MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF THE PIG ANALOG OF CD59 - RELEVANCE TO XENOTRANSPLANTATION

In this work, we report the cloning of the cDNA for the porcine analog ue of human CD59, Degenerate primers, derived from the N-terminal sequ ence of pig erythrocyte CD59, were used to obtain the corresponding cD NA sequence, From this sequence, gene-specific primers were designed a nd used to amplify the 3' and 5' ends of the cDNA using the rapid ampl ification of cDNA ends (RACE) method, The complete 768-bp cDNA so obta ined consisted of a 84-bp 5' untranslated region, a 26-amino-acid NH2- signal peptide, a 98-amino-acid coding region, including putative N-gl ycosylation 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 ana lysis revealed several distinct CD59 transcripts, and a variability in expression levels of the different transcripts in the panel of tissue s screened, Stable expression of Dig CD59 in a CD59-negative human cel l line conferred protection against lysis by complement from pig and s everal other species, Separate expression of pig and human CD59 at sim ilar levels in the same cell line allowed a direct functional comparis on between these two analogues, Pig CD59 and human CD59 showed similar activity in inhibiting lysis by complement from all species tested; i n particular, expressed pig CD59 efficiently inhibited lysis by human complement, The relevance of these data to current work in the enginee ring of pig organs for xenotransplantation is discussed.