MOLECULAR-CLONING, CHROMOSOMAL LOCALIZATION, EXPRESSION, AND FUNCTIONAL-CHARACTERIZATION OF THE MOUSE ANALOG OF HUMAN CD59

We have previously described the isolation and cloning of the rat anal ogue of the human complement inhibitor CD59 (hCD59). Using the rat CD5 9 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indica ted that they contained an open reading frame encoding a 124 amino aci d protein. Comparisons with the known sequences of hCD59 and rCD59 sug gested 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 i ncluding putative N-glycosylation sites and a glycosyl phosphatidylino sitol (GPI) anchoring signal, and similar to 0.8 kb 3'-untranslated fl anking region. Reverse transcriptase PCR revealed the presence of mCD5 9 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chrom osome 2, a region that includes areas syntenous with the location of t he human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59- negative human cell line conferred protection against lysis by complem ent from rodent, human, and several other species, confirming that mCD 59 was the functional analogue of hCD59 and that function was not spec ies restricted. The expressed protein was glycosyl phosphatidy-linosit ol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs rais ed against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutra lized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a mole cular mass of 22 to 24 kDa.