conferred protection against lysis by complement from pig and several other species. Separate expression of pig and human CD59 at similar levels in the same cell line allowed a direct functional comparison between these two analogues. Pig CD59 and human CD59 showed similar activity in inhibiting lysis by complement from all species tested; in particular, expressed pig CD59 efficiently inhibited lysis by human complement. The relevance of these data to current work in the engineering of pig organs for xenotransplantation is discussed.
Hinchliffe
S J
SJ
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.
Rushmere
N K
NK
Hanna
S M
SM
Morgan
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D018957:CD59 Antigens / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D003169:Complement Inactivator Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000276:immunology; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013552:Swine / Q000235:genetics* / Q000276:immunology*; D014162:Transfection; D014181:Transplantation Immunology; D014183:Transplantation, Heterologous
D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D018076:DNA, Complementary