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PLENARY PAPER
From the Departments of Medicine and Biochemistry,
University of Utah School of Medicine, Salt Lake City.
Functional consequences of 12 mutations Subnormal activity of uroporphyrinogen
decarboxylase (URO-D) in hepatocytes is responsible for the most common
form of porphyria in humans, porphyria cutanea tarda
(PCT).1,2 PCT occurs with a prevalence of 1 to 5 per
25 0001,2 and is clinically characterized by skin
fragility, bullous lesions, and hypertrichosis on sun-exposed areas.
Photosensitivity is mediated by uroporphyrin and by partially decarboxylated porphyrins that accumulate in the liver, circulate in
the plasma, and are excreted in the urine.
Mutations at the URO-D locus, transmitted as an autosomal
dominant trait, are found in approximately one third of patients with
PCT.1,2 PCT associated with URO-D mutations is
designated familial PCT (F-PCT). Patients with F-PCT display
approximately half-normal URO-D activity and half-normal
concentration of URO-D protein in all tissues. More than 30 different
URO-D mutations have been identified in unrelated
patients.3-14 Most carriers of mutant URO-D
alleles do not express clinical phenotypes unless additional factors
are present that further reduce the activity of URO-D in the
liver.15,16 Factors shown to be important include alcohol
abuse, exposure to the hepatitis C virus, use of medicinal estrogens,
and development of hepatic siderosis (often caused by mutation of the
hemochromatosis gene).15,16 Depletion of hepatic iron
stores through phlebotomy therapy corrects the clinical and biochemical
manifestations of F-PCT.2
URO-D catalyzes the sequential decarboxylation of the 4 acetate side
chains of uroporphyrinogen to form coproporphyrinogen. Isomer I or
isomer III of uroporphyrinogen may serve as substrate, but only
coproporphyrinogen III is a substrate for the next enzyme in the
pathway, coproporphyrinogen oxidase. The mechanism of decarboxylation is unknown, but the enzyme does not require cofactors or prosthetic groups for activity. Human URO-D is a homodimer with a monomeric molecular weight of approximately 41 000 d.17 The protein
is encoded by a single gene located on chromosome 1, 1p34.18,19 The URO-D gene spans 3.5 kb and contains 10 exons.20 The crystal structure of human
URO-D has been determined at 0.16 nm (1.6 Å) resolution.21 Structurally, URO-D is a member of the
Patients
Sequencing of URO-D loci
Expression of URO-D proteins Missense mutations were reconstructed in an Escherichia coli expression system. Mutations were introduced into a wild-type URO-D cDNA using the pAlter (Promega, Madison, WI) site-directed mutagenesis system.17 Mutated URO-D genes were subcloned into an expression plasmid, allowing incorporation of a histidine tag at the amino terminus. Recombinant proteins were then purified using metal-chelate affinity chromatography (Qiagen, Valencia, CA),17 yielding 2 to 4 mg highly purified URO-D from a 1-L bacterial culture. Recombinant proteins were approximately 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. Wild-type URO-D protein from this expression system had the same specific activity as URO-D purified from human red blood cells.17Crystallization and structural analysis Three mutant URO-D proteins (G156D, F232L, I260T) were purified and concentrated to 6 mg/mL in 50 mM Tris, pH 7, 10% glycerol, 1 mM -mercaptoethanol. These proteins were crystallized by slow equilibration in a buffer containing methylpentanediol as
described.17 Crystals were isomorphous with those used
previously to determine the crystal structure of URO-D.21
Radiographic diffraction data were collected from crystals maintained
at 100°K. Diffraction data from home and synchrotron
radiographic sources were processed and scaled with Denzo and
Scalepack (Hill Research, Charlottesville, VA).24 The
structure of native URO-D (PDB code, 1URO)21 was used as
the starting point to phase diffraction data obtained from crystals of
mutant proteins. Models were refined with X-Plor25 and
rebuilt with the program O (Table
2).26
Enzymatic activity assays for URO-D Enzymatic activities of recombinant proteins were assayed as previously described.23 Reaction products were separated and quantified by high-performance liquid chromatography as described.22Growth of Epstein-Barr virus-transformed lymphocytes Lymphocytes were isolated from venous blood and transfected with Epstein-Barr virus (EBV) as described by Pelloquin et al.27 Transformed cells were cultured in suspension at 37°C, 5% CO2, in RPMI 1640 with L-glutamine, 20% fetal calf serum and 50 µg/mL gentamicin (Life Technologies, Frederick, MD). Cells were passaged twice a week. Steady- state levels of URO-D protein in transformed cell lines were determined by Western blotting using a monospecific polyclonal rabbit anti-URO-D antibody.28 An antibody to the B56 subunit of-phosphatase29 was used to normalize for protein load.
