Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1

P. Edward Purdue, Yoshikazu Takada, Christopher J. Danpure

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Abstract

We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C. J., P. J. Cooper, P. J. Wise, and P. R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly→Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro→Leu and Ile→Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.

Original languageEnglish
Pages (from-to)2341-2351
Number of pages11
JournalJournal of Cell Biology
Volume111
Issue number6 PART 1
Publication statusPublished - Dec 1990
Externally publishedYes

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Peroxisomes
Mitochondria
Alleles
Mutation
Amino Acid Substitution
Heterozygote
Point Mutation
Oligonucleotides
Complementary DNA
Alanine-glyoxylate transaminase
Primary hyperoxaluria type 1
Polymerase Chain Reaction
Enzymes
Population
Proteins

ASJC Scopus subject areas

  • Cell Biology

Cite this

Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1. / Purdue, P. Edward; Takada, Yoshikazu; Danpure, Christopher J.

In: Journal of Cell Biology, Vol. 111, No. 6 PART 1, 12.1990, p. 2341-2351.

Research output: Contribution to journalArticle

Purdue, P. Edward ; Takada, Yoshikazu ; Danpure, Christopher J. / Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1. In: Journal of Cell Biology. 1990 ; Vol. 111, No. 6 PART 1. pp. 2341-2351.
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abstract = "We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C. J., P. J. Cooper, P. J. Wise, and P. R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly→Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro→Leu and Ile→Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10{\%}. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.",
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