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• W2024293713 abstract "Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M. The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells. Thermal stability profiles and degradation time courses of PAH expressed inE. coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system. Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates. PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay. In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein. The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations. Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M. The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells. Thermal stability profiles and degradation time courses of PAH expressed inE. coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system. Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates. PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay. In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein. The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations. phenylalanine hydroxylase phenylketonuria polyacrylamide gel electrophoresis binding domain activating domain maltose-binding protein Mammalian phenylalanine hydroxylase (PAH,1 phenylalanine 4-monooxygenase, E.C. 1.14.16.1) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the hydroxylation of phenylalanine to tyrosine. Defects in the human phenylalanine hydroxylase gene (GenBankTM cDNA reference sequence U49897, MIM 261600) cause phenylketonuria (PKU), a recessive disorder that if not treated from birth leads to variable degrees of mental retardation. PKU is in many ways regarded as a “model genetic disease,” as clinical and biochemical characteristics are well defined, an effective treatment has been successfully implemented, both the gene and the enzyme are well characterized, mutations have been identified, genotype-phenotype correlations have been established, and an animal model has been produced (1Levy H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1811-1813Crossref PubMed Scopus (54) Google Scholar). Although PKU is a classical monogenic disorder, the associated features are complex, as pointed out by Scriver and Waters (2Scriver C.R. Waters P.J. Trends Genet. 1999; 15: 267-272Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). From the genetic point of view, more than 380 mutations have been described associated to different populations (3Nowacki P. Byck S. Prevost L. Scriver C.R. Nucleic Acids Res. 1998; 26: 220-225Crossref PubMed Scopus (45) Google Scholar). After defining the mutational spectrum of PKU in several populations, the aim of the researchers has been the study of the genotype-phenotype correlations (4Kayaalp E. Treacy E. Waters P.J. Byck S. Nowacki P. Scriver C.R. Am. J. Hum. Genet. 1997; 61: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Gulberg P. Rey F. Zschocke J. Romano V. Francois B. Michiels L. Ullrich K. Hoffmann Burgard P. Schmidt H. Meli C. Riva E. Dianzani I. Ponzone A. Rey J. Guttler F. Am. J. Hum. Genet. 1998; 63: 71-79Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6Desviat L.R. Perez B. Gamez A. Sanchez A. Garcia M.J. Martinez-Pardo M. Marchante C. Boveda D. Baldellou A. Arena J. Sanjurjo P. Fernandez A. Cabello M.L. Ugarte M. Eur. J. Hum. Genet. 1999; 7: 386-392Crossref PubMed Scopus (52) Google Scholar). These studies have addressed the assessment of the severity of the mutations by in vitro expression analysis and examination of the phenotype in homozygous or functional hemizygous patients. Up to now 57 PKU mutations have been expressed in at least one in vitro system (7Waters P. Parniak M Nowacki P. Scriver C.R. Hum. Mutat. 