RNA levels by ribonuclease protection assays Total RNA was extracted from 1 × 107 EBV-transformed lymphocytes using Trizol reagent (Life Technologies). URO-D transcript levels were determined from 30 µg total cellular RNA using the Ambion ribonuclease protection assay system (RPA II; Austin, TX). An antisense RNA probe to URO-D mRNA protected a 376-base fragment at the 5' end of the message and was used in conjunction with a control probe that protected a 125-base region of human-actin mRNA. Probes were hybridized with the sample
RNA for 6 hours, then digested for 1 hour with RNase reagents supplied
with the Ambion RPA II system. Protected fragments were then separated on a 5% polyacrylamide gel. Gels were dried and exposed to film for 30 minutes to 4 days to obtain proper exposures. The relative level of
URO-D mRNA was quantified using densitometry measured with a
BioRad Molecular Imager FX equipped with the Quantity One software
package (BioRad, Hercules, CA).
Immunoblot analysis Protein was extracted from 2 × 106 EBV-transformed cells after centrifugation (1000g, 15 minutes) and 2 washes in 1 × phosphate-buffered saline (PBS) (10 × PBS = 1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4, 14 mM KH2PO4, pH 7.5). Cells were sonicated for 2 minutes on ice and then spun at 10 000g for 10 minutes at 4°C. Supernatants were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Filters were blocked with 5% nonfat dry milk, 1 × PBS, and 0.1% Tween 20 for 30 minutes, followed by 2-hour incubation with a rabbit anti-human URO-D antibody28 diluted 1:8000 and a rabbit anti-B56 phosphatase 2A subunit antibody29 diluted 1:2500 at room temperature. Blots were washed 3 × 15 minutes with 1 × PBS and 0.1% Tween 20 at room temperature. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG)-diluted 1:5000 in the blocking buffer. Blots were developed using the Renaissance Western Chemiluminescence reagent (New England Nuclear, Boston, MA) and exposure to film. The relative level of URO-D protein was quantified by densitometry as described above.
Identification of mutations URO-D gene sequences from patient DNA samples were compared to the published sequence of URO-D (GenBank accession number AF047383). All mutations were confirmed by repeating both the PCR and the sequencing procedures. We identified 12 different mutations in 14 unrelated patients with F-PCT (Table 3). Six of the 12 mutations have been reported elsewhere, and 2 were detected in more than one proband (Table 3). The mutations resulted in altered restriction enzyme digestion patterns with one exception, a mutation characterized by insertion of a thymidine in codon 218 (Glu218insT). These mutations were easily detected by digesting one of the PCR products (Table 3, Figure 1). Detection of the Glu218insT mutation required sequencing the exon 7-9 PCR product (Table 1). Representative results of restriction fragment length polymorphisms for 10 of the mutations are shown in Figure 1.
Catalytic activity of recombinant URO-D mutants The 10 missense mutations were engineered into an E coli expression system. Seven of the recombinant proteins were stable and soluble. The other 3 proteins were expressed but were detected only in inclusion bodies and could not be solubilized. Enzymatic activity of the soluble purified proteins ranged from approximately 29% to 94% of wild-type URO-D (Table 3).Steady-state levels of URO-D mRNA and protein in transformed lymphocytes EBV-transformed cell lines were generated for only 10 of the mutations because lymphocytes from the probands with the Phe232Leu and Leu253Gln mutations failed to transform. Total RNA was prepared from cells and assayed for steady-state levels of URO-D mRNA using a ribonuclease protection assay (see "Patients and methods"). The quantity of URO-D mRNA in cells from patients with PCT was compared with cell lines derived from 6 nonporphyric patients with hemochromatosis. In the cell line with the insertion mutation (Glu218insT), the URO-D mRNA level was approximately half normal, indicating that transcription of the wild-type allele was not up-regulated (Figure 2). All other mutant cells lines contained normal levels of URO-D mRNA, indicating that transcripts were generated from both wild-type and mutant alleles. The splice mutant generated a stable, truncated transcript as previously reported.30 URO-D protein levels varied from 19% to 75% of control values (Figure 2). Subnormal amounts of URO-D protein in the presence of normal levels of URO-D mRNA indicated that mutant proteins were probably unstable or insoluble.
