Characterization and comparative functional analysis in yeast of a Schistosoma mansoni Rho1 GTPase gene.
Low-molecular weight GTP-binding proteins (LMWGPs) of the Ras superfamily are believed to play a role in Schistosoma mansoni female development and egg production. Here we describe the characterization of a novel S. mansoni gene (SMRHO1), highly homologous to Rho-type LMWGPs from several other organisms and encoding a polypeptide with 193 amino acids and an estimated molecular mass of 21.8 kDa. SMRHO1 complemented a Saccharomyces cerevisiae rho1 null mutant strain even in restrictive temperature and calcium concentration, in contrast with the human RHOA GTPase that was not able to provide complementation in such conditions. Comparison of the amino acid sequence of the alpha3-helix loop7 regions of the two proteins allowed the identification of the proline 96 and threonine 100 amino acid residues of human RHOA as the most probable determinants of the complementation differences. We generated SMRHO1 mutants (smrho1(E97P), smrho1(L101T) and smrho1(E97P,) L101T) by site directed mutagenesis and reproduced the conditional lethality phenotype at high temperature, providing strong evidence that the related amino acid positions (Gln(101) and Ile(105)) in the Rho1 GTPase are indeed important for regulation of the cell wall synthesis performed by this protein in yeast. The observation that specific amino acid positions seem to be important for the different functions performed by the Rho GTPases leads to the idea that SMRHO1 might be a useful target in the development of new anti-schistosomiasis drugs, although it does share high sequence homology with the human RhoA GTPase.
Santos
Túlio Marcos
TM
Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, Belo Horizonte CEP 30161-970, MG, Brazil.
Machado
Carlos Renato
CR
Franco
Glória Regina
GR
Pena
Sérgio Danilo Junho
SD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D005690:Galactose / Q000378:metabolism; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D020741:rho GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins; D005690:Galactose