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P E Purdue

Publications and source records attributed to P E Purdue.

30 records · Page 2Linked to original sources
PubMed1993

Enzymological and mutational analysis of a complex primary hyperoxaluria type 1 phenotype involving alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity. Unlike normal individuals in whom the AGT is confined to the peroxisomal matrix, the immunoreactive AGT in these three patients was distributed approximately equally between the peroxisomes and mitochondria. The peroxisomal AGT appeared to be aggregated into amorphous core-like structures in which no other peroxisomal enzymes could be identified. Mutational analysis of the AGT gene showed that two of the three patients were compound heterozygotes for two previously unrecognized point mutations which caused Gly41-->Arg and Phe152-->Iso amino acid substitutions. The third patient was shown to be a compound heterozygote for the Gly41-->Arg mutation and a previously recognized Gly170-->Arg mutation. All three patients were homozygous for the Pro11-->Leu polymorphism that had been found previously with a high allelic frequency in normal populations. It is suggested that the Phe152-->Iso and Gly170-->Arg substitutions, which are only eighteen residues apart and located in the same highly conserved internal region of 58 amino acids, might be involved in the inhibition of peroxisomal targeting and/or import of AGT and, in combination with the Pro11-->Leu polymorphism, be responsible for its aberrant mitochondrial compartmentalization. On the other hand, the Gly41-->Arg substitution, either in combination with the Pro11-->Leu polymorphism or by itself, is predicted to be responsible for the intraperoxisomal aggregation of the AGT protein.

Adult↗
PubMed1992

Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the marmoset and rabbit genes.

In mammals, the subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT) is species dependent, with the proportion of AGT targeted to mitochondria varying between 0% and greater than 90%, the remainder being located in the peroxisome. In order to extend our studies on the molecular evolution of intracellular targeting of AGT, we have investigated the organization and expression of the AGT genes of rabbit, which has all of its AGT located in the peroxisome, and marmoset, which has approximately 50% of its AGT located in the peroxisome and 50% in the mitochondrion. Southern-blot analysis indicates that, in both of these species, AGT is encoded by a single-copy gene, as has previously been shown for human (all AGT in the peroxisome) and rat (50% AGT in the peroxisome and 50% in the mitochondrion). Comparison of the cDNA sequences encoding marmoset, rabbit, human and rat AGT, combined with transcript mapping and in vitro mitochondrial protein-import analysis, has provided a molecular explanation for the differential targeting of AGT in these species. As in the rat, marmoset AGT is synthesized in two forms, via the use of alternative transcription and translation-initiation sites. These two forms of AGT differ only in the presence or absence of a 22-amino-acid amino-terminal peptide, which acts as a cleavable mitochondrial-targeting sequence, directing the longer form of AGT to mitochondria. The shorter form of AGT, lacking the mitochondrial-targeting sequence, is presumed to be localized in the peroxisomes. In humans and rabbits, similar but distinct evolutionary mutational events within the AGT gene have resulted in exclusion of the region encoding the mitochondrial-targeting sequence from the open reading frame, explaining the exclusive peroxisomal localization of AGT in these species. We discuss the impact of these results on our understanding of both the evolution of species dependence of AGT subcellular distribution and the recent identification of amino acid changes in human AGT which result in mistargeting of this protein to mitochondria.

Alanine Transaminase↗
PubMed1992

A glycine-to-glutamate substitution abolishes alanine:glyoxylate aminotransferase catalytic activity in a subset of patients with primary hyperoxaluria type 1.

We have synthesized and sequenced alanine:glyoxylate aminotransferase (AGT; HGMW-approved symbol for the gene--AGXT) cDNA from the liver of a primary hyperoxaluria type 1 (PH1) patient who had normal levels of hepatic peroxisomal immunoreactive AGT protein, but no AGT catalytic activity. This revealed the presence of a single point mutation (G----A at cDNA nucleotide 367), which is predicted to cause a glycine-to-glutamate substitution at residue 82 of the AGT protein. This mutation is located in exon 2 of the AGT gene and leads to the loss of an AvaI restriction site. Exon 2-specific PCR followed by AvaI digestion showed that this patient was homozygous for this mutation. In addition, three other PH1 patients, one related to and two unrelated to, but with enzymological phenotype similar to that of the first patient, were also shown to be homozygous for the mutation. However, one other phenotypically similar PH1 patient was shown to lack this mutation. The mechanism by which the glycine-to-glutamate substitution at residue 82 causes loss of catalytic activity remains to be resolved. However, the protein sequence in this region is highly conserved between different mammals, and the substitution at residue 82 is predicted to cause significant local structural alterations.

