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• W2022877773 abstract "Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity. Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity. hereditary tyrosinemia type 1 fumarylacetoacetate hydrolase fumarylacetoacetate circular dichroism polymerase chain reaction Type 1 hereditary tyrosinemia (HT1,1 OMIM 276700) is an autosomal recessive disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH, EC 3.7.1.2), the last enzyme involved in the tyrosine catabolic pathway. FAH catalyzes the hydrolysis of fumarylacetoacetate into fumarate and acetoacetate (1Lindblad B. Lindstedt S. Steen G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4641-4645Crossref PubMed Scopus (426) Google Scholar). FAH is mainly expressed in mammalian liver. It is also expressed, in lesser amounts, in cells from a wide range of tissues such as kidneys, adrenal glands, lungs, heart, bladder, intestine, stomach, pancreas, lymphocytes (2Tanguay R.M. Valet J.P. Lescault A. Duband J.L. Laberge C. Lettre F. Plante M. Am. J. Hum. Genet. 1990; 47: 308-316PubMed Google Scholar), skeletal muscle, placenta, fibroblasts, chorionic villi (3Berger R. Van Faassen H. Taanman J.W. De Vries H. Agsteribbe E. Pediatr. Res. 1987; 22: 394-398Crossref PubMed Scopus (29) Google Scholar), and some glial cells of the mammalian brain (4Labelle Y. Puymirat J. Tanguay R.M. Biochim. Biophys. Acta. 1993; 1180: 250-256Crossref PubMed Scopus (20) Google Scholar). A deficiency of FAH causes the accumulation of succinylacetone (1Lindblad B. Lindstedt S. Steen G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4641-4645Crossref PubMed Scopus (426) Google Scholar), maleylacetoacetate, and fumarylacetoacetate (FAA), the latter presenting a mutagenic potential (5Tanguay R.M. Jorquera R. Poudrier J. St-Louis M. Acta Biochim. Pol. 1996; 43: 209-216Crossref PubMed Scopus (55) Google Scholar, 6Jorquera R. Tanguay R.M. Biochem. Biophys. Res. Commun. 1997; 232: 42-48Crossref PubMed Scopus (103) Google Scholar, 7Jorquera R. Tanguay R.M. FASEB J. 1999; 13: 2284-2298Crossref PubMed Scopus (57) Google Scholar). Both an acute and a chronic form of the disease have been described on the basis of the clinical severity and/or the age at diagnosis. The acute form occurs early in infancy and causes severe liver damage leading to liver failure and death. The chronic form manifests itself later in infancy or childhood with symptoms such as progressive liver cirrhosis, renal tubular dysfunction, and a high incidence of hepatocellular carcinoma (8Mitchell G.A. Grompe M. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A. Sly W. Valle D. The Molecular and Metabolic Bases of Inherited Diseases. 8th Ed. McGraw Hill, New York2001: 1777-1805Google Scholar). HT1 is the most severe of the diseases associated with the enzymes of the tyrosine catabolic pathway. Although its prevalence worldwide is low (1:120,000 births), it shows a high incidence in some populations such as that of the Saguenay-Lac-St-Jean region (Canada), where it affects 1:1,846 newborns indicating a carrier frequency of 1:20 inhabitants (9De Braekeleer M. Larochelle J. Am. J. Hum. Genet. 1990; 47: 302-307PubMed Google Scholar). The high incidence of HT1 in this region is presumably the result of a founder effect involving mostly the IVS12+5g → a splice mutation (10Grompe M. St-Louis M. Demers S. Al-Dhalimy M. Leclerc B. Tanguay R.M. N. Engl. J. Med. 1994; 331: 353-357Crossref PubMed Scopus (111) Google Scholar, 11Poudrier J. St-Louis M. Lettre F. Gibson K. Prévost C. Larochelle J. Tanguay R.M. Prenat. Diagn. 1996; 16: 59-64Crossref PubMed Google Scholar). The human FAH gene is localized to the q23-q25 region of chromosome 15 (12), contains 14 exons, and covers ∼35 kilobases of DNA (13Labelle Y. Phaneuf D. Leclerc B. Tanguay R.M. Hum. Mol. Genet. 