Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of...

We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.