Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Translation is enhanced after silent nucleotide substitutions in A+T- rich sequences of the coding region of CD46 cDNA.

Specific sequences in the coding region of CD46 (membrane cofactor protein) transcripts have been shown to have a marked effect on translation. Two A+T-rich regions of CD46 cDNA were altered by mutation without changing the CD46 amino acid sequence (silent nucleotide substitution). In one region, the A+T content was reduced from 78% to 55% and in the other a putative polyadenylation addition sequence was disrupted. In each example, mutated sequences transfected into COS-7 cells produced significantly more soluble or cell surface protein (up to a 20-fold increase) than wild-type sequences. The amount of cellular plasmid DNA and CD46 mRNA was not increased, suggesting that the effect was not due to increased transfection efficiency, or transcript synthesis or stability. Biosynthetically labelled transfected cells showed an increase in translation rate but cell-free in vitro translation studies demonstrated that wild-type and mutated transcripts were translated with similar efficiency. The data show that translation of CD46 is affected by specific mRNA coding sequences, 400-540 bases from the initiation codon, and suggest that these sequences require the structural integrity of the cell to exert their effect.

Engineering of recombinant soluble CD46: an inhibitor of complement activation.

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies. Following a single-step immunoaffinity purification, the protein resolved as a single band of approximately 56,000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rsCD46 (51 micrograms/ml) protected Chinese hamster ovary (CHO) cells from lysis initiated by a high titre rabbit anti-CHO antibody and complement from rabbit or human. The protection was specifically mediated by rsCD46 because the monoclonal antibody M177, which blocks interaction between CD46 and C3b/C4b, abrogated the protection. The results demonstrate that rsCD46 is effective as a fluid-phase regulator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Administration of a bolus injection of rsCD46 was effective at delaying hyperacute graft rejection. These data suggest that rsCD46 may have a role as a therapeutic agent.

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro.

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35 . More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsD55 and rsCD35 to regulate human complement activation mediated by the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46+ rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCd55 and rsCD35, may be useful a therapeutic agent.

Different membrane cofactor protein (CD46) isoforms protect transfected cells against antibody and complement mediated lysis.

The need for organ transplantation, especially of kidneys, exceeds the availability of human donors and the possibility of xenotransplantation from suitable animals is now being addressed. The immediate barrier to success is hyperacute graft rejection, resulting from naturally occurring xenoreactive antibodies and the activation of complement. It is proposed that the intensity of the hyperacute response can be reduced by providing additional regulatory molecules to limit activation of the complement cascade, initially as transfected gene products in cultured cells as an in vitro model and eventually as a transgene in potential donor animals, such as pigs. Limiting the activity of C3b reduces the production of the C3a, C4a and C5a anaphylotoxins, thus curtailing not only the immediate C3b-mediated lytic pathway but also the later effects of a cellular inflammatory response including endothelial and platelet cell activation. To develop and assess the first part of this strategy, we have transfected several cDNA's encoding isoforms of CD46 (membrane cofactor protein). At least four different CD46 isoforms are commonly expressed in almost all human cells, and we have compared two of these and a third form to determine if they mediate different functions. After transfection, CD46-expressing CHO-K1 cells were selected with methionine sulphoximine and identified using monoclonal antibodies. Transfectants with suitable CD46 expression were assayed for primary CD46 function using a lysis assay dependent on the reaction of antibody and complement. In this in vitro model of hyperacute rejection, normal human sera containing natural xenoreactive antibodies were shown to lyse CHO cells, but only in the presence of complement.(ABSTRACT TRUNCATED AT 250 WORDS)