1993; 2: 941-946Crossref PubMed Scopus (57) Google Scholar, 14Awata H. Endo F. Tanoue A. Kitano A. Nakano Y. Matsuda I. Biochim. Biophys. Acta. 1994; 1226: 168-172Crossref PubMed Scopus (26) Google Scholar). At this time, 34 mutations have been reported (8Mitchell G.A. Grompe M. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A. Sly W. Valle D. The Molecular and Metabolic Bases of Inherited Diseases. 8th Ed. McGraw Hill, New York2001: 1777-1805Google Scholar, 15St-Louis M. Tanguay R.M. Hum. Mutat. 1997; 9: 291-299Crossref PubMed Scopus (82) Google Scholar, 16Bergman A.J.I.W. van der Berg I.E.T. Brink W. Poll-The B.T. Ploos van Amstel J.K. Berger R. Hum. Mutat. 1998; 12: 19-26Crossref PubMed Scopus (35) Google Scholar). These include 18 missense mutations, 10 splice mutations, 5 nonsense mutations, and 1 silent mutation. These mutations are evenly spread along the FAH gene but with a slightly higher frequency in some parts of exons 8 and 13. The human FAH enzyme has been purified to homogeneity (2Tanguay R.M. Valet J.P. Lescault A. Duband J.L. Laberge C. Lettre F. Plante M. Am. J. Hum. Genet. 1990; 47: 308-316PubMed Google Scholar, 17Hsiang H.H. Sim S.S. Mahuran D.J. Schmidt D.E. Biochemistry. 1972; 11: 2098-2102Crossref PubMed Scopus (24) Google Scholar), and the crystal structure of recombinant mouse FAH has recently been reported (18Timm D.E. Mueller H.A. Bhanumoorthy P. Harp J.M. Bunick G.J Struct. Fold. Des. 1999; 7: 1023-1033Abstract Full Text Full Text PDF Scopus (73) Google Scholar). FAH represents a new class of metalloenzymes that possess a unique α/β fold. The crystal structure of FAH and its active site should prove particularly helpful in understanding the effects of mutations on both the structure and the activity of the enzyme. To determine the effects of missense mutations on the structure and activity of FAH, we used site-directed mutagenesis to generate mutant FAHs and examined the expression and enzymatic activity of mutant proteins in a bacterial expression system and by transient expression after transfection in mammalian cells. Circular dichroism spectra were measured for the wild-type FAH and four variants containing HT1-associated amino acid substitutions, and structural descriptions of HT1-associated amino acid substitutions were made based on crystal structure of murine FAH (18Timm D.E. Mueller H.A. Bhanumoorthy P. Harp J.M. Bunick G.J Struct. Fold. Des. 1999; 7: 1023-1033Abstract Full Text Full Text PDF Scopus (73) Google Scholar). Eight missense mutations (all previously reported in HT1 patients) were analyzed: N16I (19Phaneuf D. Lambert M. Laframboise R. Mitchell G. Lettre F. Tanguay R.M. J. Clin. Invest. 1992; 90: 1185-1192Crossref PubMed Scopus (77) Google Scholar), F62C (14Awata H. Endo F. Tanoue A. Kitano A. Nakano Y. Matsuda I. Biochim. Biophys. Acta. 1994; 1226: 168-172Crossref PubMed Scopus (26) Google Scholar), A134D (13Labelle Y. Phaneuf D. Leclerc B. Tanguay R.M. Hum. Mol. Genet. 1993; 2: 941-946Crossref PubMed Scopus (57) Google Scholar, 20Rootwelt H. Høie K. Berger R. Kvittingen E.A. Hum. Mutat. 1996; 7: 239-243Crossref PubMed Scopus (29) Google Scholar, 21Rootwelt H. Chou J. Gahl W.A. Berger R. Coskun T. Brodtkorb E. Kvittingen E.A. Hum. Genet. 1994; 93: 615-619Crossref PubMed Scopus (21) Google Scholar), C193R (22Ploos van Amstel J.K. Bergman A.J.I.W. van Beurden E.A.C.M. Roijers J.F.M. Peelen T. van den Berg I.E.T. Poll-The B.T. Kvittingen E.A. Berger R. Hum. Genet. 1996; 97: 51-59Crossref PubMed Google Scholar), D233V (20Rootwelt H. Høie K. Berger R. Kvittingen E.A. Hum. Mutat. 1996; 7: 239-243Crossref PubMed Scopus (29) Google Scholar, 23Rootwelt H. Berger R. Gray G. Kelly D.A. Coskun T. Kvittingen E.A. Am. J. Hum. Genet. 1994; 55: 653-658PubMed Google Scholar), W234G (24Hahn S.H. Krasnewich D. Brantly M. Kvittingen E.A. Gahl W.A. Hum. Mutat. 