1998; 11: 4-17Crossref PubMed Scopus (79) Google Scholar), and the data obtained have allowed in most cases the prediction of the biochemical phenotype based on the genotype. Nevertheless, some discrepancies have been detected in the genotype-phenotype correlations, especially in patients bearing mutations that result in decreased immunoreactive protein and consequently decreased activity when expressed in vitro(4Kayaalp E. Treacy E. Waters P.J. Byck S. Nowacki P. Scriver C.R. Am. J. Hum. Genet. 1997; 61: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Gulberg P. Rey F. Zschocke J. Romano V. Francois B. Michiels L. Ullrich K. Hoffmann Burgard P. Schmidt H. Meli C. Riva E. Dianzani I. Ponzone A. Rey J. Guttler F. Am. J. Hum. Genet. 1998; 63: 71-79Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6Desviat L.R. Perez B. Gamez A. Sanchez A. Garcia M.J. Martinez-Pardo M. Marchante C. Boveda D. Baldellou A. Arena J. Sanjurjo P. Fernandez A. Cabello M.L. Ugarte M. Eur. J. Hum. Genet. 1999; 7: 386-392Crossref PubMed Scopus (52) Google Scholar). This is the case of many missense mutations, which are broadly referred to as causing PAH enzyme instability. There are now several reports documenting increased instability and susceptibility toward aggregation and degradation of PKU mutant proteins (8Bjorgo E. Knasppskog P.M. Martı́nez A. Stevens R. Flatmark T. Eur. J. Biochem. 1998; 257: 1-10Crossref PubMed Scopus (69) Google Scholar, 9Waters P.J. Parniak M.A. Akerman B.R. Jones A.O. Scriver C.R. J. Inherit. Metab. Dis. 1999; 22: 208-212Crossref PubMed Scopus (35) Google Scholar, 10Waters P.J. Parniak M.A. Akerman B.R. Scriver C.R. Mol. Genet. Metab. 2000; 69: 101-110Crossref PubMed Scopus (47) Google Scholar), and recently, the thermodynamic stability of native wild-type PAH has been examined (11Chehin R. Thorolfsson M. Knappskogg P.M. Martı́nez A. Flatmark T. Arrondo J.L. Muga A. FEBS Lett. 1998; 422: 225-230Crossref PubMed Scopus (41) Google Scholar, 12Kleppe R. Uhlemann K. Knappskog P.M. Haavik J. J. Biol. Chem. 1999; 274: 33251-33258Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) analyzing the contribution of instability to PKU compared with other reasons for reduced activity. Currently, knowledge of the three-dimensional structure of PAH is also available (13Erlandsen H. Fusetti F. Martinez A. Hough E. Flatmark T. Stevens R.C. Nat. Struct. Biol. 1997; 4: 995-1000Crossref PubMed Scopus (175) Google Scholar, 14Fusetti F. Erlandsen H. Flatmark T. Stevens R.C. J. Biol. Chem. 1998; 273: 16962-16967Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15Kobe B. Jennings I.G. House C.M. Michell B.J. Goodwill K.E. Santarsiero B.D. Stevens R.C. Cotton R.G.H. Kemp B.E. Nat. Struct. Biol. 1999; 6: 442-448Crossref PubMed Scopus (193) Google Scholar), providing essential information to understand the effect of different mutations on the architecture of the protein. The enzyme is structured in three domains, a flexible N-terminal regulatory domain (residues 1–110), a α-helical rich catalytic domain (residues 111–410), and an oligomerization domain (residues 411–452), which includes a tetramerization motif at the extreme C-terminal end (residues 428–452).The aim of this work has been to provide more information about the effect of three point mutations (L348V, S349L, and V388M) on PAH function, structure, and subunit interaction. Mutation L348V has been reported to have 25–33% residual activity in COS cells (3Nowacki P. Byck S. Prevost L. Scriver C.R. Nucleic Acids Res. 1998; 26: 220-225Crossref PubMed Scopus (45) Google Scholar) and is an example of inconsistent genotype-phenotype correlations, as it is associated with different phenotypes in functionally hemizygous patients (5Gulberg P. Rey F. Zschocke J. Romano V. Francois B. Michiels L. Ullrich K. Hoffmann Burgard P. Schmidt H. Meli C. Riva E. Dianzani I. Ponzone A. Rey J. Guttler F. Am. J. Hum. Genet. 1998; 63: 71-79Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). V388M is one of the most frequent mutations in Spain, and we have shown that it retains 43% activity in COS cells (16Desviat L.R. Pérez B. De Lucca M. Cornejo V. Schmidt B. Ugarte M. Am. J. Hum. Genet. 