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C J Danpure

Publications and source records attributed to C J Danpure.

At least 19 recordsLinked to original sources
PubMed2001

Crystallization and preliminary crystallographic analysis of human alanine:glyoxylate aminotransferase and its polymorphic variants.

The human hereditary disease primary hyperoxaluria type 1 is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). In this study, the crystallization and preliminary crystallographic analysis of C-terminal His-tagged human AGT expressed in Escherichia coli is reported. At least two crystal forms were obtained using similar conditions for three different polymorphic variants, namely AGT, AGT[P11L] and AGT[P11L, I340M]. Complete data have been collected for all three AGT variants. The crystals of AGT[P11L] belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell parameters a = b = 90.81, c = 142.62 A, and diffract to a resolution of 2.8 A.

Crystallization↗
PubMed2000

The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase.

Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.

Amino Acid Sequence↗
PubMed2000

Functional synergism between the most common polymorphism in human alanine:glyoxylate aminotransferase and four of the most common disease-causing mutations.

The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT). Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated. In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT. The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E. coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation. These results are discussed in the light of the high allelic frequency ( approximately 20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.

Amino Acid Substitution↗
PubMed2000

Genetic disorders and urolithiasis.

A recent analysis of the McKusick's On-Line Mendelian Inheritance in Man (OMIM) database revealed over 30 genetic or putatively genetic conditions in which urolithiasis contributes to the disease pathology at least to some extent. There is wide clinical, biochemical, and genetic heterogeneity in many of these conditions.

Calcium Oxalate↗
PubMed2000

Molecular adaptation of alanine:glyoxylate aminotransferase targeting in primates.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is targeted to different organelles (mitochondria and/or peroxisomes) in different species. Possibly under the influence of dietary selection pressure, the subcellular distribution of AGT has changed on at least eight occasions during the evolution of mammals. AGT targeting is dependent on the variable use of two alternative transcription and translation initiation sites which determine whether or not the region encoding the N-terminal mitochondrial targeting sequence is contained within the open reading frame. In the present study, we sequenced the 5' region of the AGT gene, including both ancestral translation start sites, for 11 anthropoid primates and compared the results with data already available for two others. We show that while the more 3' of the two translation start sites is maintained in all species, the more 5' site has been lost in six species (five of seven catarrhines and one of six platyrrhines). In addition, the remaining two catarrhines, which have maintained the 5' translation start site, are predicted to have lost mitochondrial targeting by a different mechanism, possibly loss of the more 5' transcription start site. Analysis of the relative frequencies of nonsynonymous and synonymous mutations in the region encoding the extant or ancestral mitochondrial targeting sequences led us to suggest that there has been recent strong positive selection pressure to lose, or decrease the efficiency of, mitochondrial AGT targeting in several anthropoid lineages, and that the loss of mitochondrial targeting in this group of mammals is likely to have occurred on at least four, and possibly five, separate occasions.

5' Untranslated Regions↗
PubMed1999

Effect of N-terminal alpha-helix formation on the dimerization and intracellular targeting of alanine:glyoxylate aminotransferase.

The unparalleled peroxisome-to-mitochondrion mistargeting of alanine:glyoxylate aminotransferase (AGT) in the hereditary disease primary hyperoxaluria type 1 is caused by the combined presence of a common Pro11 --> Leu polymorphism and a disease-specific Gly170 --> Arg mutation. The Pro11 --> Leu replacement generates a functionally weak N-terminal mitochondrial targeting sequence (MTS), the efficiency of which is increased by the additional presence of the Gly170 --> Arg replacement. AGT dimerization is inhibited in the combined presence of both replacements but not when each is present separately. In this paper we have attempted to identify the structural determinants of AGT dimerization and mitochondrial mistargeting. Unlike most MTSs, the polymorphic MTS of AGT has little tendency to adopt an alpha-helical conformation in vitro. Nevertheless, it is able to target efficiently a monomeric green fluorescent (GFP) fusion protein, but not dimeric AGT, to mitochondria in transfected COS-1 cells. Increasing the propensity of this MTS to fold into an alpha-helix, by making a double Pro11 --> Leu + Pro10 --> Leu replacement, enabled it to target both GFP and AGT efficiently to mitochondria. The double Pro11 --> Leu + Pro10 --> Leu replacement retarded AGT dimerization in vitro as did the disease-causing double Pro11 --> Leu + Gly170 --> Arg replacement. These data suggest that N-terminal alpha-helix formation is more important for maintaining AGT in a conformation (i. e. monomeric) compatible with mitochondrial import than it is for the provision of mitochondrial targeting information. The parallel effects of the Pro10 --> Leu and Gly170 --> Arg replacements on the dimerization and intracellular targeting of polymorphic AGT (containing the Pro11 --> Leu replacement) raise the possibility that they might achieve their effects by the same mechanism.