Alanine↗
PubMed1992

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.

Amino Acid Sequence↗
PubMed1991

Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation.

In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment of this initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.

Alanine Transaminase↗
PubMed1991

An intronic duplication in the alanine: glyoxylate aminotransferase gene facilitates identification of mutations in compound heterozygote patients with primary hyperoxaluria type 1.

We report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.

Alanine Transaminase↗
PubMed1991

Characterization and chromosomal mapping of a genomic clone encoding human alanine:glyoxylate aminotransferase.

We have previously reported the isolation of a genomic clone encoding human liver-specific peroxisomal alanine:glyoxylate aminotransferase (AGT, EC 2.6.1.44), the deficient enzyme in primary hyperoxaluria type 1 (PH1) (P. E. Purdue, Y. Takada, and C. J. Danpure, J. Cell Biol. 111: 2341-2351, 1990). This clone has now been characterized, revealing that the coding sequence is distributed among 11 exons covering 10 kb. The nucleotide sequences of each exon have been determined, confirming that this clone corresponds to previously characterized AGT cDNA (Y. Takada, N. Kaneko, H. Esumi, P. E. Purdue, and C. J. Danpure, Biochem. J. 268: 517-520, 1990). In addition, to provide sequence data for the design of exon-specific PCR primers, the intron sequences immediately flanking each exon have been determined. Furthermore, in an attempt to identify putative transcriptional control sequences we have determined the sequence of 1.25 kb directly upstream of the cDNA 5' end. The results of genomic Southern blotting indicate that human AGT is probably encoded by a single copy gene, and a combination of in situ hybridization and PCR analysis of rodent/human somatic cell hybrids suggests that this gene is located on chromosome 2q36-q37. The gene symbol AGXT has been assigned for this locus.

Alanine Transaminase↗
PubMed1990

Human peroxisomal L-alanine: glyoxylate aminotransferase. Evolutionary loss of a mitochondrial targeting signal by point mutation of the initiation codon.

The amino acid sequence of human hepatic peroxisomal L-alanine: glyoxylate aminotransferase 1 (AGTI) deduced from cDNA shows 78% sequence identity with that of rat mitochondrial AGTI, but lacks the N-terminal 22 amino acids (the putative mitochondrial targeting signal). In humans this signal appears to have been deleted during evolution by a point mutation of the initiation codon ATG to ATA. These data suggest that the targeting defect in primary hyperoxaluria type 1, in which AGT1 is diverted from the peroxisomes to the mitochondria, could be due to a point mutation that reintroduces all or part of the mitochondrial signal sequence.

Alanine Transaminase↗
PubMed1990

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

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.

Alanine Transaminase↗
PubMed1990

Subcellular distribution of hepatic alanine:glyoxylate aminotransferase in various mammalian species.

The subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT1) enzyme activity and immunoreactive protein has been determined in the livers of a variety of mammalian species (marmoset, baboon, cat, rabbit, pig, sheep) using the techniques of post-embedding protein A-gold immunoelectron microscopy and isopycnic density gradient centrifugation. In baboon and rabbit livers, AGT1 was exclusively peroxisomal. Morphometric analysis of the distribution of immunoreactive AGT1 protein in cat liver showed that 91-92% was located in the mitochondria with 8-9% in the peroxisomes. In marmoset liver, 59% of AGT1 was found in the mitochondrial matrix and 41% in the peroxisomal matrix. The results from pig and sheep were ambiguous, because of the low levels of expression of AGT1. The implications of these data in terms of the metabolic roles of AGT1 (gluconeogenesis and glyoxylate detoxification) and in terms of the evolutionary acquisition and loss of functional mitochondrial and peroxisomal targeting sequences are discussed.

Alanine Transaminase↗