The molecular basis of inherited hemolytic anemia of Basenji dogs due to R-type pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency is a naturally-occurring autosomal recessive disease of Basenji dogs. The glycolytic enzyme defect causes chronic hemolytic anemia and persistent reticulocytosis similar to the disease in human beings.

Erythrocyte PK activity of normal dogs and of Basenji dogs with PK deficiency was characterized by ion-exchange chromatography, substrate kinetics, immunological reactivity and electrophoretic mobility. The findings suggest that the M₂-type PK isozyme is the major form of PK activity in erythrocytes of PK deficient dogs in contrast to normal dogs which have only R-type PK activity. The R-type monomer is readily detected by Western blot in erythrocyte lysates from normal dogs but R-type PK activity is not detectable in lysates from PK deficient dogs using a heterologous antibody to rat L-type PK.

Pyruvate kinase gene transcriptional activity in erythroid cells of PK deficient dogs was evaluated to determine if a failure in the normal maturational switch from M₂-type PK to R-type expression could explain the observed isozyme expression in mature PK deficient erythrocytes. Transcripts for both R- and M₂-type PK are detectable in reticulocytes of PK deficient dogs by dot blot hybridization whereas only R-type transcript is detected in reticulocytes of normal dogs. These results suggest that a mutation in the pyruvate kinase L gene is present in PK deficient dogs resulting in no detectable protein product despite the presence of transcriptional activity.

The R-type PK cDNA from normal dogs and from PK deficient Basenjis was cloned using the polymerase chain reaction. Sequence information derived from wild type and mutant clones revealed a single nucleotide deletion, ΔC⁴³³, in the mutant cDNA. This point deletion results in a reading frameshift with subsequent premature translational termination due to the appearance of the nonsense codon TGA at nucleotides 499-501 of the coding sequence. The deduced amino acid sequence predicts a truncated mutant protein devoid of all residues contributing to the catalytic site of the wild type protein and thus lacking enzymatic activity. The absence of erythrocytic Rtype PK activity necessitates the anomalous expression of M₂-type PK in maturing erythroid cells of PK deficient Basenjis.