Structure of recombinant mutants Three URO-D mutants (Gly156Asp, Phe232Leu, Ile260Thr) were produced in sufficient quantity for crystallization. Radiographic structures were compared with the wild-type structure to determine the structural effects of the mutations (Table 2). All mutant structures contained a common structural change, the disorder of a surface loop between HB and H1 (Figure 3A, residues 58-70).21 The structural explanation for this result was not apparent because this loop was remote from any of the mutations.
The most dramatic effect of a point mutation was seen with glycine 156, an absolutely conserved residue located on strand 3 of the Crystal structures of the Phe232Leu and Ile260Thr mutants revealed no significant local perturbations surrounding the mutations, even though recombinant mutant proteins had reduced catalytic activity (Table 3). In the native structure, Phe232 is partially solvent accessible. In spite of only a minor structural change, the activity of recombinant Phe232Leu URO-D was reduced to 43% of wild type (Table 3). In the Ile260Thr structure, the extra carbon atom in isoleucine is replaced by a water molecule that forms a hydrogen bond to the threonine side chain. This relatively conservative mutation results in only a modest reduction of catalytic activity (Table 3). Other URO-D mutations Thirty URO-D mutations have been reported elsewhere.3-14 Ten of these mutant alleles are predicted to generate shorter proteins as a result of a premature stop codon or a variation in a splice site.3-13 The remaining 20 previously reported mutations are missense mutations listed in Table 4 and mapped onto the URO-D monomer in Figure 3C. The structural location of these mutations and the predicted structural effects are described in Table 4.
The structure of URO-D consists of a TIM (triosephosphate
isomerase) barrel with 8 central Two of the mutants we identified encode truncated proteins. In the Glu218insT mutation, the insertion of a thymidine in codon 218 introduces a stop codon (TGA) in place of the wild-type codon (GAG) for glutamic acid. EBV-transformed cells from the patient with this mutation were from the only cell line studied that had approximately half-normal levels of URO-D mRNA (Figure 2). This finding suggests that the mutant transcript is rapidly degraded through a nonsense-mediated decay mechanism.31,32 The exon 6 splice mutation, previously described,7,30 produces a truncated but stable mRNA. Deletion of exon 6 does not disrupt the reading frame, but the mutant protein is catalytically inactive and unstable.30 An EBV-transformed line from the proband with this mutation contained normal amounts of URO-D mRNA but half-normal amounts of protein (Figure 2). Three mutations identified in this study Three missense mutations (Ala80Ser, Gly156Asp, Phe232Leu) yielded
recombinant URO-Ds with catalytic activities of approximately 40% or
less. The Ala80Ser mutation was previously detected by Brady et
al,12 but properties of the mutant enzyme were not determined. A second mutation at the same residue, Ala80Gly, has been
described by McManus et al,9 who reported the catalytic activity of recombinant Ala80Gly URO-D to range from 19% to 39% of
normal, values similar to the 33% activity we found for recombinant Ala80Ser URO-D. Ala80 is located at the N-terminal end of the S2
The crystal structure of the Gly156Asp mutant revealed that this mutation perturbed nearby residues on the surface of the active site cleft (Figure 3B), thereby providing a plausible explanation for the reduced activity. In contrast, the crystal structure of the Phe232Leu mutant shows that this mutation, distant from the active site, did not alter the structure. It is unclear why the substitution of a leucine at this position led to reduced activity; leucine occupies this position in the URO-D of Caulobacter cresentus.21 Two mutations caused only a modest reduction of catalytic activity in the recombinant proteins Leu253Gln and Ile260Thr. Residue 253 is located at the transition between helix 4 and strand 5. The sequence of URO-D in this region varies considerably between species but is conserved in mammals.21 In contrast to our finding of 75% normal activity in purified recombinant Leu253Gln URO-D, McManus