1995; 57: 337-342PubMed Google Scholar), although normal levels of mutant immunoreactive proteins were detected. This apparent catalytic effect is discrepant with the fact that V388M affects residues located outside the active site in the three-dimensional structure of the PAH enzyme (13Erlandsen H. Fusetti F. Martinez A. Hough E. Flatmark T. Stevens R.C. Nat. Struct. Biol. 1997; 4: 995-1000Crossref PubMed Scopus (175) Google Scholar). On the contrary, S349L is located lining the active site, but expression both in COS cells and in Escherichia coli rendered a highly unstable protein (17De Lucca M. Pérez B. Desviat L.R. Ugarte M. Hum. Mutat. 1998; 11: 354-359Crossref PubMed Scopus (12) Google Scholar). To clarify and extend these results, we have used several complementary expression systems (eukaryotic and prokaryotic) and experimental conditions (co-overexpression with chaperonins, different growth temperatures), providing evidence that the mutations affect directly the folding and stability of the protein. Additionally, we observe that the S349L mutation also affects the catalytic properties of the enzyme, which is attributable to the fact that serine 349 is involved in the iron binding site. Mutant PAH subunit interaction has been examined by the two-hybrid system in mammalian cells, and altered oligomerization is documented by size exclusion chromatography of mutant PAH expressed in the E. coli system. The different experimental approaches employed allow the demonstration of a major folding defect of the mutations causing protein instability, which can be modulated experimentally, revealing a possible mechanism to account for the existing inconsistencies in the genotype-phenotype correlations.EXPERIMENTAL PROCEDURESExpression analysis was performed in COS cells using the pRc/CMV expression vector (Invitrogen), as described previously (16Desviat L.R. Pérez B. De Lucca M. Cornejo V. Schmidt B. Ugarte M. Am. J. Hum. Genet. 1995; 57: 337-342PubMed Google Scholar), and inE. coli using pMALc2 (Biolabs) where PAH is cloned as a fusion protein with MBP under the control of an inducible promoter (17De Lucca M. Pérez B. Desviat L.R. Ugarte M. Hum. Mutat. 1998; 11: 354-359Crossref PubMed Scopus (12) Google Scholar, 18Martinez A. Knappskog P.M. Olafsdottir S. Doskeland A.P. Eiken H.G. Svebak R.M. Bozzini M. Apold J. Flatmark T. Biochem. J. 1995; 306: 589-597Crossref PubMed Scopus (163) Google Scholar). Mutations were introduced in the PAH cDNA sequence by site-directed mutagenesis using the Gene Editor kit from Promega. COS cells (4 × 106 or 6 × 105), grown at 37 or 27 °C were transfected with the Lipofectin reagent (Life Technologies, Inc). The plasmid pGroESL bearing the GroES and GroEL genes and the chloramphenicol resistance marker was from DuPont. The plasmids pMALc2-PAH wild-type or pMALc2-PAH mutant were cotransformed with pGroESL into E. coli JM109, and the colonies were selected using LB plates with ampicillin (0.1 mg/ml) and chloramphenicol (0.1 mg/ml). Cells were grown at 37 °C, and expression of MBP·PAH fusion proteins and of GroES and GroEL was induced by the addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside. At the same time, 0.2 mm ferrous ammonium sulfate was added. Bacteria were harvested 16–21 h after induction and disrupted by sonication in Na-Hepes 20 mm, NaCl 0.2M, pH 7.0, with 1 mg/ml lysozyme and 0.2 mm Pefabloc. After centrifugation, the supernatant (crude protein extract) was used to measure PAH residual activity by monitoring conversion of 14C-Phe to 14C-Tyr (17De Lucca M. Pérez B. Desviat L.R. Ugarte M. Hum. Mutat. 