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Hereditary hemolytic anemia caused by diverse point mutations of pyruvate kinase gene found in Japan and Hong Kong.

We identified four distinct point mutations in homozygous pyruvate kinase (PK) variants in Japanese and Chinese patients with chronic nonspherocytic hemolytic anemia. All gene abnormalities were missense mutations that caused single amino acid substitutions. 1261A (Q421K) and 1436A (R436H), which were identified in PK Sendai and PK Shinshu, had been found in unrelated Japanese and Amish PK variants, respectively. The clinical severity and extremely low residual erythrocyte PK activity of PK Shinshu as well as of the Amish PK might be caused partly by aberrant splicing, because the 1436A mutation changes a nucleotide at the last nucleotide in the exon 10. Recently, we diagnosed a 42-year-old Japanese woman with chronic nonspherocytic hemolytic anemia as having a homozygous PK deficiency. DNA sequencing of the variant PK gene showed a homozygous missense mutation at 1403GCT-->GTT, resulting in a single amino acid substitution from 468la-->Val. The gene mutation is likely to impair the allostericity of this enzyme, speculated from the tertiary structure. A homozygous missense mutation in PK Hong Kong, a boy of a non-Han southern Chinese minority group, was identified in exon 7 of the human L-PK gene, 941ATT-->ACT, resulting in a single amino acid substitution from 314lle-->Thr. The R-PK activity is expected to be severely affected, because the mutated amino acid residue is located between the 313 Lys and the 315 Glu, which are very important for acid-base catalysis and magnesium binding, respectively. Both the R- and M2-type PK were shown by polyacrylamide gel electrophoresis of the PK Hong Kong erythrocyte lysate, and this is the first report of a homozygous individual whose erythrocytes contain the immature (M2)-type isozyme.

Adult↗

Chicken ovalbumin upstream promoter-transcription factor II, a new partner of the glucose response element of the L-type pyruvate kinase gene, acts as an inhibitor of the glucose response.

Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.

Animals↗

Modulating erythrocyte chimerism in a mouse model of pyruvate kinase deficiency.

In vivo selection may provide a means to increase the relative number of cells of donor origin in recipients with hemopoietic chimerism. We have tested whether in vivo selection using chemical inducers of dimerization (CIDs) can direct the expansion of transduced normal donor erythrocytes in recipients with chimerism using a mouse model of pyruvate kinase deficiency. Marrow cells from normal CBA/N mice were transduced with a vector (F36Vmpl(GFP)) that promotes cell growth in the presence of CIDs. Transduced cells were then transplanted into minimally conditioned, pyruvate kinase-deficient recipients (CBA-Pk-1(slc)/Pk-1(slc)) to establish stable chimerism. CID administration resulted in expansion of normal donor erythrocytes and improvement of the anemia. The preferential expansion of normal erythrocytes also resulted in a decrease in erythropoietin levels, reducing the drive for production of pyruvate kinase-deficient red blood cells. CID-mediated expansion of genetically modified erythrocytes could prove a useful adjunct to transplantation methods that achieve erythroid chimerism.

Anemia↗

Human erythrocyte pyruvate kinase deficiency: the use of a kinetic study of mutant enzymes for the detection of heterozygotes.

Erythrocyte pyruvate kinase (PK) deficiency was detected in a boy of dutch origin. Immunologic, electrophoretic, and kinetic studies of the enzymes of propositus and the members of his family demonstrated that the boy was heterozygote for two different mutant PK alleles. The mutant enzyme, for which his mother was heterozygote, was characterized by a lower immunologic specific activity, a decreased affinity for the substrate phospho-enol-pyruvate, and a loss of homotropic interactions toward this substrate, an increased affinity toward the allosteric inhibitor MgATP2-, a decreased affinity for the activator fructose-1,6-diphosphate, and a lowered pH optimum. Electrophoresis, Km app. for MgADP, and the reactivity toward the purine nucleotide substrate analogues were normal. The mutant enzyme for which his father was heterozygote was characterized by a decreased affinity for the substrate phospho-enol-pyruvate and a loss of homotropic interactions toward this substrate. All other parameters mentioned above were normal. The use of a kinetic study of mutant enzymes for the detection of heterozygotes is discussed.

Adolescent↗

The use of concomitant serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) for carrier detection in Duchenne's muscular dystrophy through discriminant analysis.

Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) determinations have been carried out in a sample of 30 obligate carriers for the DMD gene and 50 normal adult control females. In all the subjects under study blood samples have been collected 3 times on 3 independent occasions and the means of these 3 determinations were considered for both CPK and PK activities in the statistical analysis. Discriminant analysis has shown that in the group of 30 obligate carriers the estimated proportions of misclassification using either serum CPK, PK or both enzymes were fourn to be: 10/30 (33.3%) for CPK alone, 6/30 (20%) for PK alone and 5/30 (16.6%) for both enzymes. It is concluded that although a small proportion of carriers still remain undetected the concomitant use of PK and CPK determinations enhances the capability of detecting carriers for the Duchenne gene mainly when compared with CPK alone.

Clinical Enzyme Tests↗

Structure of pyruvate kinase and similarities with other enzymes: possible implications for protein taxonomy and evolution.

The structure determination of pyruvate kinase shows that each subunit of the tetrameric molecule consists of three domains. The largest of these domains has a remarkable similarity to the structure of triosephosphate isomerase. Another domain shows similarities to many other nucleotide binding proteins. We discuss these similarities and their implications for current arguments on protein taxonomy and evolution.

Animals↗

Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1alpha subunit of the PDC. This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phospho-His intermediate. The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Furthermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1alpha. Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser-kinase activity.

Amino Acid Sequence↗

Identification of a novel promoter mutation in the human pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia.

Using direct sequencing we analysed the pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia due to PK deficiency. A novel promoter mutation -249delA relative to the translation initiation site and the common 1529A mutation in exon 11 of the gene could be identified. Reverse transcription (RT)-PCR analysis combined with restriction digestion revealed that the -249delA mutation leads to a reduction in the amount of mRNA produced from this allele to about 6% of normal. We assume that both mutations would account for the PK deficiency in the compound heterozygous patient.

Anemia, Hemolytic↗

Pyruvate kinase variants characterized by the methods recommended by the International Committee for Standardization in Haematology.

Seven new pyruvate kinase (PK) variants were characterized by the recently established recommended methods by the International Committee for Standardization in Haematology (ICSH). The seven cases were all true homozygotes as evidenced by consanguineous marriages of the parents. All of them were Japanese. These variants were designated PK Tokyo, Nagasaki, Sapporo, Maebashi, Itabashi, Fukushima, and Aizu. Low substrate affinity for phosphoenolpyruvate and thermal instability seem to play a major role in causing clinical manifestation of chronic hemolysis. Product inhibition of PK by ATP may also play an additional role in some cases.

Adolescent↗

A new pyruvate kinase variant (PK Osaka) demonstrated by partial purification and condensation.

A qualitative variant of erythrocyte and liver pyruvate kinases (PK Osaka) was detected in a family in which two siblings have extremely low PK activity by semipurification with DEAE cellulose chromatography and subsequent concentration of the enzyme solutions. This was previously reported to be a quantitative variant based on studies of crude tissue preparations. The molecular aberrations were characterized by slow mobility upon electrophoresis, abnormal kinetics for phosphoenolpyruvate, and low affinity for anti-human erythrocyte PK serum. The mutant PK L was similar both electrophoretically and immunologically to PK R.

Child, Preschool↗

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons.

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N-terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N-terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

Amino Acid Sequence↗

A pyruvate kinase variant in different mouse transplanted tumors.

Mouse transplanted tumors, in contrast to normal tissues, contain a pyruvate kinase (PK) variant sensitive to the inhibitory action of L-cysteine and less sensitive to saturated fatty acids than the normal enzyme. In selected normal and tumor materials two fractions of PK were separated. Fraction A (20-30% (NH4)2SO4 saturation) dominated in normal liver, and fraction B (50-60% (NH4)2SO4 saturation) in skeletal muscles and Ehrlich ascites tumor. Only this fraction B from tumor material was sensitive to L-cysteine, and seems to contain a tumor-specific PK variant which might be considered as a marker of neoplastic transformation in a broad spectrum of mouse experimental tumors.

Animals↗

Identification of mutants of pyruvate kinase from red blood cells by means of trypsinization, electrophoresis, kinetic properties and immunological methods.

