RESUMO
Ethylmalonic aciduria is a common biochemical finding in patients with inborn errors of short chain fatty acid beta-oxidation. The urinary excretion of ethylmalonic acid (EMA) may stem from decreased oxidation by short chain acyl-CoA dehydrogenase (SCAD) of butyryl-CoA, which is alternatively metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general population, respectively. One hundred and thirty-five patients from Germany, Denmark, the Czech Republic, Spain, and the United States were selected for this study on the basis of abnormal EMA excretion ranging from 18 to 1185 mmol/mol of creatinine (controls < 18 mmol/mol of creatinine). Among them, we found a significant overrepresentation of the variant allele. Eighty-one patients (60%) were homozygous for the A625 allele, 40 (30%) were heterozygous, and only 14 (10%) harbored the wild-type allele (G625) in homozygous form. By overexpressing the wild-type and variant protein (G209S) in Escherichia coli and COS cells, we showed that the folding of the variant protein was slightly compromised in comparison to the wild-type and that the temperature stability of the tetrameric variant enzyme was lower than that of the wild type. Taken together, the over-representation and the biochemical studies indicate that the A625 allele confers susceptibility to the development of ethylmalonic aciduria.
Assuntos
Acil-CoA Desidrogenases/genética , Erros Inatos do Metabolismo Lipídico/enzimologia , Malonatos/metabolismo , Acil-CoA Desidrogenase , Animais , Sítios de Ligação , Linhagem Celular Transformada , Chlorocebus aethiops , DNA/análise , Expressão Gênica , Variação Genética , Erros Inatos do Metabolismo Lipídico/genética , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , RNA MensageiroRESUMO
The cDNA sequence and serological data for HLA-B73 are reported. Anti-B73 sera are found relatively frequently, considering the rarity of the antigen. It was noted early that in some cases the antibodies in sera of multiparous women did not react with the eliciting cells (fathers) and thus all behaved as a naturally occurring antibody. We report on 18 B73 antisera found during the screening of 55,000 Danish sera. Only one of the 17 stimulators typed also had the B73 tissue type. Ten of the stimulators had antigens from the B7 CREG (B7, B22, B27, B42, B67, B73), whereas none of the responders had such tissue types. In seven cases the serum was not able to react with the stimulator's lymphocytes in a cytotoxicity assay and in four cases the stimulator lymphocytes could not deplete the anti-B73 activity from the serum in absorption experiments. The cDNA of B73 was expressed correctly in COS cells and was recognized on the cell surface by a monospecific serum. The alpha 1 alpha 2 domains of B73 are most similar to those of the HLA-B22 family. Interestingly, the alpha 3 and transmembrane domains of HLA-B73 are not standard human domains, but are most similar to the corresponding domains of some gorilla and chimpanzee HLA-B genes.
Assuntos
DNA Complementar/genética , Genes MHC Classe I , Antígenos HLA-B/genética , Isoanticorpos/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Chlorocebus aethiops , Dinamarca , Feminino , Frequência do Gene , Antígenos HLA-B/química , Antígenos HLA-B/imunologia , Teste de Histocompatibilidade , Humanos , Isoanticorpos/isolamento & purificação , Transplante de Rim , Masculino , Programas de Rastreamento , Modelos Moleculares , Dados de Sequência Molecular , Paridade , Paternidade , Gravidez , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Trofoblastos/imunologiaRESUMO
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest that the MCAD mutations can be modulated by chaperones, a phenomenon that may influence the manifestation of the MCAD disease.
Assuntos
Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenase , Acil-CoA Desidrogenases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Escherichia coli/genética , Expressão Gênica/genética , MutaçãoRESUMO
In light of the recently determined structure of elongation factor Tu, and taking into account chemical studies mapping functional sites, a number of residues have been selected for site-directed mutagenesis studies. Gly94, Gly126, His66, His118, Lys89 and Asp90 have each been point-mutated. Preliminary in vitro characterization data are presented.