1998; 11: 354-359Crossref PubMed Scopus (12) Google Scholar). Briefly, the standard reaction mixture performed in a 50-μl final volume contained 150–300 μg of total protein, catalase (10 μl at a concentration 100 units/μl), 14C-Phe (0.5 μCi, >450 mCi/mmol), and 10 μl of 1 mm6-methyltetrahydropterin (synthetic cofactor, added last), in 20 mm Na-Hepes, 0.2 m NaCl, pH 7. After 1 h at 37 °C, the reaction was stopped by boiling for 5 min and centrifugation at 10,000 × g for 5 min. A 6-μl sample of the supernatant was spotted onto a TLC plate, developed two times in chloroform:methanol:amonia (55:35:10), dried, and visualized by autoradiography.Wild-type and mutant fusion proteins were purified in an amylose column equilibrated with 20 mm Na-Hepes, 0.2 m NaCl, pH 7.0, and eluted with buffer containing 10 mm maltose (18Martinez A. Knappskog P.M. Olafsdottir S. Doskeland A.P. Eiken H.G. Svebak R.M. Bozzini M. Apold J. Flatmark T. Biochem. J. 1995; 306: 589-597Crossref PubMed Scopus (163) Google Scholar). PAH activity was also measured in the purified fraction, using 30–60 μg of protein. Size exclusion chromatography of the purified fusion proteins was performed at 4 °C following the conditions described (18Martinez A. Knappskog P.M. Olafsdottir S. Doskeland A.P. Eiken H.G. Svebak R.M. Bozzini M. Apold J. Flatmark T. Biochem. J. 1995; 306: 589-597Crossref PubMed Scopus (163) Google Scholar) and using a HiLoad Superdex 200HR column (1.6 cm × 60 cm) prepacked from Amersham Pharmacia Biotech. The fast protein liquid chromatography system, UV monitor, and recorder were all from Amersham Pharmacia Biotech. Assignation of the different enzyme forms to the peaks obtained in the chromatograms was done by comparison with previously published elution positions of tetramers and dimers of the fusion protein (18Martinez A. Knappskog P.M. Olafsdottir S. Doskeland A.P. Eiken H.G. Svebak R.M. Bozzini M. Apold J. Flatmark T. Biochem. J. 1995; 306: 589-597Crossref PubMed Scopus (163) Google Scholar) and by molecular mass value estimation using calibration curves obtained by running the following standard proteins, obtained from Amersham Pharmacia Biotech: ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). Blue dextran and acetone were used to determine the void volume (V0 = 44.06 ml) and the exclusion volume (VT = 114.9 ml), respectively.The cleavage of PAH protein from the MBP fusion partner was performed with Xa factor (ratio protein:Xa factor 1:100) at 4 °C for 3 h. The reaction was further incubated at 4 or 37 °C, and aliquots were removed at different times up to 24 h. After SDS-PAGE and Coomassie Blue staining, MBP and PAH proteins were quantified by laser densitometry.The TnT-T7 transcription-translation system from Promega was used for pulse-chase experiments. The wild-type and mutant PAH cDNAs cloned in the pRc/CMV vector were amplified using a sense primer that introduces the T7 promoter and the consensus Kozak sequences close to the ATG initiation codon (5′-TAATACGACTCACTATAGGGAGCCACCATGTCCACTGCGGTCCTGGAA-3′). Five microliters of the polymerase chain reaction product from the wild type and mutant cDNAs were mixed with the reticulocyte lysate and [35S]methionine-cysteine (14.3 mCi/ml). After a 35-min incubation at 30 °C the reaction was stopped with excess cold methionine, RNase (1 mg/ml), and DNase (1 mg/ml). The whole reaction was incubated at 37 °C, and aliquots were removed at different times between 1 and 8 h. All samples were separated by denaturing polyacrylamide gel electrophoresis, and the labeled PAH protein was quantitated by laser densitometry after fluorography.Thermal stability profiles were performed with purified fusion proteins MBP·PAH expressed in E. coli. Aliquots (20 μl, containing 30–60 μg of purified protein) were incubated at different temperatures for 10 min and chilled on ice. PAH enzyme activity was subsequently measured as described above.For two-hybrid analysis we have used the Mammalian Matchmaker two-hybrid assay kit (CLONTECH). The plasmid pGL-G5 (kindly provided by P. Stäheli) was used as reporter vector containing the luciferase gene under the control of the Gal4 promoter. The full-length human PAH cDNA was excised from phPAH247 (19Kwok S.C.M. Ledley F.D. DiLella A.G. Robson K.J.H. Woo S.L.C. Biochemistry. 