et al9 reported the identical mutant and found a marked reduction in activity of the mutant enzyme in a crude bacterial extract. The reason for this discrepancy is unclear. Although residue 253 is remote from the active site, the mutation results in the substitution of a hydrophilic glutamine residue for the hydrophobic, aliphatic leucine, a change that might have significant structural effects. The crystal structure of the Ile260Thr mutant revealed that the void generated by removal of the terminal isoleucine methyl is filled by incorporation of a buried water molecule, a change that has little effect on the overall structure but reduces catalytic activity by 40%. Two missense mutations yielded recombinant URO-D with near normal catalytic activity, Val134Gln and Glu167Lys. Both have been previously described. Meguro et al4 also found near normal activity for the Val134Gln recombinant protein. The substitution of a glutamine for valine at position 134 resulted in the insertion of a polar residue in a hydrophobic pocket on helix 2 at a location remote from the active site. Catalytic activity of the Glu167Lys has not been previously measured, but the recombinant mutant URO-D has been shown to be rapidly degraded.6 Glutamic acid normally at position 167 links residues in a surface loop. The charge reversal resulting from substitution of a lysine would alter the structure of this surface loop and might target the protein for degradation. URO-D is a dimer in the crystal21 and in
solution.17 Formation of the dimer interface is critically
important Patients with F-PCT are heterozygous for mutant URO-D alleles and theoretically could produce 3 dimeric proteins: the wild-type homodimer, the mutant homodimer, and a mutant-wild-type heterodimer. The observation that URO-D activity in erythrocyte lysates from patients with F-PCT usually approximates 50% is consistent with the conclusion that most clinically relevant mutations yield unstable or unfolded proteins that are unable to form dimers. It is possible that the occasional detection of URO-D activity of 40% or less13 reflects formation of mutant-wild-type heterodimers with reduced catalytic activity or altered stability. Normal levels of URO-D mRNA were detected in EBV-transformed lines (with the exception of the mutation causing the premature stop codon), yet URO-D protein levels varied widely. This is surprising in view of the half-normal protein levels found in red blood cells, suggesting that the capacity of the proteolytic machinery capable of recognizing mutant URO-D genes differs between these 2 cell types. URO-D is not a rate-limiting step in the porphyrin biosynthetic pathway,1,2 and most carriers of mutant URO-D alleles do not express a clinical phenotype. Homozygosity for URO-D null alleles is lethal,33 but homozygosity or compound heterozygosity for mutant alleles that encode URO-D genes with some residual activity can be tolerated.1,2 In homozygotes or compound heterozygotes, the porphyric phenotype is pronounced and is usually evident in early childhood.2 The incidence of mutant URO-D alleles in the population is unknown, but it is unlikely to approach the approximately 30% incidence we and others observe in patients with PCT.2,15 The conclusion that heterozygosity for mutant URO-D alleles is a risk factor for the development of PCT15 is supported by the observation that heterozygous mice are more sensitive to porphyrinogenic stimuli than wild-type animals.33 In heterozygous mice and humans displaying porphyric phenotypes, hepatic URO-D protein level is half-normal, but catalytic activity is reduced to approximately 20%.33-35 This finding strongly suggests that an inhibitor of hepatic URO-D is generated when the porphyric phenotype becomes manifest. The mechanism by which mutant URO-D alleles cause predisposition to the development of a URO-D inhibitor remains unresolved.
Submitted June 4, 2001; accepted July 25, 2001.
Supported by National Institutes of Health grants R-37 DK20503, RO-1 GM56775, MO-1 RR00064, and P-30 CA42014.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: John D. Phillips, Division of Hematology, University of Utah School of Medicine, 50 North Medical Dr, Salt Lake City, UT 84132; e-mail: john.phillips{at}hsc.utah.edu.
