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.     

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

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.     

Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1

A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.     

Persistent equine arteritis virus infection in HeLa cells

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.     

Multiple mechanisms of action of pyridoxine in primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is a rare hereditary calcium oxalate kidney stone disease caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). About one third of patients are responsive to pharmacological doses of pyridoxine (vitamin B6), but its mechanism of action is unknown. Using stably transformed Chinese Hamster Ovary (CHO) cells expressing various normal and mutant forms of AGT, we have shown that pyridoxine increases the net expression, catalytic activity and peroxisomal import of the most common mistargeted mutant form of AGT (i.e. Gly170Arg on the background of the polymorphic minor allele). These multiple effects explain for the first time the action of pyridoxine in the most common group of responsive patients. Partial effects of pyridoxine were also observed for two other common AGT mutants on the minor allele (i.e. Phe152Ile and Ile244Thr) but not for the minor allele mutant AGT containing a Gly41Arg replacement. These findings demonstrate that pyridoxine, which is metabolised to pyridoxal phosphate, the essential cofactor of AGT, achieves its effects both as a prosthetic group (increasing enzyme catalytic activity) and a chemical chaperone (increasing peroxisome targeting and net expression). This new understanding should aid the development of pharmacological treatments that attempt to enhance efficacy of pyridoxine in PH1, as well as encouraging a re-evaluation of the extent of pyridoxine responsiveness in PH1, as more patients than previously thought might benefit from such treatment.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.     

Characterization of five new mutants in the carboxyl-terminal domain of human apolipoprotein E: No cosegregation with severe hyperlipidemia

Assessment of the apolipoprotein E (apoE) phenotype by isoelectric focusing of both hyperlipidemic and normolipidemic individuals identified five new variants. All mutations were confined to the downstream part of the APOE gene by using denaturing gradient gel electrophoresis (DGGE). Sequence analysis revealed five new mutations causing unique amino acid substitutions in the carboxyl-terminal part of the protein containing the putative lipid-binding domain. Three hyperlipoproteinemic probands were carriers of the APOE*2(Val236→Glu) allele, the APOE*3(Cys112→Arg; Arg251→Gly) allele, or the APOE*1(Arg158→Cys; Leu252→Glu) allele. DGGE of the region encoding the receptor-binding domain was useful for haplotyping the mutations at codons 112 and 158. Family studies failed to demonstrate cosegregation between the new mutations and severe hyperlipoproteinemia, although a number of carriers for the APOE*3(Cys112→Arg; Arg251→Gly) allele and the APOE*1(Arg158→Cys; Leu252→Glu) allele expressed hypertriglyceridemia and/or hypercholesterolemia. Two other mutant alleles, APOE*4⁻ (Cys112→Arg; Arg274→His) and APOE*4⁺(Ser296→Arg), were found in normolipidemic probands. The lack of cosegregation of these new mutations with severe hyperlipoproteinemia suggests that these mutations do not exert a dominant effect on the functioning of apoE.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Four of the Most Common Mutations in Primary Hyperoxaluria Type 1 Unmask the Cryptic Mitochondrial Targeting Sequence of Alanine:glyoxylate Aminotransferase Encoded by the Polymorphic Minor Allele

The gene encoding the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT, EC. 2.6.1.44) exists as two common polymorphic variants termed the “major” and “minor” alleles. The P11L amino acid replacement encoded by the minor allele creates a hidden N-terminal mitochondrial targeting sequence, the unmasking of which occurs in the hereditary calcium oxalate kidney stone disease primary hyperoxaluria type 1 (PH1). This unmasking is due to the additional presence of a common disease-specific G170R mutation, which is encoded by about one third of PH1 alleles. The P11L and G170R replacements interact synergistically to reroute AGT to the mitochondria where it cannot fulfill its metabolic role (i.e. glyoxylate detoxification) effectively. In the present study, we have reinvestigated the consequences of the interaction between P11L and G170R in stably transformed CHO cells and have studied for the first time whether a similar synergism exists between P11L and three other mutations that segregate with the minor allele (i.e. I244T, F152I, and G41R). Our investigations show that the latter three mutants are all able to unmask the cryptic P11L-generated mitochondrial targeting sequence and, as a result, all are mistargeted to the mitochondria. However, whereas the G170R, I244T, and F152I mutants are able to form dimers and are catalytically active, the G41R mutant aggregates and is inactive. These studies open up the possibility that all PH1 mutations, which segregate with the minor allele, might also lead to the peroxisome-to-mitochondrion mistargeting of AGT, a suggestion that has important implications for the development of treatment strategies for PH1.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Genetic polymorphism of CYP4A11 and CYP4A22 genes and in silico insights from comparative 3D modelling in a French population

The CYP4A subfamily is known to ω-hydroxylate the endogenous arachidonic acid into 20-hydroxyeicosatetranoic acid, which has renovascular and tubular functions. The aim of this work was to report a comprehensive investigation of the CYP4A11 and CYP4A22 genetic polymorphisms in a French population. Using PCR-SSCP and sequencing strategies, a total of 26 sequence variations were identified comprising 3 missense mutations for CYP4A11 (Ser404Phe, Phe434Ser and Arg505His) and 7 missense mutations for CYP4A22 (Arg126Trp, Gly130Ser, Asn152Tyr, Val185Phe, Cys231Arg, Leu428Pro and Leu509Phe). In comparison with SNPs reported in the database (dbSNP) of the National Center for Biotechnology information (NCBI), 6 and 3 novel polymorphisms were identified in CYP4A11 and CYP4A22, respectively. The potential impact of the amino acid substitutions on the structure and/or catalytic activity of the enzymes has been estimated by the construction and validation of the CYP4A 3D models. These results could be helpful for further investigations of the potential role of CYP4A variants in the genetic susceptibility to cardiovascular diseases in humans such as arterial hypertension.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

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.     

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.     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

Studies on peptides. LXXXI. Application of a new arginine derivative, NG-mesitylene-2-sulfonylarginine, to the synthesis of substance P and neurotensin.

A new devised arginine derivative, NG-mesitylene-2-sulfonylarginine, Arg(Mts), was employed for the synthesis of hypothalamic substance P and neurotensin. The former was obtained in 74% yield by treatment of the protected undecapeptide amide, Z - Arg(Mts) - Pro - Lys(Z) - Pro - Gln - Gln - Phe - Phe - Gly - Leu - Met(O)-NH2, with methanesulfonic acid in the presence of anisole followed by reduction of the sulfoxide with 2-mercaptoethanol. The latter was obtained in 54% yield by the similar treatment of the protected tridecapeptide ester, Z - Pyr - Leu - Tyr - Glu(OBzl) - Asn - Lys(Z) - Pro - Arg(Mts) - Arg(Mts) - Pro - Tyr - Ile - Leu - OBzl, with methanesulfonic acid. As scavenger, a mixture of anisole-thioanisole-o-cresol (1:1:1, by vol.) was employed to suppress the side reaction, O-mesitylene-2-sulfonation of the Tyr residue.