1985; 24: 556-561Crossref PubMed Scopus (236) Google Scholar) withSmaI and EcoRI and subcloned into pBluescript. To introduce the normal PAH as fusion protein to the GAL4 (binding domain, BD) and VP16 (activating domain, AD) proteins, the PAH cDNA was excised from pBluescript with BamHI and XbaI and ligated with the AD and BD vectors previously digested with the same enzymes. Both plasmids with the normal cDNA were sequenced using a fmol sequencing kit (Promega) to confirm the in-frame cloning. The GeneEditor in vitro site-directed mutagenesis system from Promega was used to introduce the L348V, S349L, and V388M mutations in the PAH sequence. COS cells were plated in 6-well plates at a density of 4 × 105 cells/well. In each transfection 1 μg of each plasmid (reporter vector, AD-PAH and BD-PAH vectors) was introduced using Lipofectin reagent (Life Technologies, Inc.). The cells were harvested after 72 h, and luciferase activity was measured. In the transfection experiments using pRc/CMVPAH or in the two-hybrid system, PAH proteins were detected by Western blot using PH8 anti-PAH monoclonal antibody (20Hufton S.E. Jennings I.G. Cotton R.G.H. Biochim. Biophys. Acta. 1998; 1382: 295-304Crossref PubMed Scopus (20) Google Scholar).For the homology modeling, three-dimensional models of L348V, S349L, and V388M were built on the basis of the human phenylalanine hydroxylase coordinates determined by x-ray crystallography (13Erlandsen H. Fusetti F. Martinez A. Hough E. Flatmark T. Stevens R.C. Nat. Struct. Biol. 1997; 4: 995-1000Crossref PubMed Scopus (175) Google Scholar, 14Fusetti F. Erlandsen H. Flatmark T. Stevens R.C. J. Biol. Chem. 1998; 273: 16962-16967Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar,15Kobe B. Jennings I.G. House C.M. Michell B.J. Goodwill K.E. Santarsiero B.D. Stevens R.C. Cotton R.G.H. Kemp B.E. Nat. Struct. Biol. 1999; 6: 442-448Crossref PubMed Scopus (193) Google Scholar) (Protein Data Bank accession codes 1PAH and 2PAH). In the respective models, the formerly introduced alanine residues at the corresponding mutation sites were exchanged for the correct residues. The models were optimized for stereochemistry and refined by energy minimization using X-PLOR (21Brunger A.T. X-PLOR Version 3.1, a System for X-ray Crystallography and NMR. Yale University Press, New Haven1992Google Scholar). The energy-minimized structures were heated at 1000 K and were further refined with a slow cooling simulated annealing molecular dynamics protocol using X-PLOR.RESULTSAs described in a preliminary report, we have overexpressed and affinity purified PAH protein as fusion protein with MBP (17De Lucca M. Pérez B. Desviat L.R. Ugarte M. Hum. Mutat. 1998; 11: 354-359Crossref PubMed Scopus (12) Google Scholar). PAH with the S349L mutation produced an unstable protein undetectable in SDS-PAGE. The expression of this mutation in eukaryotic cells showed that the mutant enzyme was not detected by Western blot, and consequently no residual activity in bacteria nor in COS cells could be measured (17De Lucca M. Pérez B. Desviat L.R. Ugarte M. Hum. Mutat. 1998; 11: 354-359Crossref PubMed Scopus (12) Google Scholar). In this work we have extended the expression analysis to two other mutations, L348V and V388M, which have been previously reported to retain residual activity in COS cells, 25–33% for L348V (3Nowacki P. Byck S. Prevost L. Scriver C.R. Nucleic Acids Res. 1998; 26: 220-225Crossref PubMed Scopus (45) Google Scholar) and 43% for V388M (16Desviat L.R. Pérez B. De Lucca M. Cornejo V. Schmidt B. Ugarte M. Am. J. Hum. Genet. 1995; 57: 337-342PubMed Google Scholar). Based on the emerging view that many missense mutations in human disease cause defective folding resulting in protein instability (22Bross P Corydon T.J. Andresen B.S. Jorgensen M.M. Bolund L. Gregersen N. Hum. Mutat. 1999; 14: 186-198Crossref PubMed Scopus (191) Google Scholar), we have tested this hypothesis using different experimental approaches known to prevent missfolding of proteins expressed in eukaryotic and prokaryotic systems.Prokaryotic Expression StudiesIn prokaryotes, PAH was expressed as a fusion protein with MBP. Expression of the mutant proteins compared with the wild type resulted in a variable but lower yield of fusion protein. When we performed the co-overexpression of the plasmid pGroESL, there was a considerable increase in the amount of fusion protein, both for wild type and mutant PAH, although the effect was more pronounced for the mutant proteins, especially for PAH harboring S349L, which as described before, was undetectable without chaperonin coexpression (Fig. 1). Thus, high levels of GroES and GroEL have a clear stabilizing effect on the mutant proteins, revealing a primary defect in folding and/or oligomer assembly. The increase in mutant protein correlated with an increase in residual catalytic activity with chaperonin coexpression, except for the S349L mutation, for which no enzyme activity is rescued (Table I).Table IExpression analysis of PKU mutations in different systemsPAHExpression in E. coli (−GroESL)Expression in E. coli (+GroESL)Expression in COS cellsTwo hybrid system interactionActivityOligomeric state (proportion)ActivityOligomeric state (proportion)37 °C27 °CActivityProteinActivityProtein%%%%%%%Wild-type100Aggregates (27%)100Aggregates (20%)100100100100100Tetramers (60%)Tetramers (66%)Dimers (13%)Dimers (14%)L348V25Aggregates (35%)55Aggregates (53%)3870778273Tetramers (28%)Tetramers (34%)Dimers (37%)Dimers (13%)S349L0No protein0Aggregates (100%)0009010V388M40Aggregates (59%)82Aggregates (34%)4398788291Tetramers (18%)Tetramers (50%)Dimers (23%)Dimers (17%)Data obtained from the expression analysis of wild-type and mutant forms of PAH in E. coli with and without chaperonins, in COS cells at different temperatures and in a two-hybrid system. Oligomeric state and proportion (percentage of total) of fusion proteins in E. coli were estimated by size exclusion chromatography (see Fig. 2 and “Experimental Procedures”). In the two-hybrid assay, the relative strength of interaction between PAH subunits was measured as luciferase activity, 100% interaction is the luciferase activity when wild-type PAH cDNA is present on both activating and binding domain; in the rest of the assays, the mutations are present on the activation domain. The data represent the mean of three independent experiments. Open table in a new tab The oligomeric state of the fusion proteins was analyzed by size exclusion chromatography. It has previously been described that the oligomeric structure of PAH is similar as fusion protein with MBP and as isolated enzyme (18Martinez A. Knappskog P.M. Olafsdottir S. Doskeland A.P. Eiken H.G. Svebak R.M. Bozzini M. Apold J. Flatmark T. Biochem. J. 1995; 306: 589-597Crossref PubMed Scopus (163) Google Scholar, 23Knappskog P.M. Flatmark T. Aarden J.M. Haavik J. Martinez A. Eur. J. Biochem. 1996; 242: 813-821Crossref PubMed Scopus (82) Google Scholar), and our results also show that the wild-type fusion protein is resolved into three main components, a fraction of high molecular mass aggregates, eluting at the column void volume, a major fraction corresponding to tetramers, and a minor component of dimers. With chaperonin coexpression, there is no substantial change in the oligomeric profile of the wild-type protein (Fig. 2). Regarding the mutant proteins, L348V and V388M fusion proteins show a much lower proportion of tetramers and increased amounts of aggregated forms. When V388M is coexpressed with GroES and GroEL, a major peak corresponding to the tetrameric form is observed with a concomitant decrease in aggregated forms. For L348V with chaperonins the proportion of tetramers also increases, although there is still a considerable amount of aggregates (Fig. 2). In contrast, the S349L protein