1. Wyckoff EE, Kushner JP. Heme biosynthesis, the porphyrias, and the liver. In: Arias IM,Boyer JL,Fausto N,Jakoby WB,Schachter DA,Shafritz DA, eds. The Liver: Biology and Pathobiology. 3rd ed. New York, NY: Raven Press; 1994:505-527. 2. Kappas A, Sassa S, Galbraith RA, Nordmann Y. The porphyrias. In: Scriver CR,Beaudet AL,Sly WS,Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. Vol 2. 7th ed. New York, NY: McGraw-Hill; 1995:2103-2160. 3. Moran-Jimenez MJ, Ged C, Romana M, et al. Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria. Am J Hum Genet. 1996;58:712-721[Medline] [Order article via Infotrieve]. 4. Meguro K, Fujita H, Ishida N, et al. Molecular defects of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria. J Invest Dermatol. 1994;102:681-685[CrossRef][Medline] [Order article via Infotrieve].
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de Verneuil H, Grandchamp B, Beaumont C, Picat C, Nordmann Y.
Uroporphyrinogen decarboxylase structural mutant (Gly281 6. Romana M, Grandchamp B, Dubart A, et al. Identification of a new mutation responsible for hepatoerythropoietic porphyria. Eur J Clin Invest. 1991;21:225-229[Medline] [Order article via Infotrieve].
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Garey JR, Hansen JL, Harrison LM, Kennedy JB, Kushner JP.
A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda.
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9.
McManus JF, Begley CG, Sassa S, Ratnaike S.
Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria.
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1996;88:3589-3600 10. Christiansen L, Ged C, Hombrados I, et al. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis: identification and characterization of six novel mutations associated with familial PCT. Hum Mutat. 1999;14:222-232[CrossRef][Medline] [Order article via Infotrieve]. 11. McManus JF, Begley CG, Sassa S, Ratnaike S. Three new mutations in the uroporphyrinogen decarboxylase gene in familial porphyria cutanea tarda [abstract]. Hum Mutat. 1999;13:412[Medline] [Order article via Infotrieve]. 12. Brady JJ, Jackson HA, Roberts AG, et al. Co-inheritance of mutations in the uroporphyrinogen decarboxylase and hemochromatosis genes accelerates the onset of porphyria cutanea tarda. J Invest Dermatol. 2000;115:868-874[CrossRef][Medline] [Order article via Infotrieve]. 13. Mendez M, Sorkin L, Rossetti MV, et al. Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles. Am J Hum Genet. 1998;63:1363-1375[CrossRef][Medline] [Order article via Infotrieve]. 14. Mendez M, Rossetti MV, Siervi AD, del Carmen Batlle AM, Parera V. Mutations in familial porphyria cutanea tarda: two novel and two previously described for hepatoerythropoietic porphyria. Hum Mutat. 2000;16:269-270[Medline] [Order article via Infotrieve].
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Bulaj ZJ, Phillips JD, Ajioka RS, et al.
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20.
Romana M, Dubart A, Beaupain D, et al.
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© 2001 by The American Society of Hematology.
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  A. K. Aarsand, H. Boman, and S. Sandberg Familial and Sporadic Porphyria Cutanea Tarda: Characterization and Diagnostic Strategies Clin. Chem., April 1, 2009; 55(4): 795 - 803. [Abstract] [Full Text] [PDF]
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 R. S. Ajioka, J. D. Phillips, R. B. Weiss, D. M. Dunn, M. W. Smit, S. C. Proll, M. G. Katze, and J. P. Kushner Down-regulation of hepcidin in porphyria cutanea tarda Blood, December 1, 2008; 112(12): 4723 - 4728. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
10 missense, 1 splicing
defect, and 1 frameshift mutation
8/
2Fo-Fc) covering the refined structure of
Gly156Asp URO-D. The Gly156Asp structure is shown in yellow, and the
wild-type URO-D structure is shown in green. The Asp side chain at
position 156 causes the movement in nearby residues, 85 to
87, toward the active site cleft (right side of figure). Two water
molecules (as depicted by the red difference density 
3
and 
angles that
favored only glycine with no obvious space to accommodate a larger side
chain. The Met165Arg mutation was previously described by Mendez et
al,
Glu) in a case of porphyria.
Science.
1986;234:732-734