Enzymopathies of pyruvate kinase (PK) are characterized by polymorphism. 11 different mutants of PK were detected in 12 analysed cases. The frequency of double heterozygotes of the PK deficiency is very high. The mutant forms have been identified by electrophoretic, kinetic and immunologic methods in red blood cells of patients and members of their families. The effect of trypsin has greatly increased the sensitivity of the differentiation procedures. Differences in activity and quality of the proteolytic systems in liver and kidney in comparison to red blood cells are responsible for inactivation and degradation of PK mutants in both organs. Homozygotes and double heterozygotes show clinic manifestations. The rigidity of red blood cells increases with the severity of the nonspherocytic haemolytic anaemia.

Erythrocytes↗

Pyruvate kinase deficiency in dog and human erythrocytes: effects of energy depletion on cation composition and cellular hydration.

Pyruvate kinase-(PK) deficient human reticulocytes incubated 4 hr under conditions where mitochondrial oxidative phosphorylation is inhibited become ATP-depleted. Concomitantly, these energy-depleted red blood cells (RBC) lose massive amounts of potassium and water. PK-deficient reticulocyte-rich RBC from a Basenji dog, incubated under the same conditions, also manifest ATP-depletion and K-loss. In contrast to the cation changes in human cells, however, K-loss in PK-deficient dog reticulocyte-rich RBC is balanced by an equivalent Na gain, and consequently these canine erythrocytes do not undergo any change in cellular hydration. Potassium depletion and dehydration, which may be related to membrane injury in human RBC, do not appear to be involved in the hemolysis of PK-deficient dog erythrocytes.

Adenosine Triphosphate↗

Conversion of pyruvate kinase (PK) isozymes during development of normal and PK deficient erythroblasts.

Using immunofluorescent antibody techniques, conversion of pyruvate kinase (PK) isozymes during the maturation of erythroblasts was studied. In normal subjects, M2-type PK was clearly seen at the proerythroblast stage, then markedly declined with cell maturation, whereas L-type PK continued to increase. In classical type PK deficiency, M2-type PK was still clearly seen in orthochromatic erythroblasts, whereas L-type PK was scarcely detected during maturation. In other PK deficiency cases, change of L-type PK showed a similar pattern to that of normal while M2-type PK was still clearly seen at the later stage of maturation as in the classical type. In K-562 human leukemia cell line, M2-type PK declined and L-type PK increased in parallel with hemoglobin synthesis by addition of hemin, an inducer for differentiation into erythroblasts. Hemoglobin synthesis and L-type PK production appear to have interrelation during the differentiation and maturation of erythroblasts.

Erythroblasts↗

Cis-regulation of the L-type pyruvate kinase gene promoter by glucose, insulin and cyclic AMP.

The glucose/insulin response element of the L-pyruvate kinase gene is a perfect palindrome located from nt -168 to -144 with respect to the cap site. This element (L4) is partially homologous to MLTF binding sites. Its full efficiency requires cooperation with a contiguous binding site for HNF4, termed L3 and located from nt -145 to -125. In the presence of the L4 element contiguous to L3, cyclic AMP inhibits activity of the L-PK promoter while in its absence, or when the normal L4-L3 contiguity is modified, cyclic AMP behaves as a transcriptional activator that does not seem to be sequence-specific. Therefore, we propose that the mechanism of inhibition of the L-PK gene by cyclic AMP requires precise interactions between the nucleoprotein complex built up at sites L4 and L3 and other components of the L-PK transcription initiation complex.

Animals↗

Purification and properties of mouse pyruvate kinases K and M and of a modified K subunit.

The K4 and M4 isozymes of mouse pyruvate kinase were purified to homogeneity, and their physical, chemical, and kinetic properties were compared. The K isozyme is slightly larger, but a high degree of homology exists as evidenced by a similar amino acid composition, immunotitration value, and two-dimensional arginine peptide pattern after tryptic digestion. Also, the more active conformational form of the K isozyme has kinetic and chromatographic properties similar to those of the M isozyme. Only K subunit could be extracted with antibody from fresh spleen extracts, but this subunit can be cleaved to form a product with the mobility of the M subunit. The cleavage is accomplished by an endogenous enzyme and appears to be the first step in K-enzyme degradation. This product is called Kpm . KpmK hybrid could also be purified to homogeneity. This enzyme has the structure K2pmK2, and both types of subunit have activity. The Kpm form has a higher K0.5S value for phosphoenolpyruvate and a lower K0.5S value for ADP than does either the K or the M type. However, the Kpm and M subunits otherwise have very similar properties and it is speculated that the Kpm subunit is an M-type precursor.

Amino Acids↗