Amino Acid Sequence↗
PubMed1998

Evolution of alanine:glyoxylate aminotransferase intracellular targeting: structural and functional analysis of the guinea pig gene.

The distribution of alanine:glyoxylate aminotransferase 1 (AGT) within liver cells has changed many times during mammalian evolution. Depending on the particular species, AGT can be found in mitochondria or peroxisomes, or mitochondria and peroxisomes. In some cases significant cytosolic AGT is also present. In the livers of most rodents, AGT has what is thought to be the more 'ancestral' distribution (i.e. mitochondrial and peroxisomal). However, AGT is distributed very differently in the guinea pig, being peroxisomal and cytosolic. In this study, we have attempted to determine the molecular basis for the loss of mitochondrial AGT targeting and the apparent inefficiency of peroxisomal targeting of AGT in the guinea pig. Our results show that the former is owing to the evolutionary loss of the more 5' of two potential transcription and translation initiation sites, resulting in the loss of the ancestral N-terminal mitochondrial targeting sequence from the open reading frame. Guinea pig AGT is targeted to peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) peroxisomal import machinery, even though its C-terminal tripeptide, HRL, deviates from the standard consensus PTS1 motif. Although HRL appears to target AGT to peroxisomes less efficiently than the classical PTS1 SKL, the main reason for the low efficiency of AGT peroxisomal targeting in guinea pig cells (compared with cells from other species) lies not with guinea pig AGT but with some other, as yet undefined, part of the guinea pig peroxisomal import machinery.

Amino Acid Sequence↗
PubMed1997

A unique molecular basis for enzyme mistargeting in primary hyperoxaluria type 1.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is normally targeted to the peroxisomes in human liver cells. However, in a third of patients suffering from the autosomal recessive disease primary hyperoxaluria type 1 (PH1), AGT is mistargeted to the mitochondria. Such organelle-to-organelle mistargeting is without parallel in human genetic disease. AGT mistargeting results from the combination of a common Pro11-->Leu polymorphism and a rare Gly170-->Arg mutation. The former generates a functionally weak mitochondrial targeting sequence (MTS) while the latter, in combination with the former, increases the efficiency of this MTS by slowing the rate at which AGT dimerises. The fact that the intracellular compartmentation of AGT can be determined, at least in part, by its oligomeric status highlights the fundamental differences in the molecular requirements for protein import into two intracellular organelles--the peroxisomes and mitochondria.

Amino Acids↗
PubMed1997

Variable peroxisomal and mitochondrial targeting of alanine: glyoxylate aminotransferase in mammalian evolution and disease.

Under the putative influence of dietary selection pressure, the subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT) has changed on many occasions during the evolution of mammals. Depending on the particular species, AGT can be found either in peroxisomes or mitochondria, or in both peroxisomes and mitochondria. This variable localization depends on the differential expression of N-terminal mitochondrial and C-terminal peroxisomal targeting sequences by the use of alternative transcription and translation initiation sites. AGT is peroxisomal in most humans, but it is mistargeted to the mitochondria in a subset of patients suffering from the rare hereditary disease primary hyperoxaluria type 1. Mistargeting is due to the unlikely combination of a normally occurring polymorphism that generates a functionally weak mitochondrial targeting sequence and a disease-specific mutation which, in combination with the polymorphism, inhibits AGT dimerization. The mechanisms by which AGT can be targeted differentially to peroxisomes and/or mitochondria highlight the different molecular requirements for protein import into these two organelles.

Alanine Transaminase↗
PubMed1997

A vertical (pseudodominant) pattern of inheritance in the autosomal recessive disease primary hyperoxaluria type 1: lack of relationship between genotype, enzymic phenotype, and disease severity.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of alanine:glyoxylate aminotransferase (encoded by the AGXT gene). Primary hyperoxaluria type 1 is characterized by the elevated urinary excretion of oxalate and glycolate, and the deposition of insoluble calcium oxalate in the renal parenchyma and urinary tract. In the present study, we investigated an unusual family containing four affected individuals in two different generations. Based on our genetic, enzymic, metabolic, and clinical analyses, we have come to the following conclusions. First, although the pattern of inheritance of PH1 is usually horizontal (ie, all patients in the same generation), as expected for an autosomal recessive disease, it can sometimes show a vertical (pseudodominant) pattern of inheritance (ie, patients in more than one generation) due to the segregation within a family of three, rather than two, mutant AGXT alleles. Second, affected members of such a family can manifest very different clinical phenotypes both within and between generations. Although the clinical differences between generations might be at least partly due to differences in AGXT genotype, differences can equally occur within the same generation in individuals who possess the same AGXT genotype. Finally, individuals with PH1 at the level of the AGXT genotype might remain asymptomatic and undiagnosed for many years. The consequences of these findings for the clinical management and genetic counseling of families with PH1 are profound and wide-ranging.

Adolescent↗
PubMed1996

Molecular basis of the variable mitochondrial and peroxisomal localisation of alanine-glyoxylate aminotransferase.

The molecular basis of the variable species-specific peroxisomal and/or mitochondrial targeting of the enzyme alanine-glyoxylate aminotransferase 1 (AGT) has been studied in human fibroblasts by confocal immunofluorescence microscopy after intranuclear microinjection of various human, rabbit, marmoset, and feline AGT cDNA constructs. The expression of full-length human and rabbit AGT cDNA led to an exclusively peroxisomal distribution of AGT. However, the distribution of feline and marmoset AGT depended on the cDNA construct injected. In both species, injection of the short cDNAs (from transcripts that occur naturally in marmoset liver but not in feline liver) led to an exclusively peroxisomal distribution. However, injection of the long cDNAs (from transcripts that occur naturally in both species) led to most of the AGT being targeted to the mitochondria and only a small, yet significant, fraction to the peroxisomes. Reintroduction of the 'ancestral' first potential translation initiation site into human AGT cDNA led to an 'ancestral' distribution of AGT (i.e. both mitochondrial and peroxisomal). Deletion of the second potential translation start site from the long feline cDNA led to a distribution that was almost entirely mitochondrial, which suggests that most peroxisomal AGT encoded by the long cDNA results from internal translation initiation from this site with the consequent loss of the N-terminal mitochondrial targeting sequence. Expression of rabbit cDNA and the short marmoset and feline cDNAs in cells selectively deficient in the import of peroxisomal matrix proteins showed that peroxisomal AGT in all these species is imported via the peroxisomal targeting sequence type 1 (PTS1) import pathway. The almost complete functional dominance of the N-terminal mitochondrial targeting sequence over the C-terminal PTS. which was not due to any direct interference of the former with peroxisomal import, was maintained even when the unusual PTS1 of AGT (KKL in human) was replaced by the prototypical PTS1 SKL. The results demonstrate that the major determinant of alanine-glyoxylate aminotransferase subcellular distribution in mammals is the presence or absence of the mitochondrial targeting sequence rather than the peroxisomal targeting sequence. Various strategies have arisen during the evolution of mammals to enable the exclusion of the mitochondrial targeting sequence from the newly synthesised polypeptide, all of which involve the use of alternative transcription and/or translation initiation sites.

Alanine Transaminase↗
PubMed1996

Strategies for the prenatal diagnosis of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a potentially lethal autosomal recessive disorder of glyoxylate metabolism caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Over the past 13 years, various strategies have been adopted for its prenatal diagnosis, including (1) glyoxylate metabolite analysis of amniotic fluid in the second trimester; (2) AGT enzyme assay, immunoassay, and immuno-electron microscopy of fetal liver biopsies also in the second trimester; and (3) linkage and mutation analysis of DNA isolated from chorionic villus samples in the first trimester. These methods have evolved in parallel with our increased understanding of the molecular aetiology and pathogenesis of the disease. Although the usefulness of metabolite analysis remains unproven, all the other methods have been successfully applied to the prenatal diagnosis of PH1. In this review, examples of the use of the available methodologies are provided, and their pros and cons are discussed with reference to specific cases.

Alleles↗
PubMed1996

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1.

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.

Alanine Transaminase↗
PubMed1995

How can the products of a single gene be localized to more than one intracellular compartment?

Protein-targeting sequences are specific for each intracellular compartment, so that most proteins are found at only one location within the eukaryotic cell. Increasingly, however, examples are being found of proteins that occur and function in more than one cellular compartment. In some cases, the multicompartmentalized isoforms are encoded by the same gene. Several mechanisms have evolved to enable such genes to encode and differentially express multiple types of topogenic information. These mechanisms include alternative forms of transcription initiation, translation initiation, splicing and post-translational modification.

Journal Article↗