Acid hydrolases in leukocytes and platelets of normal subjects and in patients with Gaucher's and Fabry's disease.

Lymphocytes, monocytes, neutrophilic granulocytes and platelets were each separated to greater than 95% purity from six normal subjects, three patients with Gaucher's disease, two heterozygotes for Gaucher's disease, and one patient with Fabry's disease. Activities of the following acid hydrolases were determined: "acid" (pH 4.0) beta-glucosidase, pH 5.0 beta-glucosidase, alpha-galactosidase, alpha-arabinosidase, alpha-mannosidase, alpha-glucosidase, beta-glucuronidase, beta-galactosidase, beta-hexosaminidase, and acid phosphatase. Enzymatic activity varied greatly with cell type and the enzyme being measured; the importance of assaying pure preparations especially for heterozygote detection is emphasized. Gaucher's disease patients' cells were found to be deficient in the pH 4.0 acid beta-glucosidase, variable in the pH 5.0 beta-glucosidase, and normal in all other acid hydrolases tested, including acid phosphatase, the activity of which is known to be elevated in plasma. Blood cells of a patient with Fabry's disease were deficient in alpha-galactosidase and normal in all other acid hydrolases tested.

Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

Glutathione reductase: stimulation in normal subjects by riboflavin supplementation.

Glutathione reductase of hemolyzates from clinically normal subjects is activated by the addition of flavin-adenine dinucleotide. One-half maximum stimulation could be achieved by approximately 0.02 micromolar flavin-adenine dinucleotide; prior addition of adenosine triphosphate, adenosine diphosphate, or adenosine mnonophosphate prevented activation. Stimulation of glutathione reductase activity of red cells of normal subjects occurred when they were given 5 milligrams of riboflavin daily for 8 days. The degree of stimulation in vitro by flavin-adenine dinucleotide and in vivo by riboflavin was inversely proportional to dietary intake of riboflavin. The variety of clinical disorders which have been associated with glutathione reductase deficiency may have, as a common denominator, abnormalities in flavin-adenine dinucleotide formation.

Gaucher disease: new molecular approaches to diagnosis and treatment.

Gaucher disease is characterized by the accumulation of glucocerebroside, leading to enlargement of the liver and spleen and lesions in the bones. It is caused by an inherited deficiency of the enzyme glucocerebrosidase. Many mutations exist, but four of these account for over 97% of the mutations in Ashkenazi Jews, the population group in which Gaucher disease is the most common. Although there is a strong relation between the mutations and disease manifestations, genetic counseling is made difficult by the fact that within each genotype there is considerable variability in the severity of the disease. Intravenous infusion of glucocerebrosidase is an effective treatment, but the availability of enzyme replacement therapy is limited by its high cost. Marrow transplantation is also effective in treating the disease, but is rarely performed because of the risks involved. In the future gene transfer may become the treatment of choice.

Pyridoxal kinase: decreased activity in red blood cells of Afro-Americans.

The mean pyridoxal kinase activity in red blood cells of American blacks was approximately 50 percent lower than that of American whites. Lymphocytes, granulocytes, and cultured skin fibroblasts from black and white donors contained identical pyridoxal kinase activity. The pyridoxal kinase of blacks was indistinguishable from that of whites with respect to heat stability, chromatographic mobility on microgranular diethylaminoethyl cellulose, Michaelis-Menten constant for pyridoxine, and susceptibility to inhibition by 4-deoxypyridoxine. The difference of the activity of this enzyme in whites and in blacks is much greater than any previously observed biochemical difference between the races.

Hexokinase isoenzymes in human erythrocytes.

The electrophoretic mobility of hexokinase from human erythrocytes and other tissues was studied with a new method that depends on the fluorescence of reduced nicotinamide-adenine dinucleotide phosphate for detecting enzyme activity on starch gel. The hexokinase of cord-blood erythrocytes has slightly different electrophoretic properties from that of adult red cells. Type I enzyme is split into type I(A) and type I(F); the latter is more intense in cord blood; in hemolyzates of adult blood, the activity of the two bands is usually about equal. No type II enzyme was found in cord blood. The double type I band was present in red cells from adult rabbits.

Electrophoretic variation of galactose-1-phosphate uridyltransferase.

A specific method for starch-gel electrophoresis of galactose-1-phosphate uridyltransferase has been developed. Electrophoresis of red-cell hemolyzate from normal subjects and subjects homozygous for the Duarte variant has shown that the Duarte variant has a slightly faster electrophoretic mobility than the normal enzyme under the various conditions used. Molecular-weight estimation on Sephadex G-200 indicates that this observed difference in electrophoreticmobility of the Duarte variant is not due to difference in molecular size. Both enzymes have a molecular weigh of approximately 85,000.

