Identification and Functional Characterization of a New Gene Encoding the Mouse Terminal Complement Inhibitor CD59

Abstract

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

Analysis of the promoters and 5′-UTR of mouse Cd59 genes, and of their functional activity in erythrocytes

“…19,20 In order to determine the location of functionally essential sequences required for maximal promoter activity of mCd59a or mCd59b, we mapped the promoter regions by deletion analysis combined with luciferease gene reporter assays in NIH 3T3 cells. Several luciferase reporter constructs were generated by inserting different fragments of mCd59a or mCd59b genomic DNA into a pGL3-basic vector upstream from the firefly luciferase reporter gene; in both cases the mCd59 genomic DNA inserts were from sequences upstream of exon 1 of the respective mCd59 gene (Figure 1).…”

“…We have previously reported the presence of mCd59b mRNA in different tissues, 21 a finding disputed by Baalasubramanian et al 25 We reasoned that the presence of mCd59b in tissues other than testes and bone marrow could be investigated by a mouse expressed sequence tag (EST) database search. A BLAST EST database search using the whole mouse mCd59b cDNA sequence 20 found that several ESTs from different tissues matched the mCd59b sequence with over 97% similarity in the region of exons 2 to 4. In contrast, the region corresponding to exon 1 mCd59b in different ESTs showed no homology with the mCd59b sequence used for the search.…”

“…[19][20][21] In both genes, the first exon contains only 5 0 -untranslated region (5 0 -UTR), the second exon contains the remainder of the 5 0 -UTR and the beginning of the coding sequence, and the third and fourth exons contain the rest of the coding sequence and the 3 0 -untranslated regions. 20 The elucidation of the mouse Cd59 genomic structure allowed the targeted deletion of the Cd59 genes. 21,22 Holt et al generated mCd59a…”

The role of complement system in ocular diseases including uveitis and macular degeneration

“…It controls complement activation at the critical step of C3 convertase formation (Kim et al, 1995a;Takizawa, et al, 1994;Molina, et al, 1992). Tissue distribution studies have shown that both Crry and CD59 are widely distributed proteins in rodents (Li, et al, 1993;Powell, et al, 1997;Qian, et al, 2000). DAF and MCP have also been identified in rodents (Spicer, et al, 1995;Miwa, et al, 1998).…”

Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis

“…In the mouse, the situation is complicated by a gene duplication event, creating two forms of CD59, the first described, termed CD59a being broadly distributed whereas the second, CD59b is likely expressed only in testis. [26][27][28] We have recently generated CD59a-deficient mice (CD59aÀ/À) by targeted gene deletion. 29 These mice are healthy apart from mild intravascular haemolysis and haemoglobinuria.…”