1995; 6: 66-73Crossref PubMed Scopus (8) Google Scholar), Q279R (25Kim S.K. Kupke K.G. Ierardi-Curto L. Holme E. Greter J. Tanguay R.M. Poudrier J. D'Astous M. Lettre F. Hahn S.H. Levy H. J. Inherit. Metab. Dis. 2000; 23: 791-804Crossref PubMed Scopus (29) Google Scholar), and R341W, the so-called pseudo-deficiency mutation (26Kvittingen E.A. Børresen A.L. Stokke O. van der Hagen C.B. Lie S.O. Clin. Genet. 1985; 27: 550-554Crossref PubMed Scopus (9) Google Scholar, 27Rootwelt H. Brodtkorb E. Kvittingen E.A. Am. J. Hum. Genet. 1994; 55: 1122-1127PubMed Google Scholar). There is still no clear relation between the genotype and the phenotype in HT1, which varies from an acute to a more chronic form. Mutations analyzed in this study were chosen in a manner to include mutations affecting residues in different parts of the FAH structure and involving mostly residues conserved from Caenorhabditis elegans to Homo sapiens. Some of the mutations were studied for specific reasons. For example, the C193R mutation was analyzed to determine whether the cysteine residue at position 193 was essential to the structural integrity of the enzyme. Other mutations were examined because they were located in the enzyme's active site (D233V and W234G). The molecular basis of the R341W mutation, described as a pseudo-deficiency mutation, was investigated because of the unusual phenotype observed in homozygote individuals who show no symptoms of the disease. Finally, the Q279R mutation was studied because it represented a new mutation for which the molecular basis had not yet been described. Many of the mutated proteins were found to be inactive, probably as a result of misfolding of the mutated FAH. A human FAH cDNA clone was obtained by amplification of cDNA reverse-transcribed from mRNA of a normal liver (patient 8688). The amplification reaction mixture (50 μl) contained 5 μl of cDNA, 5 μl of 10× PCR buffer (Expand Long Template PCR System, Roche Molecular Biochemicals), 500 μm dNTPs, 200 ng of primers TANR130 and RT025, and 2.5 units of recombinant Taq-Pwo polymerase (Roche Diagnostics). TANR130 primer (5′-CAT GTC CTT CAT CCC GGT GGC-3′) is situated at the very beginning of the 5′-coding end of FAH cDNA, a cytosine has been added in front of the start ATG codon of the FAH gene. RT025 (5′-GGG AAT TCT GTC ACT GAA TGG CGG AC-3′) is located in the 3′-noncoding end of the gene. The 5′ extremity of RT025 is not complementary to the cDNA but contains an EcoRI restriction site used to clone the amplification product in a vector. The reaction mixture was covered with 50 μl of mineral oil and incubated at 95 °C for 5 min, 53 °C for 5 min, and 72 °C for 40 min to allow the synthesis of the second strand of the cDNA. The PCR performed on a DNA Thermal Cycler (model N801–0150, PerkinElmer Life Sciences) included 35 cycles of the following program: 40 s at 95 °C, 1 min at 53 °C, and 2 min at 72 °C. A final extension of 15 min at 72 °C was done to complete the elongation. The amplification product was digested with NcoI (2,700 units/ml, Amersham Pharmacia Biotech) and blunted with nuclease S1 (343,400 units/ml, Amersham Pharmacia Biotech). After this first enzymatic digestion, the product was digested with EcoRI to obtain a cohesive end at the 3′ extremity. The same enzymatic digestions were performed on the pET30 vector (Novagene). The digested amplification product was then ligated to the pET30 vector using the T4 DNA ligase (400,000 units/ml, New England Biolabs), and the pET30FAH construction was fully sequenced on an ABI 373XL. Site-directed mutagenesis was performed on the pET30FAH vector using the Quick Change™ site-directed mutagenesis kit (Stratagene). PCR reactions (50 μl) contained 10 mm KCl, 10 mm(NH4)2SO4, 20 mmTris-HCl (pH 8.8), 2 mm MgSO4, 0.1% Triton® X-100, 0.1 mg/ml nuclease-free bovine serum albumin, 50 ng of pET30FAH vector, 125 ng of each complementary primer (Table I), 250 μm