RESUMO
BACKGROUND AND OBJECTIVES: Most secreted proteins, including coagulation factor X (FX), are synthesized with a signal peptide, which is necessary for targeting the nascent polypeptide into the endoplasmic reticulum. Characterization of naturally occurring mutations may provide insights into the functional roles of the amino acids in the signal peptide. DESIGN AND METHODS: A 52-year old male patient with type I FX deficiency was studied. Mutations were searched for by FX gene (F10) sequencing. The wild-type and the mutant FX proteins were expressed in transfected cells and then immunological assays were performed. Pulse-chase experiments and cell-free expression studies were conducted to determine the cellular fate of the mutant FX molecules. RESULTS: The patient we studied was homozygous for a substitution of arginine for serine at codon -30 in the signal sequence of F10. Immunoassays detected low FX antigen levels in both the conditioned media and lysates of the cells expressing the mutant protein. Pulse-chase analysis showed that only trace amounts of the mutant FX protein were detectable in the conditioned media, and that the mutant molecules did not accumulate inside the cells either. The results of cell-free expression studies showed that although the transcription and translation of the mutant construct were normal, no post-translational processing, such as N-linked glycosylation, occurred in the presence of microsomes. INTERPRETATION AND CONCLUSIONS: These findings suggest that substitution of a neutral polar amino acid, serine by arginine, in the hydrophobic core of FX signal peptide severely impairs the ability of the protein to enter the endoplasmic reticulum and results in FX deficiency.
Assuntos
Retículo Endoplasmático/metabolismo , Deficiência do Fator X/genética , Fator X/genética , Mutação de Sentido Incorreto , Transporte Proteico , Substituição de Aminoácidos , Sistema Livre de Células , Células Cultivadas , Consanguinidade , Fator X/química , Fator X/metabolismo , Deficiência do Fator X/metabolismo , Hemorragia Gastrointestinal/etiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Pessoa de Meia-Idade , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Relação Estrutura-Atividade , TransfecçãoRESUMO
OBJECTIVE: To identify the gene mutation in a Chinese pedigree of type I hereditary protein C deficiency. METHODS: The plasma levels of protein C activity (PC:A), protein C antigen (PC:Ag), protein S activity, and anti-thrombin activity (AT:A) of the propositus, male, aged 7, and 11 members of the pedigree were detected using ELISA and chromogenic assay respectively. All of the nine exons and intron-exon boundaries of protein C gene of the propositus were analyzed by direct sequencing of the corresponding amplified PCR products in DNA from the propositus. Restriction enzyme site analysis was used to confirm the mutation. RESULTS: The plasma concentrations of protein C activity and antigen of the propositus were 26% and 1.43 g/L respectively. The PC:Ag and PC:A of his father were normal. Decreased PC:A level was seen in his mother and 4 of his maternal pedigree. PS:A and AT:A were all normal in all of the pedigree members. A C5498T heterozygous mutation in exon 3, resulting in the substitution of Arg for Trp at the 15th amino acid, was identified in the propositus and 8 of his relatives. This mutation was confirmed by restriction enzyme site analysis. Mutations C/T at position 2405, A/G at position 2418, and A/T at position 2583 in the protein C promoter region were confirmed in the propositus and all members of the pedigree. CONCLUSION: C5498T heterozygous mutation in exon 3 of protein C gene, first reported in China, leads to type I hereditary.
Assuntos
Mutação , Deficiência de Proteína C/genética , Proteína C/genética , Criança , Feminino , Humanos , Masculino , Linhagem , Proteína C/análiseRESUMO
OBJECTIVE: To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM. METHODS: The retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM. RESULTS: Human FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%. CONCLUSION: PAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.
Assuntos
Fator VIII/genética , Vetores Genéticos/genética , Plasmídeos , Poliaminas/farmacologia , Retroviridae/genética , Animais , Dendrímeros , Camundongos , Células NIH 3T3RESUMO
OBJECTIVE: To investigate the mechanisms of two novel missense mutations of factor XIIIA subunit gene (Arg77-->Cys,Ser413-->Trp) in the pathogenesis of hereditary factor XIII deficiency. METHODS: Site-directed mutagenesis was conducted to obtain 2 mutant human XIII A recombinant plasmids, mut-PCI/FXIIIA. Normal wild type factor XIII A recombinant plasmid, wt-PCI/FXIIIA, and mut-PCI/FXIIIA, were transfected into cultured COS7 cells line, renal fibroid cell of African green monkey using Superfect reagent respectively, The expression levels of DNA, RNA and protein of human factor XIII, both wild type and mutant, were detected by PCR, RT-PCR and Western blotting. Pulse-chase experiment was used to look into the changing of factor XIII A in the cytoplasm. Factor XIIIA activity was assayed by Biotin-pentylamine incorporation technique. RESULTS: The mRNA levels of the two mutants in transfected cells were similar to that of the wild type factor XIIIA. But the amount of mutant factor XIIIA protein and its activity in cells decreased markedly, even disappeared. Pulse-chase experiment revealed that at the two mutants existed chase time 0.5 h and 1 h considerable amounts in cells and then disappeared rapidly later. CONCLUSION: The 2 mutations of the factor XIIIA cause the instability, degradation, and rapid disappearance of FXIIIA in cytoplasm, thus resulting in hereditary factor XIII deficiency.