Hexose-6-phosphate dehydrogenase found in human liver.

Starch-gel electrophoresis of extracts of human liver revealed the presence of a new hexose-6-phosphate dehydrogenase that was slower-moving at pH 8.6 than the sex-linked glucose-6-phosphate dehydrogenase. When the gel plate was stained, galactose-6-phos phate being used as a substrate, this enzyme band stained intensely, but the sex-linked glucose-6-phosphate dehydro genase failed to stain. This new human enzyme may well be homologous with the autosomally inherited glucose-6-phosphate dehydrogenase of the deer mouse (Peromyscus maniculatus), re ported by Shaw and Barto.

Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies.

Increases or decreases of red cell glutathione reductase (GR) have been described in connection with many clinical abnormalities. We find that GR activity as measured in hemolysates represents only a portion of the available GR activity. The addition of small amounts of flavin adenine dinucleotide (FAD), but not of flavin mononucleotide or riboflavin, activates the GR of hemolysates. 1 muM FAD results in a maximal activation within 10 min; gradually increasing activation occurs at much lower, for example, 20 mmuM FAD concentrations. Once FAD has activated GR, dilution or dialysis does not reverse activation of the enzyme. Activation of GR by FAD can be inhibited by adenosine triphosphate (ATP), and to a lesser extent by adenosine diphosphate (ADP) and adenosine monophosphate (AMP), if these adenine nucleotides are added before the addition of FAD, but only to a slight extent if FAD is added before the adenine nucleotides. The addition of FAD to GR does not alter its electrophoretic mobility but produces intensification of the bands. The administration of 5 mg of riboflavin daily produces marked stimulation of red cell GR activity within only 2 days. After cessation of riboflavin administration, the GR activity again begins to fall. The degree of stimulation of GR activity by riboflavin is inversely correlated with the level of dietary riboflavin intake. The base line GR activity of normal individuals is directly correlated with the level of dietary riboflavin intake. The previously unexplained variations of glutathione reductase in health and disease must be reevaluated in light of the state of riboflavin nutrition and metabolism of the subject.

Molecular study of pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia.

DNA analysis was performed on 30 unrelated patients with hereditary nonspherocytic hemolytic anemia (HNSHA) who had been found to be pyruvate kinase (PK) deficient by enzyme assay. 19 different mutations were identified among 58 of the 60 alleles at risk. 13 of these were missense mutations that caused single amino acid changes. Included were the following nucleotide substitutions: 401A, 464C, 993A, 1022C, 1076A, 1178G, 1179A, 1373A, 1378A, 1456T, 1484T, 1493A, 1529A. The remaining six mutations were as follows: two nonsense mutations, 721T and 808T; a nucleotide deletion, 307C; a nucleotide insertion, 1089GG; a three nucleotide in frame deletion, 391-392-393 and a deletion of 1149 bp from the PKLR gene that resulted in the loss of exon 11. All the patients were studied for two polymorphic sites, nucleotide (nt) 1705 A/C and a microsatellite in intron 11, to better understand the origin of the mutations. The 1529A mutation, which is the most common mutation in the European population, was found in 25 alleles. With a single exception this mutation was in linkage disequilibrium with both of the polymorphic markers, i.e., found with 1705C and 14 repeats in the microsatellite. This finding is consistent with a single origin of this common mutation. Other mutations occurring more than once were of much lower frequency than the 1529A mutation.

The characterization of gene mutations for human glucose phosphate isomerase deficiency associated with chronic hemolytic anemia.

DNA was isolated from four unrelated glucose phosphate isomerase-deficient patients. Seven new mutations in the coding region were found: 247 C-->T, 671 C-->T, 818 G-->A, 833 C-->T, 1039 C-->T, 1459 C-->T, and 1483 G-->A. Three patients were compound heterozygotes, and one patient was a homozygote of 247 C-->T/247 C-->T. Six mutations were found to involve highly conserved amino acids of glucose phosphate isomerase, suggesting that these residues are crucial for the maintenance of biological activity. Two polymorphic sites were also identified, 489 A-->G and 1356 G-->C, which do not produce a change in the amino acid sequence.

Developmental changes in glucose transport of guinea pig erythrocytes.

