ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case.
Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half-life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.
Nishiyama
K
K
Department of Biochemistry, Hamamatsu University School of Medicine, Shizuoka, Japan.
Funai
T
T
Yokota
S
S
Ichiyama
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000410:Alanine Transaminase / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002474:Cell-Free System; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology* / Q000235:genetics; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D008830:Microbodies / Q000378:metabolism; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000637:Transaminases; D000410:Alanine Transaminase; C019635:Alanine-glyoxylate transaminase; C022638:serine-pyruvate aminotransferase