RESUMO
Polymerase chain reaction (PCR) primers based on the cysteine proteinase-like active site regions of the Plasmodium falciparum serine repeat antigen (SERA) were used to identify related sequences within the genome of P. vivax. Molecular cloning and sequence analysis of approximately 25 kb of P. vivax genomic DNA revealed a cluster of five repeated SERA-like genes (V-SERA-1-5), each encoding a cysteine proteinase-related protein. In addition to DNA sequence homology, significant similarities in deduced intron/exon organizations were also observed. The characteristic polyserine sequence found in SERA was not present in any of the deduced V-SERA sequences. Instead, in this region of the five genes, considerable sequence differences were found, suggesting the potential for antigenic variation in the V-SERA molecules. In common with SERA, however, the codon at the position corresponding to the active site cysteine residue of active mammalian and plant cysteinyl proteinases was found to be that of a serine residue in each of the V-SERA genes. Furthermore, in four of the five genes, including the expressed V-SERA-5 gene, the codon for the active site histidine residue was changed to that of a leucine residue. These critical differences reinforce the concept that a biological activity other than proteolysis is likely to be the primary function of the proteins encoded by this family of genes.
Assuntos
Antígenos de Protozoários/genética , Genes de Protozoários , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA de Protozoário/genética , Dados de Sequência Molecular , Família Multigênica , Plasmodium vivax/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.