of each dNTP, and 2.5 units of Pfu DNA polymerase. The PCR conditions used were 30 s at 95 °C for DNA melting, followed by 12 cycles of elongation (each cycle consisting of 30 s at 95 °C, 1 min at 55 °C, and 13.5 min at 68 °C). The DNA in the reaction mixtures was digested 1 h at 37 °C using 10 units ofDpnI (Stratagene) and was used to transform competent bacterial cells. Clones containing the site-directed mutations were verified by sequencing (results not shown) to ensure that no other mutations were present in the gene.Table IPCR primers and their location in the FAH geneMutantOligonucleotide sequence (5′ → 3′)Location in FAHntFAH-N16ICCC CAT CCA CAt CCT GCC CTA CGG C47FAH-F62CCAA ACA CCA GGA TGT CTg CAA TCA GCC TAC ACT C185FAH-A134DCTC TCG GCA GCA TGa TAC CAA CGT CGG AAT C401FAH-C193RGTA TAT GGT GCC cGC AAG CTC TTG GAC577FAH-D233VGGT CCT TAT GAA CGt CTG GAG TGC ACG AG698FAH-W234GCCT TAT GAA CGA CgG GAG TGC ACG AG700FAH-R341WCTG CAA CCT GtG GCC GGG GGA1021The mutated nucleotides are in bold lowercase letters. nt, nucleotides. Open table in a new tab The mutated nucleotides are in bold lowercase letters. nt, nucleotides. The wild-type and mutated pCEP4FAH mammalian expression vectors were constructed by inserting the fragments of pET30FAH vectors digested with KpnI (10,000 units/ml, New England Biolabs) andHindIII (20,000 units/ml, New England Biolabs) and containing the wild-type or mutant FAH gene intoKpnI-HindIII-cut pCEP4 vector (Invitrogen). Thefah fragments were ligated to digested pCEP4 vector using T4 DNA ligase (40,000 units/ml, New England Biolabs). The wild-type vector pET30FAH and mutant vectors were transformed in the GJ1158 strain of Escherichia coli. The cells were grown to an A600 of 0.4–0.5 at 37 °C in Luria-Bertani medium with NaCl omitted, containing 10 g of tryptone and 5 g of yeast extract per liter (pH 7.0), with 50 μg/ml kanamycin. The temperature was then lowered to 30 °C, 0.3m NaCl was added to the medium, and cultures were further incubated for 5 h (28Bhandari P. Gowrishankar J. J. Bacteriol. 1997; 179: 4403-4406Crossref PubMed Google Scholar). Cells were harvested and pellets resuspended in 20 ml of 10 mm phosphate buffer (pH 7.3). Each sample was introduced into a French press cell and lysed at 800 p.s.i. of pressure. Afterward, samples were centrifuged at 10,000 × g for 30 min at 4 °C. Following this centrifugation, the soluble supernatant was recovered and protein concentration was determined using the Bio-Rad protein assay. For CD measurements, the recombinant proteins were purified by affinity chromatography through their His tag on nickel-nitrilotriacetic acid superflow columns (Qiagen). Samples for CD measurements were dialyzed overnight against a 1000-fold excess volume of 10 mm sodium phosphate (pH 7.0) at 4 °C. Wild-type FAH remains soluble in excess of 1 mg/ml under these conditions; however, precipitation was noted for several of the variants and was removed by centrifugation at 14,000 × g. The yield of soluble N16I, F62C, and W234G from the bacterial expression system was not sufficient for making CD measurements. Concentrations of supernatants of the dialyzed samples were determined by scanning UV absorbance spectra using an extinction coefficient of 1.3 ml·mg−1·cm−1 at 280 nm. Less than 3% of the C193R sample remained soluble following dialysis, preventing further CD analysis of this sample. Samples were diluted to 0.12 mg/ml, and CD spectra were recorded as an average of 12 scans at room temperature using a Jasco J720 instrument and a 1-mm light path. Base-line spectra were recorded using individual dialysis solutions as a blank for each sample. Mean residue ellipticities were calculated using a mean residue weight of 110. Final protein concentrations were calculated using photomultiplier tube voltages and standard curves based on serial dilutions of wild-type FAH. Data