CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a “hydrophobic strip” suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.
The murine coronavirus polymerase gene is 22 kb in length with the potential to encode a polyprotein of approximately 750 kDa. The polyprotein has been proposed to encode three proteinase domains which are responsible for the processing of the polyprotein into mature proteins. The proteolytic activity of the first proteinase domain has been characterized and resembles the papain family of cysteine proteinases. This proteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein designated p28. To identify the cleavage site of this papain-like cysteine proteinase, we isolated the peptide adjacent to p28 and determined the amino terminus sequence by Edman degradation reaction. We report that proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleavage site, we introduced a total of 42 site-specific mutations at the residues spanning the P5 to P3' positions and assessed the effects of the mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position or Arg-246 at the P2 position resulted in a dramatic decrease of proteolytic activity, and the mutations of Arg-243 at P5 position also led to considerable reduction in p28 cleavage. In contrast, the substitutions of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 (P2'), and Pro-250 (P3') had little or no effect on the amount of p28 that was released. This work had identified Gly-247-Val-248 as the cleavage site for the release of p28, the amino-terminal protein of the murine coronavirus polymerase polyprotein. Additionally, we conclude that the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murine coronavirus.
The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.
CD59 is a widely distributed membrane-bound glycoprotein that inhibits the formation of the cytolytic membrane attack complex (MAC) of complement on host cells. CD59 from different species varies in its capacity to inhibit heterologous complement, and this species selective function of CD59 contributes to the phenomenon of homologous restriction. Here, we demonstrate that human CD59 is not an effective inhibitor of rat complement, although rat CD59 inhibits rat and human complement equally well. By constructing human-rat CD59 chimeric proteins, we have mapped the residues important in conferring human CD59 species selectivity to two regions; 40-47 and 47-66 in the primary structure. Analysis of a model of the molecular surface of human CD59 revealed that residues 40-66 mapped to a region in the three-dimensional structure that surrounds residues previously identified as important for CD59 function.
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