Membrane cofactor protein (CD46) of complement. Processing differences related to alternatively spliced cytoplasmic domains.

Membrane cofactor protein (MCP, CD46), a widely distributed regulatory protein, inhibits complement activation on host cells and serves as a measles virus receptor. Most cells express four isoforms (with one of two cytoplasmic tails, CYT-1 or CYT-2). Previously, we noted that MCP precursors had variable intracellular processing. Therefore, we characterized the intracellular transport of individual MCP isoforms. Transfectants were used for pulse-chase analyses. MCP isoforms bearing CYT-1 chased into their mature, surface forms with a half-life (t1/2) of 10-13 min while those with CYT-2 required 35-40 min. The precursor of a tail-less mutant possessed a t1/2 of 160-165 min. Chimeras were constructed that added both tails in opposite orientation onto the isoform (i.e. CYT 1 + 2 or CYT 2 + 1). Chimera 1 + 2 precursor processed with a t1/2 of 35-37 min, similar to CYT-2. Chimera 2 + 1 had a t1/2 of 15-19 min, more closely resembling CYT-1. Thus, in both cases the carboxyl-terminal tail controlled the processing rate. Deletions were made in the beginning, middle, and carboxyl terminus of CYT-1. Deletion of the first or middle six amino acids had no effect on the processing rate. However, deletion of the terminal tetrapeptide (FTSL) slowed the rate to 30-32 min, suggesting that this sequence facilitates exit from the endoplasmic reticulum.

Expression of human decay accelerating factor or membrane cofactor protein genes on mouse cells inhibits lysis by human complement.

Mouse cells expressing the human complement regulatory proteins decay accelerating factor (DAF) or membrane cofactor protein (MCP) were produced both by hybridoma technology and by transfection with the appropriate cDNAs. The expression of either or both of these products protected the mouse cell from lysis by human (though not rabbit) complement in the presence of naturally occurring human anti-mouse antibody. This effect could be abrogated by the addition of monoclonal antibody against DAF or MCP. These data suggested that the production of animals transgenic for human complement regulatory proteins should in principle be similarly protected from hyperacute xenograft rejection.

Membrane cofactor protein of the complement system: alternative splicing of serine/threonine/proline-rich exons and cytoplasmic tails produces multiple isoforms that correlate with protein phenotype.

Membrane cofactor protein (MCP) is a complement regulatory protein that is expressed on human cells and cell lines as two relatively broad species with Mr of 58,000-68,000 and 48,000-56,000. The structure of a previously reported cDNA clone indicated that MCP was a type 1 membrane glycoprotein and a member of the regulators of complement activation gene/protein cluster. However, it did not provide an explanation for the unusual phenotypic pattern of MCP. Therefore, in parallel with an analysis of the gene, additional cDNAs were cloned and characterized. Six different MCP cDNA classes were identified. All encode the same 5' untranslated signal peptide, four SCRs, transmembrane domain, and basic amino acid anchor. However, they differ in the length and composition of an extracellular serine/threonine/proline (STP)-rich area, a site of heavy O-glycosylation, and cytoplasmic tail. Analysis of the MCP gene demonstrated that the variation in cDNA structure was a result of alternative splicing. Peripheral blood cells and cell lines predominantly expressed four of the six isoforms. These varied by the presence or absence of an STP-rich segment of 15 amino acids (STPB) and by the use of one of two cytoplasmic domains. Analysis by polymerase chain reaction, Northern blots, and transfection indicated that the predominance of MCP cDNA isoforms with STPB correlated with the high molecular weight protein phenotype, while the predominance of isoforms without STPB correlated with the lower molecular weight phenotype. The expression in a single cell of four distinct protein species with variable STP-rich regions and cytoplasmic tails represents an interesting example of the use of alternative splicing to provide variability in a mammalian protein.