The developmental changes in the capacity for D-glucose transport of guinea pig erythrocyte membranes were compared to alterations in the electrophoretic pattern of erythrocyte membrane components. Guinea pig erythrocytes lose their D-glucose carrier functions during development. Good correlation was observed between the loss of glucose uptake and apparent decrease of the zone 4.5 of Coomassie Blue-stained membrane proteins on electrophoresis. Reconstitution of membrane preparations in liposomes resulted in a parallel change in the D-glucose uptake and D-glucose penetration of intact erythrocytes. This suggests that the decrease of D-glucose transport capacity during development is caused by the loss of one or more protein components from the erythrocyte membranes.

Binding, internalization, and degradation of mannose-terminated glucocerebrosidase by macrophages.

Mannose-terminated glucocerebrosidase (alglucerase; Ceredase) was designed for enzyme replacement therapy in Gaucher disease to take advantage of mannose receptor-mediated endocytosis by macrophages. To provide a rational basis for designing enzyme replacement therapy protocols, we examined the in vitro binding, uptake, and degradation of alglucerase by murine and human macrophages. Both were found to have approximately 500,000 mannose-dependent receptors for alglucerase per cell with a Kd of 10(-7) M at 0 degrees C. In contrast, the number of binding sites for mannose-bovine serum albumin (mannose-BSA), the classical ligand for the mannose receptor, was only approximately 20,000 with a Kd of 10(-8) M. Alglucerase was also bound in a mannose-dependent manner by cells that lack the capacity to bind mannose-BSA, such as Cos-1 cells, endothelial cells, and peripheral blood monocytes. The fact that the binding of alglucerase by macrophages was mediated principally by a receptor distinct from the classical mannose receptor that binds mannose-BSA was confirmed by differential inhibitors, viz., alpha-methyl-glucoside, fucose, and mannose-BSA, and by its independence on Ca2+. Uptake of alglucerase by macrophages at 37 degrees C was concentration dependent and half maximal at 10(-6) M. However, at a concentration of 10(-7) M, only 0.5% of the added alglucerase was incorporated into macrophages and approximately 50% of the alglucerase taken up was quickly released into the medium. Endothelial cells also manifest mannose-dependent binding and uptake of alglucerase and may therefore account for a large proportion of the infused alglucerase. Our data suggest that only a small amount of the alglucerase administered is effectively delivered to macrophages and that a more efficiently targeted enzyme might have a marked therapeutic advantage over mannose-terminated glucocerebrosidase.

Mannose metabolism in the human erythrocyte.

The metabolism of mannose by human erythrocytes has been investigated. Phosphorylation of mannose is achieved by an enzyme with electrophoretic mobility on starch gel indistinguishable from the glucose-phosphorylating enzyme. Mannose phosphorylation is competitively inhibited by glucose; glucose phosphorylation is competitively inhibited by mannose. The K(i) values of inhibition are similar to the K(m) values for uninhibited phosphorylation. The normal average mannose-phosphorylating activity was found to be 0.69 U/g of Hb; the normal average glucose-phosphorylating activity was found to be 0.64 U/g of Hb. The ratio of mannose-phosphorylating activity to glucose-phosphorylating activity of a hemolysate prepared from the red cells of a subject with hexokinase deficiency was found to be within the normal range.Phosphomannose isomerase (PMI) activity of the red cells was found to average 0.064 U/g of Hb at its pH optimum of 5.9 with a mannose-6-phosphate (Man-6-P) concentration of 5 mmoles/liter. The enzyme activity in young cells was greater than activity in old cells. When human erythrocytes are incubated with mannose rapid accumulation of Man-6-P occurs, a finding indicating that PMI and not hexokinase is the limiting enzyme in the over-all conversion of mannose to fructose by the red cell. The ratio of mannose utilization to glucose utilization in hexokinase-deficient cells was greater than normal, as has been reported previously. These cells were found to have greatly increased PMI activity, presumably because of their young mean cell age. Consequently, Man-6-P accumulated only approximately one-third as rapidly as normal in hexokinase-deficient cells incubated with mannose. It is believed that the more rapid utilization of mannose relative to glucose by intact hexokinase-deficient cells may be explained on the basis of the regulatory effect of the PMI reaction on the rate of mannose utilization.

The reduction of glyceraldehyde by human erythrocytes. L-hexonate dehydrogenase activity.

