Echinococcus multilocularis: identification and molecular characterization of a Ral-like small GTP-binding protein.
In mammals, Ral (Ras-like) GTPases have been implicated in the regulation of several cellular key processes such as oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics. Here we provide, for the first time, molecular data on a Ral homologue from a parasitic helminth. We have cloned and characterized the complete cDNA molecule and the chromosomal locus encoding a novel GTP binding protein, EmRal, of the human parasite Echinococcus multilocularis. The encoded protein contained all highly conserved amino acid residues of the protein family at corresponding positions and shared significant sequence homologies with human RalA (53% identity) and RalB (54%). Upon heterologous expression of EmRal in Escherichia coli, the recombinant protein was able to bind GTP, thus indicating functionality of the Echinococcus factor. Using an in vitro prenylation assay, the purified protein was shown to be geranylgernylated, but not farnesylated, in both rabbit reticulocyte and Echinococcus cell extracts. The EmRal mRNA was found to be processed via trans-splicing and, using RT-PCR and virtual Northern blot experiments, expression of the factor could be demonstrated for the larval stages metacestode and protoscolex during an infection of the intermediate host. The data presented herein provide a solid basis for further investigations on Ras-Ral signaling mechanisms in Echinococcus.
Spiliotis
Markus
M
Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Strasse 2, D-97080, Germany.
Brehm
Klaus
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D004446:Echinococcus / Q000737:chemistry* / Q000235:genetics; D005786:Gene Expression Regulation; D005849:Gerbillinae; D006801:Humans; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D018510:RNA, Helminth / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000737:chemistry; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017385:Sequence Homology; D020725:ral GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D018509:DNA, Helminth; D011106:Polyisoprenyl Phosphates; D018510:RNA, Helminth; D012333:RNA, Messenger; D020038:RNA, Spliced Leader; D020725:ral GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate