Human Molecular Genetics Advance Access originally published online on July 5, 2007
Human Molecular Genetics 2007 16(18):2209-2214; doi:10.1093/hmg/ddm172
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In vitro demonstration of intra-locus compensation using the ornithine transcarbamylase protein as model
1 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal, 2 Faculty of Medicine, University of Porto, Porto, Portugal and 3 Department of Inorganic, Physical and Material Chemistry, University of Torino, Torino, Italy
* To whom correspondence should be addressed at: Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), rua Dr Roberto Frias, s/n 4200-465 Porto, Portugal. Tel: +351 225570730; Fax: +351 225570799; Email: gsuriano{at}ipatimup.pt
Received May 16, 2007; Accepted July 2, 2007
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ABSTRACT |
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Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn defect of metabolism of the urea cycle, which causes hyperamonemia. Mutations of the OTC gene have been recognized as the genetic cause underlying the OTC deficiency. The severity of the disease is associated with the type of mutation, leading either to neonatal onset of hyperammonemia or to a later appearance of the disease. The mutation Thr125Met is associated with neonatal hyperammonemia. Recently, the disease-causing Thr125Met mutation in humans was reported as wild-type neutral allele in chimpanzees. Further analysis confirmed the presence of Met125 fixed in chimpanzees together with Thr135, representing the only two divergent positions between human and chimpanzee OTCs. Thr125 and Thr135 were identified as ancestral mammalian combination, so the Thr135Ala substitution occurred as human-specific event, whereas the substitution of Thr125Met was characteristic of the chimpanzee linage. Only when Met125 emerges in a background with the human-specific Ala135, a highly deleterious effect is observed, suggesting among other hypotheses the existence of a compensatory effect in chimpanzee. To explore this hypothesis, we built an in vitro cell model system to study the effect of the three distinct genetic backgrounds (Ala135–Thr125; Ala135–Met125 and Thr135–Met125) on the OTC protein function. We observed that the human Thr125Met mutant is inactive, whereas the chimp OTC shows an enzymatic activity comparable with the wild-type human OTC. We concluded that the presence of a threonine at position 135 in chimps rescues the deleterious effect of the methionine at position 125, in a mechanism of intra-locus compensation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ornithine transcarbamylase (OTC; EC 2.1.3.3 [EC] ) protein exerts its biological function in the mitochondria of ureatelic animals, where it catalyzes the conversion of L-ornithine and carbamyl phosphate into citrulline and phosphate. This mechanism is crucial for the elimination of otherwise toxic nitrogen compounds, which are formed upon de-amination of amino acids resulting from protein intake.
OTC deficiency (OTCD) is an X-linked inborn defect of metabolism of the urea cycle, which causes hyperamonemia (1,2). Mutations of the OTC gene have been recognized as the genetic cause underlying OTCD (3). More than 300 different mutations have been reported to date, which include point mutations, frameshift, small and gross deletions and insertions, evenly distributed along the gene (4,5). The severity of the disease appears to be strongly associated with the type of mutation, leading either to neonatal onset of hyperammonemia or to a later appearance of the disease. The mutation Thr125Met is associated with neonatal hyperammonemia, lethal in a carrier male within 14 days after birth (6). Accordingly, functional studies on liver extracts revealed complete loss of OTC activity. Recently, when the chimpanzee genome sequence was released, the disease-causing Thr125Met mutation in humans was reported as wild-type neutral allele in chimpanzees (7). We further investigated this finding and confirmed the presence of Met125 fixed in chimpanzees together with Thr135, representing the only two divergent positions between human and chimpanzee OTCs (8). Our survey of both positions in other mammals indicated Thr125 and Thr135 as ancestral mammalian combination (8), so the Thr135Ala substitution occurred as human-specific event, whereas the substitution of Thr125Met was characteristic of the chimpanzee linage. However, when the derived Met125, potentially deleterious, is associated with the ancestral Thr135 (chimps), no phenotypic effect is perceptible. Only when Met125 emerges in a background with the human-specific Ala135, a highly deleterious effect is observed, suggesting the existence of a compensatory effect in chimpanzee. To explore this hypothesis, we built an in vitro cell model system and characterized the effect of Met125 on the OTC activity on both the human and chimps backgrounds.
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RESULTS |
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Establishment of cell lines stably expressing the different OTC constructs
We chose an in vitro approach to verify the hypothesis that the human mutant Met125 allele would be compensated in chimpanzees by the Thr135 residue owing to intra-protein compensatory interactions. Chinese hamster ovary (CHO) OTC-negative cells were selected as cell model system. The human OTC complementary deoxyribonucleic acid (cDNA) was amplified from a colon cDNA library and cloned into a mammalian expression vector. We used site-directed mutagenesis to obtain the different OTC constructs (Fig. 1). Cell lines stably expressing human wild-type OTC, the T125M germline mutant, the chimp wild-type OTC and the ancestral OTC were obtained by retroviral infection followed by blasticidin selection. CHO cells retroviral infected with the empty vector (mock cells) were also established and used as negative control.
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We used both semiquantitative RT–PCR and real-time PCR to estimate the level of OTC expression in the established cell lines. The expression of the housekeeping gene GAPDH was used as reference for the RT–PCR. As shown in Figure 2A, in all cell lines but mock cells, a PCR product corresponding to the OTC cDNA was amplified. Densitometric analysis of the bands normalized to the GAPDH control suggested that all cell lines express comparable amounts of OTC mRNA.
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To confirm these findings, we estimated the OTC mRNA levels in the different established cell lines by quantitative real-time PCR. As reported in Figure 2B, human wild-type OTC-expressing cells showed 20% increased OTC mRNA levels in comparison with the other OTC-expressing cell lines.
These differences were taken into account for the protein activity measurements. Since no human OTC antibody is currently commercially available, we could not confirm protein expression by western blot analysis.
OTC activity measurements
Figure 3 shows the results of the activity measurements performed on the different OTC products. The activity assay is based on the photometric determination of the amount of a chromogen formed by condensation of the enzymatically generated citrulline with diacetyl monoxime, in the presence of catalytical amounts of Fe(III). Stability of the reaction product is strongly dependent on the time length of incubation at 95°C, on exposure to light and on the concentration of sulfuric acid in the chromogenic reagent. Under our experimental conditions, the product was stable for several hours in the dark, at 4°C. The activity calculation is normalized to a citrulline standard and allows discarding the contribution from basal levels of citrulline in lysates.
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Our results indicate that wild-type human OTC as well as the chimp wild-type OTC exhibit a comparable catalytic activity. Conversely, the human mutant Thr125Met appears inactive compared with the negative control (mock), confirming the deleterious impact of the single amino acid change on the protein activity. A clear dependence of the activity values from the quantity of OTC found in lysates was observed.
Altogether, these results support the hypothesis that the deleterious effect of the Thr125Met mutant in humans is likely compensated in chimps by compensatory mechanisms involving the second site change Ala135Thr, the only two divergent positions between human and chimp proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The human OTC gene (EC2.1.3.3) maps to Xp.21.1 and is expressed as an inactive precursor polypeptide containing an amino acid N-terminal signal sequence that is cleaved upon transport into the mitochondria, where the active homotrimeric enzyme is assembled (9,10).
As other X-linked proteins, the impairment in functionality (OTCD, MIM 311250 [OMIM] ) affects mainly males who, characteristically, manifest lethargy, vomiting and cerebral edema as a consequence of hyperammonemia (11). Often, OTCD males die during the first years of life, although some of them survive until adulthood with the adequate management of protein intake and nitrogen excretion by alternative routes (12). Females are rarely severely affected, presenting only mild symptoms associated with the hyperammonemic status, or, in some cases, being completely asymptomatic. This wide pattern of clinical consequences appears to be a direct consequence of the severity of the inherited mutations on the protein activity in most cases. In this regard, the most recent comprehensive update contained 341 mutations evenly distributed along the gene (3–5). Out of the 341 mutations, 149 were associated with neonatal onset of hyperammonemia (within the first week of life), 70 were seen in male patients with later onset of hyperammonemia and 121 were found in heterozygous females.
One of the confounding effects to the unambiguous definition of genotype–phenotype correlations is the presence of a human disease-associated mutation (Thr125Met) as wild-type (Met125) allele in chimpanzee (7,8). The Thr125Met mutation is associated with the neonatal onset of the disease in humans. Accordingly, functional studies on hepatocytes from affected carriers confirmed its deleterious effect responsible for the complete loss of protein activity. The description of human disease-associated residues present in other mammals as wild-type, apparently neutral, alleles is not uncommon (7,13,14). Gene–environment interactions could explain why the Met125 is compatible with chimpanzee’s life. Lower levels of protein intake in chimpanzees could mask the deleterious effect of this residue. In agreement with this, many OTCD carriers remain asymptomatic for a long time, and clinical manifestations occur only after high-proteic meals. However, chimps might have developed alternative routes for ammonia detoxification.
A third hypothesis, which we recently discussed (8), is epistatic interactions (compensation), by which the deleterious Met125 allele in chimpanzee would be compensated by the Thr135 allele, the only two divergent positions between human and chimp OTC. In this process, it is believed that the compensatory effect of a second-site substitution, within the same (intra-locus) or other proteins (inter-locus), may lead to the attenuation of the pathological effects imposed by otherwise disruptive residues (15,16). Supporting this hypothesis, neither gorillas nor orangutans (ancestral) share the same allele with the chimpanzee, since both carry a Thr at position 125 as humans. Furthermore, the residue Thr125 in the human OTC belongs to a domain that was shown to be pivotal for the correct assembling of the active homotrimer. It was suggested that the replacement of the Thr by a Met would disrupt this domain, thus hampering the clustering of the homotrimer (8), an effect that could be partially rescued by the presence of a Thr at position 135.
The main question to be addressed to explain the presence of the Met125 as wild-type allele in chimps is whether this protein is active or not.
To this end, we established an in vitro cell model system and stably expressed the different recombinant OTC forms, corresponding to the human, chimps and ancestral backgrounds, as well as the human mutant Thr125Met (Fig. 1). The catalytic activity of the corresponding protein products was characterized. As expected, the Thr125Met mutation completely abrogates the human OTC activity, confirming its disease-causative nature in OTCD patients. The human and chimp OTCs displayed comparable enzymatic activities, whereas the ancestral OTC resulted more active. Altogether, these results, although not ruling out the hypothesis of alternative routes for ammonia detoxification in chimps, support the idea that the deleterious effect of the Met125 in chimps is compensated by the presence of Thr at position 135, by a mechanism of intra-locus compensation.
In conclusion, in this work, we provided direct experimental evidence on the existence of mechanisms of intra-locus epistatic interactions. In this regard, of note, when the mice (Mus musculus) genome was made public (13), 27 amino acid residues directly associated with human disease found correspondence with the wild-type sequence in mouse ortholog. Later, six human disease alleles were described as the wild-type sequence in chimpanzees (7), so what we describe here for the OTC gene is likely to be a rather common mechanism during evolution.
Besides the clear impact in evolutionary biology, our data also have important implications for a broader understanding of phenotypic variability among individuals. In this regard, it is widely accepted that simple allelic Mendelian variants are not sufficient to explain the observed large phenotypic spectrum of genetic diseases, even between siblings. In particular, in an era when genomic medicine is merging with clinical practice, this stresses the need of more realistic models in which complex epistatic factors should be taken into account when studying and managing diseases and their many complications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the plasmids encoding wild-type human and chimpanzee OTC
According to the human OTC sequence available, oligonucleotide primers were designed and purchased from Sigma Genosys (forward: 5'-CACCATGCTGTTTAATCTGAGGATC-3'; reverse: 5'-TCAAAATTTAGGCTTCTGGAG-3'). A human colon cDNA library (human colon 50-stretch plus cDNA library, 1 ng/ml, colon cells pooled from 50 Caucasian males) was purchased from Clontech (Clontech Laboratories, Paolo Alto, CA, USA) and the full-length human OTC amplified by the Hot Start Method. Amplification was performed for 35 cycles using the following settings: denaturation at 95°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 1 min and 30 s per cycle. The amplified cDNA was cloned into the pLenti6/V5 Directional TOPO® vector (Invitrogen) for retroviral infection, following the manufacturer instructions. Mutant plasmids corresponding to OTCD-associated mutation T125M, to the ancestral combination T125–T135 and to the chimps combination M125–T135 were obtained by nested PCR, using specific primers carrying the desired changes (M125 forward: 5'-AAAGTCTCATGGACACGGC-3'; M125 reverse: 5'-GCCGTGTCCATGAGACTTT; T135 forward: 5'-CTAGCATGACAGATGCAGTA-3'; T135 reverse: 5'-TACTGCATCTGTCATGCTAG-3') and wild-type human OTC as DNA template.
Establishment of cell lines stably expressing the different OTC constructs
CHO DG44 dhfr– cells were chosen as cell model system. Cells were tested to exclude the presence of endogenous OTC expression before retroviral infection and cultured at 37°C under 5% CO2 in humidified air, in DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin. Retroviral infection was performed using the Virapower infection kit (Invitrogen), according to the manufacturer’s instructions. 293FT cells were chosen for viral packaging and lipofectamin transfection performed with 95% confluent cells in OPTI-Mem medium (Invitrogen). Virus-containing supernatants were collected at 48 and 72 h, respectively, upon transfection, filtered and applied, in the presence of 100 µg of polybrene, on CHO target cells grown at 60% confluence. Blasticidin selection was carried out for the following 3 weeks. To verify the level of OTC expression in the selected cells, both semiquantitative PCR and real-time PCR were performed.