Incubation of red cell suspensions with D-glyceraldehyde resulted in disappearance of glyceraldehyde and appearance of glycerol. Concomitantly, there was an increase of CO(2) formation from glucose. This indicated that the reduction of glyceraldehyde to glycerol occurred through a NADPH-linked system. Studies in hemolysates revealed the presence of an enzyme with the capacity to catalyze the reduction of glyceraldehyde to glycerol by NADPH. This enzyme was partially purified by DEAE chromatography. The elution pattern of the enzyme and its kinetic characteristics indicated that the enzyme was L-hexonate dehydrogenase (L-gulonate: NADP oxidoreductase, EC 1.1.1.19), not aldose reductase (Alditol: NADP oxidoreductase, EC 1.1.1.21), which had previously been thought present in erythrocytes. The reduction of glyceraldehyde to glycerol is one of a number of pathways for the metabolism of glyceraldehyde that have been found in red cells and/or other mammalian tissues.

Alternative splicing of human glucose-6-phosphate dehydrogenase messenger RNA in different tissues.

Different forms of glucose-6-phosphate dehydrogenase (G-6-PD) have been described in different tissues. Moreover, the directly determined amino acid sequence amino end of the red cell enzyme does not exactly match the sequence deduced from cDNA isolated from HeLa cells or lymphoblasts. We have therefore investigated the sequence of cDNA from sperm, granulocytes, reticulocytes, brain, placenta, liver, lymphoblastoid cells, and cultured fibroblasts. A novel human cDNA, which has extra 138 bases coding 46 amino acids, was isolated from a lymphoblastoid cell library. Sequencing of genomic DNA amplified by the polymerase chain reaction (PCR) revealed that the extra sequence was derived from the 3'-end of intron 7 by alternative splicing. This longer form of mRNA was also detected in sperm and granulocytes. Sequence analysis using PCR-amplified cDNA revealed that the 5'-end of the coding sequence of G6PD mRNA in reticulocytes is identical to those in other tissues.

The effect of copper on red cell enzyme activities.

Previous studies have shown a marked effect of very high levels of copper on red cell glucose-6-phosphate dehydrogenase and glutathione. When the effect of more nearly physiological levels of copper were studied, red cell hexokinase, phosphofructokinase, phosphoglyceric kinase, pyruvate kinase, and 6-phosphogluconate dehydrogenase were found to be inhibited. Inhibition was observed both when copper was added directly to hemolysates or when hemolysates were prepared from red cells from whole blood which had been incubated with copper and washed. The inhibition of red cell enzymes by copper was completely reversed by the addition of EDTA.

Erythrocyte glutathione synthetase deficiency leads not only to glutathione but also to glutathione-S-transferase deficiency.

Glutathione synthetase (GSH-S) is one of the two known hereditary causes of glutathione deficiency. We describe a family whose two children have hemolytic anemia. The children's erythrocytes lack GSH and are severely deficient in GSH-S activity. No neurologic findings or 5-oxoprolinuria were present. A concurrent deficiency of glutathione-S-transferase (GST) was also detected in the erythrocytes. Residual glutathione could be detected in the erythrocytes using a sensitive cycling assay. The deficiency was found to be most severe in reticulocyte-depleted preparations. The GSH-S activity of the erythrocytes of the parents was one-half normal, while the glutathione S-transferase activity was normal. We conclude that the primary defect is one of GSH-S. Glutathione stabilizes GST in vitro, and it is assumed that the deficiency of GST in the erythrocytes of the patients is due to the instability of this enzyme in the absence of adequate intracellular GSH levels.

High level transcription of the glucocerebrosidase pseudogene in normal subjects and patients with Gaucher disease.

Gaucher disease is due to mutations involving the glucocerebrosidase gene. A closely homologous pseudogene is located approximately 16 kD downstream from the functional gene. Sequence analysis of clones from cDNA libraries made from skin fibroblast cultures showed several independent clones with the sequence of an aberrantly processed pseudogene message. Examination of cellular RNA from lymphoblasts or fibroblasts obtained from thirteen Gaucher disease patients, one Gaucher disease heterozygote, and four normal subjects showed that the pseudogene was consistently transcribed, and that in some cases the level of transcription seemed to be approximately equal to that of the functional gene. The transcription of the pseudogene must be taken into account when attempting to detect mutations of glucocerebrosidase by the study of cDNA libraries.

A glucocerebrosidase fusion gene in Gaucher disease. Implications for the molecular anatomy, pathogenesis, and diagnosis of this disorder.

The molecular diagnosis of Gaucher disease has been difficult due to the existence of several different point mutations in the glucocerebrosidase gene and due to the presence of a tightly linked, highly homologous pseudogene. We now report the occurrence of a "Lepore-like" glucocerebrosidase fusion gene in which the 5' end is the functional gene and the 3' end is the pseudogene. This further complicates the molecular diagnosis of Gaucher disease but sheds light on the molecular anatomy of the glucocerebrosidase gene complex and on the pathogenesis of this important storage disease.