Transfected cDNA Directs Expression of Hemolytically Active Murine C4 in Cultured Mouse and Monkey Cells

Abstract

Previously cloned and sequenced full-length cDNAs for murine C4 and the closely related sex-limited protein (Sip) have been placed into an eucaryotic expression vector. Transfer of these DNA constructs transiently into monkey COS cells or stably into mouse L cells results in the expression and secretion of hemolytically active mouse C4 and mature Sip. We estimate from hemolytic activities that COS and L cells secrete 0.04 and 3%, respectively, of the C4 level found in mouse plasma. Sip expression is consistently only 10-20% that of C4 although the identical expression system is used for both. Our results show subtle but reproducible cell-type-specific differences in C4 maturation; they also indicate that surprisingly large shifts in electrophoretic mobility on SDS-polyacrylamide gels are induced by a small number of amino acid substitutions. The expression of C4 from cDNAs of known sequence provides a starting point for studies of structure/function relationships in C4 employing site-specific mutagenesis and gene transfer.

Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.

“…Expression of both wild-type and mutant Slp proteins is very poor, however (e.g., ref. 15), and our preliminary results suggest that both proteins, from both COS-1 and L cells, are not in the native conformation because they do not appear to have intact internal thioester groups. These results are equivocal, however, because the levels of expression we observed were very low and C4 expression at similar low levels also yields apparently nonnative proteins (unpublished observations).…”

“…Construction of plasmid pC427A, which consists of a full-length mouse C4 cDNA cloned into the pcD expression vector (16), and the sequence of the cDNA have been described (8). All plasmid DNAs were prepared by a preparative alkaline lysis procedure (17) (excluding the CsC1 equilibrium centrifugation procedure) followed by extraction with phenol, treatment with RNase A at 50 ,ug/ml, precipitation twice from 5.5% (wt/vol) polyethylene glycol (PEG 6000) to remove RNA (18) Transient transfections were carried out as described (26,27) by treating cells in 35-mm dishes with 2 ,ug of DNA in 0.7 ml of DEAE-dextran at 200 ,ug/ml followed by a 15% (vol/vol) glycerol shock after 6 (15,30). Immunoprecipitated products were fractionated on SDS/8.5% polyacrylamide gels, which were treated with EN3HANCE (DuPont/NEN) and autoradiographed.…”

“…Other labels are as in Fig. 1. served, of course, in particular with the murine C4 P-and -chains (15) and with the a-chains of human C4A and C4B (34,38). Nevertheless, the large shift observed with CMD3 and CMD6 raised the possibility that other unanticipated changes in the cDNA sequence may have been introduced during the mutagenesis procedure.…”

“…Hemolytic activities of the mutant C4 proteins were assayed to determine the effects of each mutation on C4 functional activity. Stably transformed murine L cells were used for these studies because they express higher levels of C4 than transiently transfected COS-1 cells (15). Cultures were radiolabeled with [35S]methionine and both C4 quantitation and hemolytic assays were carried out on the same sample of culture supernatant.…”

“…We also cannot exclude the more likely possibility of other structural defects in Slp that would render it inactive even if Cli cleavage were to occur. We have tried to address this question by placing into the pcD expression vector the wild-type Slp cDNA (10,15) and a Slp mutant in which the three deleted residues have been restored (unpublished data). Expression of both wild-type and mutant Slp proteins is very poor, however (e.g., ref.…”