smoothing has not been performed. Protein concentration-dependent differences between the CD spectra were assessed based on the proportionality between variant and the wild-type spectra in the 240–190 nm range, and by calculating the ratio of ellipticities measured at fixed wavelengths (data not shown) and measured at two minima apparent in the spectra (Fig. 2 and Table IV). Differences between the wild-type and Q279R spectra were judged to be insignificant, based on these criteria.Table IVMajor features in CD spectraSubstitutionMinimaMin1:Min2CrossoverMaximumnmnmnmWild-type225.0208.81.25201.9195.8A134D220.0209.0 4-aIndicates values that do not differ significantly between the wild-type and variant samples.1.04200.5195.8 4-aIndicates values that do not differ significantly between the wild-type and variant samples.D233V224.6 4-aIndicates values that do not differ significantly between the wild-type and variant samples.208.8 4-aIndicates values that do not differ significantly between the wild-type and variant samples.1.18201.3195.8 4-aIndicates values that do not differ significantly between the wild-type and variant samples.Q279R223.2209.6 4-aIndicates values that do not differ significantly between the wild-type and variant samples.1.23201.5 4-aIndicates values that do not differ significantly between the wild-type and variant samples.196.0 4-aIndicates values that do not differ significantly between the wild-type and variant samples.R341W222.2210.21.17201.1195.8 4-aIndicates values that do not differ significantly between the wild-type and variant samples.The positions of the major features present in the indicated CD spectra were identified using the Jascow Standard Analysis Program version 1.20. Min1:Min2 is the ratio of the minimal mean residue ellipticity values measured at the wavelengths indicated in columns two and three.4-a Indicates values that do not differ significantly between the wild-type and variant samples. Open table in a new tab The positions of the major features present in the indicated CD spectra were identified using the Jascow Standard Analysis Program version 1.20. Min1:Min2 is the ratio of the minimal mean residue ellipticity values measured at the wavelengths indicated in columns two and three. CV-1 (kidney African Green monkey cells, American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Medicorp Inc.) at 37 °C with 5% CO2. The day prior to transfection, 2 × 106 CV-1 cells were seeded in a 75-cm2 flask and grown at 37 °C. The cells were transfected with 2 μg of pCEP4FAH wild-type or mutant DNA using the FuGENE6 kit (Roche Molecular Biochemicals) according to the manufacturer's recommendations. Six hours after transfection, cells were washed twice in PBS and fresh DMEM, 10% fetal bovine serum medium was added to the cells. Forty hours after transfection, cells were washed in PBS, trypsinized and resuspended in 10 mm phosphate buffer (pH 7.3) for homogenization using a Teflon pestle and a syringe. The homogenized extract was centrifuged 15 min at 10,000 × g, both the supernatant and the pellet were recovered, and protein concentration was determined using the Bio-Rad protein assay. These samples were used for kinetics assays as well as for Western blot analysis. Nontransfected CV-1 cells showed no FAH activity (data not shown). FAH hydrolytic activity was determined as described previously using about 50 μm FAA as a substrate (2Tanguay R.M. Valet J.P. Lescault A. Duband J.L. Laberge C. Lettre F. Plante M. Am. J. Hum. Genet. 