cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.

Abstract

ABSTRACTcDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-spedflc primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino adds, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr3N -Met. Dot blot hybridization of PCR-ampifled genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the patients were homozygous for this nucleotide change and that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coil and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris-HCI, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.

Application of disease-associated differentially expressed genes – Mining for functional candidate genes for mastitis resistance in cattle

“…In humans, variants of genes coding for protein kinase that cause genetic disorders and diseases have been described. Defects in the myotonin protein kinase are the causes of myotonic dystrophy [11,12], whereas defects in the pyruvate kinase genes are the most common cause of chronic hereditary nonspherocytic haemolytic anaemia [17]. Genetic variants of DNA-dependent protein kinase that is involved in DNA double-stranded break repair and modulation of transcription are associated with severe combined immunodeficiency, type I [13].…”

Mutations in Krüppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression

“…19 The KLF1 and PKLR genes were amplified and sequenced as previously described. 20,21 Primary erythroid cell culture and expression analysis Erythroid cells were obtained using a 2-phase culture system 22 and harvested once they were double-positive for CD71 and glycophorin A. RNA was extracted, reverse transcribed to generate double-stranded cDNA (Superscript Double Stranded cDNA Synthesis Kit; Invitrogen), and labeled with dCTPCy3 as the test sample; 500 ng sonicated genomic DNA was labeled with dCTP-Cy5 as the input (Bioprime DNA Labeling System; Invitrogen). Both test and input samples were applied to a custom-designed Agilent expression array, as described.…”

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…Exon 1 is erythroidspecific whereas exon 2 is liver-specific [Kanno et al, 1992]. The red blood cell-specific mRNA is 2 kb in length and codes for a PK-R subunit of 574 amino acids [Kanno et al, 1991].…”