rescued by chaperonin coexpression is exclusively recovered as aggregates.Figure 2Size exclusion chromatography on a HiLoad Superdex column of affinity purified MBP· PAH fusion proteins expressed with or without chaperonins. The column was equilibrated and eluted with 20 mm Hepes and 0.2 m NaCl, pH 7.0, at a flow rate of 0.38 ml/min. Peak positions: thearrow points to the position of the tetramers. Retention times were 107–116 min, aggregated forms; 137–139 min, tetrameric form; and 155–158 min, dimeric form. For each MBP·PAH fusion protein, the following amount was loaded on the column: wild-type, 380 μg; wild-type (+ GroESL), 400 μg; V388M, 54 μg; V388M (+ GroESL), 170 μg; L348V, 13 μg; L348V(+ GroESL), 120 μg. For ease of comparison the sensitivity of the detector in the chromatograms of V388M and L348V without chaperonins was 5-fold greater than for the rest of the chromatograms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to impaired folding, the mutations could also be affecting the stability of the assembled enzyme. Therefore, we analyzed the thermal inactivation profiles for the L348V and V388M fusion proteins purified in the pMalc2 expression system. The curves of the mutant enzymes are clearly shifted to lower temperature, demonstrating a reduced stability (Fig. 3). The half-denaturation temperatures were 59 °C for the wild-type enzyme, 50 °C for L348V, and 51 °C for the V388M mutant protein. Similar denaturation profiles were observed if the enzymes were expressed with or without chaperonins.Figure 3Thermal inactivation profiles of normal and mutant (L348V and V388M) PAH enzymes. Aliquots of the affinity purified fusion proteins were incubated at various temperatures for 10 min and chilled on ice. PAH enzyme activity was subsequently measured. The residual enzyme activities (percentage of the maximum value obtained) are plotted versus the incubation temperature. The mean values from two independent experiments each with fusion protein expressed with (panel B) or without (panel A) chaperonins are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Another approach used to analyze the effect of the PAH mutations on the stability of the protein was performed after digestion with factor Xa of the purified normal and mutant fusion proteins. The cleaved proteins were subsequently incubated at 4 or 37 °C up to 24 h, and the Coomassie Blue-stained bands were quantified by laser densitometry. After cleavage, MBP and wild-type PAH are essentially stable up to 24 h, the ratio PAH/MBP is close to 1 up to 24 h. In contrast, immediately after cleavage, the amount of detectable mutant PAH forms, expressed as PAH/MBP ratio, is reduced to 50% (for V388M), 40% (L348V), or 20% (in the case of S349L). This remaining mutant protein is stable up to 24 h. Similar data are obtained if the fusion proteins are coexpressed with or without chaperonins (data not shown).Eukaryotic Expression StudiesTo test the relevance of these results obtained in the E. coli expression system, the mutations were also expressed in COS cells at different temperatures, 27 and 37 °C. At low temperature, S349L mutant protein could be detected by Western blot analysis, reaching near normal levels, although no activity was rescued. Both L348V and V388M showed an increase in residual activity at 27 °C, from 38 to 77% and from 43 to 78%, respectively. Western blot analysis revealed similar levels of immunoreactive protein for the wild-type and mutant proteins expressed at both temperatures (Table I).Expression by in Vitro Tran" @default.
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• W2024293713 creator A5040246496 @default.
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• W2024293713 date "2000-09-01" @default.
• W2024293713 modified "2023-10-14" @default.
• W2024293713 title "Expression Analysis of Phenylketonuria Mutations" @default.
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