1990; 47: 308-316PubMed Google Scholar). Briefly, the decrease of absorbance at 330 nm, which corresponds to hydrolysis of the FAA substrate, was measured at room temperature using a spectrophotometer (Ultrospec III (Amersham Pharmacia Biotech) or Cary Varian 100) for the CV-1 cell extracts (using 40–150 μg of total protein/assay) and the bacterial extracts (using 5–10 μg of total protein/assay). To correct for differences in transfection efficiency and expression of the mutant FAH proteins, the hydrolytic activity of FAH against FAA was expressed as nanomoles of FAA hydrolyzed/min × mg of FAH, as determined by the following equation. FAH activityEquation 1 −ΔOD(330nm)/min×reaction volume(ml)FAA molar extinction coefficient(ml/nmol)×FAH(mg) 1.35 μm−1cm−1 was used as the extinction coefficient of FAA at 330 nm, and ΔOD represents the change in optical density at 330 nm/min. The amount of FAH was measured by a quantitative Western blot assay. The cell extracts were loaded on a 12% SDS-PAGE gel along with known quantities (0.1, 0.2, 0.5, and 1 μg) of purified recombinant human FAH protein. Proteins were transferred to an Immobilon-P membrane (Millipore) for Western blot analysis (29Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The primary antibody, anti-FAH (antibody 488) (2Tanguay R.M. Valet J.P. Lescault A. Duband J.L. Laberge C. Lettre F. Plante M. Am. J. Hum. Genet. 1990; 47: 308-316PubMed Google Scholar) was used at a 1:25,000 dilution, whereas the secondary antibody, alkaline phosphatase coupled to a goat anti-rabbit IgG (Jackson Immunoresearch Laboratories), was diluted 1:50,000. A standard curve showing the integrated density of the signal on the quantity of FAH present in the sample was obtained from NIH Image program analysis by measuring the signal intensity of FAH containing samples on films. To study the structure-function of FAH, various missense mutations found in the FAH gene of different HT1 patients (TableII) were introduced in a human FAH cDNA by site-directed mutagenesis. These patients were either homoallelic or heteroallelic and exhibited different phenotypes ranging from normal to an acute form. For example, the patient heteroallelic for the Q279R and the IVS6 − 1g → t mutations showed mild clinical symptoms until 36 years but then developed hepatocellular carcinoma (25Kim S.K. Kupke K.G. Ierardi-Curto L. Holme E. Greter J. Tanguay R.M. Poudrier J. D'Astous M. Lettre F. Hahn S.H. Levy H. J. Inherit. Metab. Dis. 2000; 23: 791-804Crossref PubMed Scopus (29) Google Scholar).Table IICharacteristics of missense mutations studied in HT1 patientsMutationCharacteristicsGenotypePhenotypeN161Normal mRNA, no activityHeterozygous, N16I/U 2-aU, undetermined.AcuteF62CNo activityHomozygousND 2-bND, not described.A134DReduced mRNA, no activityHeterozygous, A134D/IVS12 + 5g → aChronic-intermediateC193RNormal mRNAHeterozygous, C193R/E364XSubacuteD233VNormal mRNAHomozygousChronic-intermediateW234GReduced mRNA, no activityHeterozygous, W234G/IVS12 + 5g → aChronicQ279RUncharacterizedHeterozygous, Q279R/IVS6 − 1g → tMildR341WPseudo-deficiency, normal mRNA reduced activityHomozygousNormal2-a U, undetermined.2-b ND, not described. Open table in a new tab The mutated FAH constructs were introduced in E. coli and the corresponding proteins induced with NaCl (28Bhandari P. Gowrishankar J. J. Bacteriol. 1997; 179: 4403-4406Crossref PubMed Google Scholar). The soluble recombinant protein fractions were analyzed by Western blot and for enzymatic activity. As shown in Fig.1 A, all mutated FAH proteins with the exception of N16I, F62C, and W234G were expressed in the soluble fraction of GJ1158 E. coli strain. The FAH harboring N16I, F62C, and W234G were expressed but were retained in the insoluble cell fraction (data not shown). Next, the hydrolytic activity of the mutated FAH proteins was measured using FAA as the substrate. As shown in Table III, all mutated FAHs with the exception of R341W and Q279R were inactive in this hydrolytic assay. R341W and Q279R showed activities equal to that of normal FAH.Table IIIEnzymatic activity of wild-type and mutated FAHFAHSpecific activity in E. coliSpecific activity in CV-1 cellsnmol × min−1 × mg−1 FAHnmol × min−1 × mg−1 