OBJECTIVETo investigate the interaction of deficiency in thrombosis-related gene in a mouse model.

METHODSTo generate mice carrying mutations in alpha-galactosidase A (Gla) and factor V Leiden (Fvl) and analyze the phenotypes, namely, tissue fibrin deposition and thrombus formation in organs.

RESULTSFibrin deposition in organs of mice carrying both mutations in Gla and Fvl was significantly increased compared with that in mice with single mutaton: [Gla(-/0) Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=(0.28+/-0.03)% vs.(0.07+/-0.007)%, P<0.01; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=(0.28+/-0.03)% vs.(0.11+/-0.02)%, P< 0.01. Meanwhile, the number of thrombi on organ sections of mice carrying both mutations in Gla and Fvl was significantly increased compared with the single mutation carrier: [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=1.9+/-0.7 vs. 0.0+/-0.0, P<0.05; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs. [Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=1.9+/-0.7 vs. 0.3+/-0.1, P<0.05.

CONCLUSIONThese observations demonstrated that there was synergistic effect in Gla and Fvl deficiency in mice. It suggested that there could be a combination of GLA deficiency and FVL or other thrombosis-related gene defect in patients with genetic severe early-onset thrombosis.

Animals ; Factor V ; genetics ; Fibrin ; metabolism ; Immunohistochemistry ; Mice ; Mutation ; Thrombosis ; genetics ; metabolism ; alpha-Galactosidase ; genetics

In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.
ABO Blood-Group System ; immunology ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes ; drug effects ; Fermentation ; physiology ; Hemagglutination ; drug effects ; Humans ; Pichia ; genetics ; metabolism ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology

Fabry's disease is an X-linked lysosomal storage disorder caused by abnormalities in the alpha-galactosidase A (GLA) gene, which leads to a GLA deficiency and to the intracellular deposition of globotriaosylceramide (Gb3) within vascular endothelium and other tissues. It manifests as progressive multiple organ dysfunctions caused by the deposition of Gb3. On the other hand, congenital agammaglobulinemia is usually caused by mutations in Bruton's tyrosine kinase (Btk) gene with X-linked dominence, suppresses B cell maturation, and causes recurrent pyogenic infections. In former reports, the distance between the loci in the Xq22 region of the human X chromosome was found to be about 69 kilobases. A 23-yr-old man diagnosed with congenital agammaglobulinemia at age 5, showed typical clinical and laboratory and histopathological findings of Fabry's disease. The genetic basis of this combination of the two syndromes was studied in this patient. Here, we report a case of Fabry's disease with congenital agammaglobulinemia.
Adult ; Agammaglobulinemia/congenital/*genetics/pathology ; Chromosomes, Human, X ; Fabry Disease/diagnosis/*genetics ; Humans ; Kidney/pathology ; Male ; Microscopy, Electron ; Sequence Analysis, DNA ; Skin/pathology ; alpha-Galactosidase/genetics/metabolism

OBJECTIVETo explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.

METHODSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.

RESULTSHuman alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.

CONCLUSIONSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.

Animals ; Antigens, Heterophile ; metabolism ; Cell Death ; Cells, Cultured ; Disaccharides ; metabolism ; Endothelial Cells ; metabolism ; Fucosyltransferases ; genetics ; metabolism ; Graft Rejection ; genetics ; Humans ; Mice ; Mice, Transgenic ; Spleen ; cytology ; Swine ; Transfection ; alpha-Galactosidase ; genetics ; metabolism

BACKGROUND/AIMS: Fabry disease is an X-linked recessive and progressive disease caused by alpha-galactosidase A (alpha-GaL A) deficiency. We sought to assess the prevalence of unrecognized Fabry disease in dialysis-dependent patients and the efficacy of serum globotriaosylceramide (GL3) screening. METHODS: A total of 480 patients of 1,230 patients among 17 clinics were enrolled. Serum GL3 levels were measured by tandem mass spectrometry. Additionally, we studied the association between increased GL3 levels and cardiovascular disease, cerebrovascular disease, or left ventricular hypertrophy. RESULTS: Twenty-nine patients had elevated serum GL3 levels. The alpha-GaL A activity was determined for the 26 patients with high GL3 levels. The mean alpha-GaL A activity was 64.6 nmol/hr/mg (reference range, 45 to 85), and no patient was identified with decreased alpha-GaL A activity. Among the group with high GL3 levels, 15 women had a alpha-GaL A genetics analysis. No point mutations were discovered among the women with high GL3 levels. No correlation was observed between serum GL3 levels and alpha-GaL A activity; the Pearson correlation coefficient was 0.01352 (p = 0.9478). No significant correlation was observed between increased GL3 levels and the frequency of cardiovascular disease or cerebrovascular disease. CONCLUSIONS: Fabry disease is very rare disease in patients with end-stage renal disease. Serum GL3 measurements as a screening method for Fabry disease showed a high false-positive rate. Thus, serum GL3 levels determined by tandem mass spectrometry may not be useful as a screening method for Fabry disease in patients with end stage renal disease.
Adult ; Aged ; Fabry Disease/blood/*diagnosis ; Female ; Humans ; Kidney Failure, Chronic/blood/*therapy ; Male ; Middle Aged ; *Renal Dialysis ; Trihexosylceramides/*blood ; alpha-Galactosidase/genetics/metabolism

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the alpha-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.
1-Deoxynojirimycin/*analogs & derivatives/metabolism ; Adolescent ; Adult ; Animals ; Asian Continental Ancestry Group/*genetics ; COS Cells ; Cercopithecus aethiops ; Fabry Disease/*enzymology/genetics ; Gene Expression ; Humans ; Male ; Middle Aged ; Mutation ; Young Adult ; alpha-Galactosidase/*genetics/*metabolism

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.
ABO Blood-Group System ; classification ; immunology ; Blood Group Incompatibility ; prevention & control ; Blood Transfusion ; methods ; Coffee ; enzymology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; immunology ; metabolism ; Humans ; Isoantigens ; drug effects ; metabolism ; Recombinant Proteins ; metabolism ; pharmacology ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology