The role of cell-substrate interaction in regulating osteoclast activation: potential implications in targeting bone loss in rheumatoid arthritis.

Analysis of tissues retrieved from the bone-pannus interface from patients with rheumatoid arthritis (RA) and studies in animal models of inflammatory arthritis provide strong evidence that osteoclasts, the cells that are essential for physiological bone resorption, are responsible for articular bone destruction in RA. However, current treatments that specifically target osteoclast-mediated bone resorption in RA have not been successful in preventing bone erosions, and new therapeutic strategies are needed. It has been noted that, although osteoclast precursors are present within the bone microenvironment at sites of pathological bone resorption, cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface and adjacent calcified cartilage. These findings provide evidence that, in addition to requirements for specific cytokines, interaction of osteoclast precursors with these mineralised matrices results in activation of specific signal pathways and the induction of unique gene products that are essential for terminal osteoclast differentiation and activation. These studies are designed to define the gene products and signalling pathways regulated by bone and calcified cartilage, to identify new molecular targets and novel therapeutic approaches for preventing osteoclast-mediated joint destruction in RA and related forms of pathological bone loss.

Peroxisome biogenesis.

Fifteen years ago, we had a model of peroxisome biogenesis that involved growth and division of preexisting peroxisomes. Today, thanks to genetically tractable model organisms and Chinese hamster ovary cells, 23 PEX genes have been cloned that encode the machinery ("peroxins") required to assemble the organelle. Membrane assembly and maintenance requires three of these (peroxins 3, 16, and 19) and may occur without the import of the matrix (lumen) enzymes. Matrix protein import follows a branched pathway of soluble recycling receptors, with one branch for each class of peroxisome targeting sequence (two are well characterized), and a common trunk for all. At least one of these receptors, Pex5p, enters and exits peroxisomes as it functions. Proliferation of the organelle is regulated by Pex11p. Peroxisome biogenesis is remarkably conserved among eukaryotes. A group of fatal, inherited neuropathologies are recognized as peroxisome biogenesis diseases; the responsible genes are orthologs of yeast or Chinese hamster ovary peroxins. Future studies must address the mechanism by which folded, oligomeric enzymes enter the organelle, how the peroxisome divides, and how it segregates at cell division. Most pex mutants contain largely empty membrane "ghosts" of peroxisomes; a few mutants apparently lacking peroxisomes entirely have led some to propose the de novo formation of the organelle. However, there is evidence for residual peroxisome membrane vesicles ("protoperoxisomes") in some of these, and the preponderance of data supports the continuity of the peroxisome compartment in space and time and between generations of cells.

Pex18p is constitutively degraded during peroxisome biogenesis.

Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Deltapex13 or Deltapex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Deltapex4), and others (Deltapex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Deltapex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeast doa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly.

Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p.

Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids. It has been reported, as well as confirmed in this study, to be a peroxisomal protein. Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL). We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor). This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting. Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL. Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway. Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own. By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting. This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p. Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p. Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp. These data demonstrate a novel, redundant targeting pathway for Eci1p.

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.

Rhizomelic chondrodysplasia punctata, a peroxisomal biogenesis disorder caused by defects in Pex7p, a peroxisomal protein import receptor: a minireview.

Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease corresponding to complementation group 11 (CG11), the second most common of the thirteen CGs of peroxisomal biogenesis disorders (PBDs). RCDP is characterized by proximal limb shortening, severely disturbed endochondrial bone formation, and mental retardation, but there is an absence of the neuronal migration defect found in the other PBDs. Plasmalogen biosynthesis and phytanic acid oxidation are deficient, but very long chain fatty acid (VLCFA) oxidation is normal. At the cellular level, RCDP is unique in that the biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybe more, peroxisomal matrix proteins fail to be imported from the cytosol. In this review, we discuss recent advances in understanding RCDP, most prominently the cloning of the affected gene, PEX7, and identification of PEX7 mutations in RCDP patients. Human PEX7 was identified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, which had previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targeting sequences (PTS2). Normal human PEX7 expression rescues the cellular defects in cultured RCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients, including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsense mutation which results in loss of the carboxyterminal 32 amino acids. Identification of RCDP as a PTS2 import disorder explains the observation that several, but not all, peroxisomal matrix proteins are mistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to be PTS2-targeted.

Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway.

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.

Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 4475-4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor.

The rhizomelic form of chondrodysplasia punctata (RCDP) is an autosomal recessive disease of peroxisome biogenesis characterized by deficiencies in several peroxisomal proteins, including the peroxisomal enzymes of plasmalogen biosynthesis and peroxisomal 3-ketoacyl thiolase. In cultured fibroblasts from patients with this disorder, both the peroxisomal targeting and proteolytic removal of the amino-terminal type 2 peroxisomal targeting sequence (PTS2) of thiolase are defective, whereas the biogenesis of proteins targeted by carboxyterminal type 1 peroxisomal targeting sequences (PTS1) is unimpaired. We have previously isolated a Saccharomyces cerevisiae peroxisomal biogenesis mutant, pex7 (formerly peb1/pas7), which demonstrates a striking similarity to the cellular phenotype of RCDP fibroblasts in that PTS1 targeting is functional, but the peroxisomal packaging of PTS2 targeted thiolase is lacking. Complementation of this mutant has led to the identification of the protein ScPex7p, a PTS2 receptor. In this paper we report cloning of the human orthologue of ScPEX7, and demonstrate that this is the defective gene in RCDP. We show that expression of human PEX7 in RCDP cells rescues PTS2 targeting and restores some activity of dihydroxyacetone phosphate acyltransferase (DHAP-AT), a peroxisomal enzyme of plasmalogen biosynthesis, and we identify the mutations responsible for loss of function of PEX7 in a compound heterozygote RCDP patient. These results imply that several peroxisomal proteins are targeted by PTS2 signals and that the various biochemical and clinical defects in RCDP result from a defect in the receptor for this class of PTS.

A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.

Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogen-deficient Chinese hamster ovary (CHO) cell line. The mutant, NZel-1, showed a dramatic (90%) reduction in the rate of biosynthesis and levels of plasmalogens, as determined using short- and long-term labeling with 32Pi. Enzymatic assays and lipid supplementation studies showed that NZel-1 was defective in a single step in the biosynthetic pathway for plasmalogens. This step, catalyzed by the peroxisomal enzyme, alkyl-dihydroxyacetone phosphate (DHAP) synthase, is responsible for the introduction of the ether bond found in plasmalogens. The activity of alkyl-DHAP synthase was reduced in whole-cell homogenates from NZel-1 to 18% of wild-type values. Unlike previously described plasmalogen-deficient mutants, NZel-1 contained peroxisomes, as confirmed by immunofluorescence microscopy and catalase release by digitonin. Peroxisomal functions, including the breakdown of very long-chain (>20 carbons) fatty acids, phytanic acid oxidation, and the acylation of DHAP, were normal. Cell fusion studies revealed that the mutation is recessive and belongs to a new complementation group. To our knowledge this is the first report describing the isolation and characterization of a mutant CHO cell line defective in plasmalogen biosynthesis which contains intact, functional peroxisomes. These cells will allow us to examine the role of ether lipids in cellular functions without complications associated with peroxisome deficiency.

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence.

We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged amino acid, asparagine, at the penultimate residue of the human catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this asparagine residue appears to constitute an important component of the catalase PTS, in that replacement with aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human catalase PTS comprises more than the COOH-terminal three amino acids, in that COOH-terminal-ANL cannot functionally replace the PTS1 signal-SKL in targeting a chloramphenicol acetyl transferase fusion protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the catalase PTS (lysine) is emphasized by the fact that substitution of this residue with a variety of other amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this lysine was replaced with arginine, suggesting that a basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PAS10 gene encoding the S. cerevisiae PTS1 receptor resulted in a cytosolic location of chloramphenicol acetyl transferase appended with the human catalase PTS, as did expression of this protein in cells from a neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PAS10 gene product (Pas10p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that this interaction is abolished by substitutions at the penultimate residue (asparagine-to- aspartate) and at the fourth residue from the COOH terminus (lysine-to-glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human catalase mutated in the COOH-terminal PTS. Consistent with this species difference, fusions between catalase A and human catalase which include the catalase A internal PTS are targeted, at least in part, to peroxisomes regardless of whether the COOH-terminal human catalase PTS is intact.

Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes.

Many yeast peroxisome biogenesis mutants have been isolated in which peroxisomes appear to be completely absent. Introduction of a wild-type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre-existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239, 1536-1538). Introduction of a wild-type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope-tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3HA) was inserted in the genome, replacing the wild-type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3pHA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold-labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.

Molecular characterization and clinical use of a polymorphic tandem repeat in an intron of the human alanine:glyoxylate aminotransferase gene.

The autosomal recessive disease primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate amino-transferase (AGT). This paper concerns the identification, characterization and clinical use of an unusual discretely polymorphic tandem repeat sequence in the fourth intron of the human AGT gene (gene locus designation AGXT). In a random Caucasian population, three alleles could be clearly recognized that consisted of either 12 (type III), 17 (type II) or approximately 38 (type I) tandemly repeated copies of a highly conserved 29/32-bp sequence with frequencies of 33%, 7% and 60%, respectively. In a random Japanese population, the allelic frequencies were markedly different (i.e. 31%, 45% and 19%, respectively). In addition, a fourth allele was identified, consisting of approximately 32 repeats (type IV), with an allelic frequency of approximately 5% in Japanese. The repetitive sequence was similar to previously identified mammalian sequences with homology to the Epstein-Barr virus IR3 repetitive element involving a 12/15-bp region GCA(GGN)GGAGGAGGG within the repeat unit. This IR3-like sequence was interspersed with a 17-bp sequence with no similarity to any currently known repetitive element. The type I and type III alleles were judged to be equivalent to a previously identified TaqI polymorphism. Two polymorphisms previously shown to be associated with the peroxisome-to-mitochondrion mistargeting of AGT in PH1 (a C154-->T point substitution in exon 1 and a 74-bp duplication in intron 1) were found to segregate exclusively with the type I intron 4 polymorphism in Caucasians, but not in Japanese. The polymorphic nature of the intron 4 tandem repeats makes them of potential use in the prenatal diagnosis of PH1, especially when coupled with the exon 1 C154-->T substitution or intron 1 duplication polymorphisms. A PH1 family, in which a fetus had been predicted previously to be either normal or a carrier by AGT enzymic analysis of a fetal liver biopsy, but who had been shown to be only partially informative with respect to the C154-->T/intron 1 polymorphisms, was analysed retrospectively. The family was completely informative for the intron 4 tandem repeat polymorphism and the carrier status of the fetus was confirmed.

Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the feline gene.

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). In vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of approximately 43 kDa, compatible with the cleavage of the N-terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N-terminal 22 amino acids was absent could not be imported into mitochondria in vitro.

Primary hyperoxaluria type 1: genotypic and phenotypic heterogeneity.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT). The disease is notable for its extensive heterogeneity at the clinical, biochemical, enzymic and molecular genetic levels. A study of 116 PH1 patients over the past 8 years has revealed four main enzymic phenotypes: (1) absence of both AGT catalytic activity and immunoreactive AGT protein (approximately 40% of patients); (2) absence of AGT catalytic activity but presence of immunoreactive protein (approximately 16% of patients); (3) presence of both AGT catalytic activity and immunoreactive protein (approximately 41% of patients), in most of which cases the AGT is mistargeted to the mitochondria instead of the peroxisomes; and (4) a variation of the mistargeting phenotype in which AGT is equally distributed between peroxisomes and mitochondria, but in which that in the peroxisomes is aggregated into matrical core-like structures (approximately 3% of patients). Various point mutations, all occurring at conserved positions in the coding regions of the AGT gene, have been identified in these patients. The five mutations discussed in the present study, which have been found in individuals manifesting all of the four major enzymic phenotypes, account for the expressed alleles in about half of all Caucasian PH1 patients. The most common mutation found so far leads to a Gly170-->Arg amino acid substitution. This mutation, in combination with a normally occurring Pro11-->Leu polymorphism, appears to be responsible for the unprecedented peroxisome-to-mitochondrion mistargeting phenotype.

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.