FAHWild-type14,000 ± 1,400253 ± 5.7 to 362 ± 11.3N16IU 3-aU, undetectable.UF62CUUA134DUUC193RUUD233VUUW234GUUQ279R15,700 ± 1,800267 ± 5.9R341W14,600 ± 150344 ± 25FAH hydrolytic activity was determined as described in the experimental section. Data are means ± S.D. (n = 3).3-a U, undetectable. Open table in a new tab FAH hydrolytic activity was determined as described in the experimental section. Data are means ± S.D. (n = 3). To check if these mutated proteins were equally expressed in a mammalian background, the FAH coding fragment of each pET30FAH construct was subcloned in the pCEP4 mammalian expression vector and transfected in simian CV-1 cells. Forty-eight hours after transfection, cells were homogenized and proteins from the soluble and insoluble fractions (10,000 × g pellet) were loaded on a 12% SDS-PAGE gel and immunoblotted with the anti-FAH antibody. As can be seen in Fig. 1 B, all mutant proteins were expressed in CV-1 cells. However, proteins containing the N16I, F62C, C193R, and W234G mutations were only present in the insoluble fraction of the cellular extract (Fig. 1 B, top), which suggests that these proteins are subject to misfolding and aggregation in mammalian CV-1 cells. This would explain their absence from the soluble fraction of cellular extracts. To assess whether the expressed proteins were localized properly in the cell, immunofluorescent staining with anti-FAH was performed on CV-1 cells 48 h after transfection. The mutated proteins found in the insoluble fraction of the cellular extracts by Western blot analysis (N16I, F62C, C193R, and W234G) were shown to form aggregates mainly in the perinuclear-Golgi region of the cell (data not shown). This observation supports the hypothesis that the presence of these mutations interferes with the proper folding of the enzyme. The other FAH variants (A134D, D233V, Q279R, and R341W) were evenly distributed in the cytoplasm, much like the wild-type protein (data not shown). Assay of the hydrolytic activity of the mutant FAHs in transfected mammalian cells gave results identical to those seen for proteins expressed in bacteria, i.e. only the R341W and Q279R proteins had hydrolytic activity (TableIII), which was similar to that of the wild-type enzyme. The specific activity of FAH expressed in bacterial cells is notably much higher than that measured in transfected mammalian CV-1 cells. The activity in these cells is comparable to that measured in pork liver (780 nmol·min−1·mg−1FAH). To assess structural perturbations caused by HT1-associated amino acid substitutions, circular dichroism (CD) spectra were measured for wild-type FAH and the A134D, D233V, Q279R, and R341W variants. Representative spectra are shown for wild-type FAH, FAH D233V, and FAH A134D in Fig.2. The wild-type FAH CD spectrum is characterized by minima at 225.0 and 208.8 nm, a crossover point at 201.9 nm, and a maximum at 196.0 nm. This spectrum is consistent with an α/β structure in solution. The FAH crystal structure contains 27% β-strand and 18% α-helical secondary structure (18Timm D.E. Mueller H.A. Bhanumoorthy P. Harp J.M. Bunick G.J Struct. Fold. Des. 1999; 7: 1023-1033Abstract Full Text Full Text PDF Scopus (73) Google Scholar). Significant differences were observed with all the HT1 variants, except Q279R (Fig. 2 and Table IV). The largest differences involved the position of the minimum between 220 and 225 nm and the intensity of the CD bands across the spectrum. The spectrum for A134D, having a minimum at 220.0 nm, enhanced negative ellipticity between 220 and 200 nm, and diminished positive ellipticity below 200 nm, deviated most from the wild-type spectrum (Fig. 2). The R341W FAH spectrum shows enhanced negative ellipticity above 201 nm and diminished positive ellipticity below 201 nm, relative to wild-type." @default.
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