Semiquantitative RT–PCR
Total RNA was extracted from semiconfluent cells, using Tripure Isolation Reagent (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. The isolated RNAs were treated with ribonuclease-free deoxyribonuclease DNase I (1 U/µl) for 1 h to eliminate contamination with genomic DNA. To prepare first-strand cDNA, total RNA was first primed with oligodeoxythymidilic [oligo(dT)] primer and then reverse-transcribed with SuperScript (GIBCO-BRL) at 37°C for 60 min. Semiquantitative RT–PCR was used to compare the expression of OTC in the different established cell lines. The housekeeping gene GAPDH was used as internal control, and GAPDH primers (predicted to amplify a 660 bp product) were added into the same RT–PCR tubes. PCRs were carried out in a volume of 25 µl containing 2 µl of cDNA, 1.5 mmol/l of MgCl2, 0.5 mmol/l of each PCR primer, 2 mmol/l of deoxynucleotide triphosphates, 2.5 U of 10x PCR buffer and 0.5 U of Taq polymerase. Samples were initially denaturated at 95°C for 5 min followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 45 s of extension at 72°C. PCR products were size-fractionated with 25 g/l agarose gel electrophoresis. The expected OTC and GAPDH bands from the same sample were densitometrically analyzed and normalized by dividing the OTC values by the corresponding GAPDH values.
Real-time PCR
Real-time PCR assays were done using QuantiTect SYBER Green PCR kit (Qiagen) with SYBR Green I as the fluorescent dye enabling real-time detection of PCR products according to the manufacturer’s protocol. OTC-specific primers (Quantitect Primer Assay Hs_OTC_1_SG cat no. QT00019509, Qiagen) were purchased from Qiagen. For quantification, the target gene was normalized to the internal standard 18S gene. Cycling was done in an ABI Prism 7000 SDS v1.1, using the following conditions: 95°C for 15 min followed by 40 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 1 min. Results of the real-time PCR data were represented as Ct values, where Ct is defined as the threshold cycle of PCR at which amplified product was first detected. To compare the different RNA samples in an experiment, we used the comparative Ct method. We compared the RNA expression in samples with that of the control in each experiment. PCR was done in triplicate for each sample. The results were expressed as mean ± SD for the relative expression levels compared with the control, and minimum values of three independent experiments were taken.
OTC activity measurements
Cells were grown until 90% confluency in 9 cm tissue culture dishes and lysed with a TRITON X114 buffer (EDTA 20 mM, pH 8.0, 100 mM HEPES, pH 8.0, Triton 114 0.5%, DTT 20 mM, Protease Inhibitor Cocktail Roche 1 tablet/50 ml buffer). Extracted proteins were quantified by following the Bradford dye-binding procedure (17).
OTC activity in lysates was measured according to the protocol described by Ceriotti (18) and modified by Pierson et al. (19).
Briefly, the following stock solutions were prepared:
- Solution A = antipyrin (4 g/l) + iron(III)sulfate (50 mg/l) in sulfuric acid 10% v/v
- Solution B = Brij35 (0.3% w/w) + 2,3-butanedione monoxime (5 g/l) in acetic acid 5% v/v
The chromogenic reagent was prepared just before use by mixing equal volumes of solutions A and B. A standard solution of citrulline 1 mM in water was freshly prepared as well.
All measurements were done in 75 mM trietanolamine (TEA) buffer, pH 6.75 (the pH was adjusted by HCl). Twenty-five microliters of lysates containing an amount of OTC ranging from 5 to 30 µg was mixed with: (i) 25 µl of 7.5 mM ornithine hydrochloride solution in TEA buffer, pH 6.75, and (ii) 25 µl of 75 mM carbamyl phosphate solution in TEA buffer, pH 6.75. The reaction mixture was incubated at 37°C in the dark for 30 min. The reaction was stopped by addition of 3 ml of chromogenic reagent, and the sample was incubated at 95°C in the dark for 15 min. The absorbance at 460 nm was read after cooling down the reaction mixture. All measurements were done in triplicate.
Basal citrulline levels were determined by mixing 25 µl of lysates with 50 µl of TEA buffer; the mixture underwent the same treatment as described earlier. Citrulline standards were prepared by mixing 25 µl of citrulline solution (1 mM) with 50 µl of TEA; the mixture underwent the same treatment as described earlier.
Activity is expressed in milliunits, namely, as nanomole citrulline produced per milliliter of lysates and per minute of incubation at 37°C. It was calculated according to the following algorithm:
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ACKNOWLEDGEMENTS |
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This work was supported by grant SFRH/BPD/14867/2003 from Fundação para a Ciência e a Tecnologia and by Programa Operacional Ciência, Tecnologia e Inovação (POCTI), research project POCI/CVT/58082/2004 and by grants CNR/GRICES (411/2007). B.B. is a recipient of a PhD fellowship from the Italian Ministero per l’Istruzione, l’Università e la Ricerca (MIUR).
Conflict of Interest statement. None declared.
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