- Email: [email protected]
A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity
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Clinical Biochemistry 41 (2008) 611 – 615
A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity Valentina Di Pietro a , Alessandra Gambacurta b , Angela Maria Amorini c , Antonino Finocchiaro a , Serena D'Urso c , Lia Ceccarelli a , Barbara Tavazzi a , Bruno Giardina a , Giuseppe Lazzarino c,⁎ a Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome “Sacro Cuore”, Rome, Italy Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, Rome, Italy Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b
c
Received 26 September 2007; received in revised form 17 January 2008; accepted 22 January 2008 Available online 7 February 2008
Abstract Objective: To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. Design and methods: Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. Results: Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. Conclusion: Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Canavan disease; N-acetylaspartate; N-acetylaspartoacylase; T677C mutation; Site-directed mutagenesis
Introduction Human aspartoacylase (hASPA; EC 3.5.1.15) deficiency is responsible for Canavan disease (CD, MIM # 271900), an autosomal recessive form of leukodystrophy characterized by spongy degeneration of the white matter in the brain, for which there is currently no effective treatment [1]. The salient clinical features of Canavan disease include atonia of neck muscles, hypotonia, hyperextension of legs and flexion of arms, blindness, severe mental defect, and megalocephaly and are manifest from early infancy. The hASPA gene, located in 17pterp13, is made up of 6 exons and encodes a 313-amino acid polypeptide chain with a molecular mass of 36 kD. The mature form of hASPA has recently been fully characterized [2] and consists of two identical subunits, each carrying a covalentlybound zinc atom essential for its catalytic activity. hASPA has evident similarities to carboxypeptidase A in both the tridimen-
⁎ Corresponding author. Fax: +39 095337036. E-mail address: [email protected] (G. Lazzarino).
sional structure of the catalytic site and the mechanism of catalysis [2]. In mammals, ASPA plays a critical role in brain metabolism, catalyzing in oligodendrocytes the deacetylation of the major Nacetylated amino acid, N-acetylaspartate (NAA) and generating free acetate and aspartate. Since abnormally high NAA values are detectable in CD-affected patients [1], determination of NAA levels in the brain, cerebrospinal fluid and urine is generally used as a clinical biochemical index to diagnose CD. Currently, there are at least 56 known mutations in the hASPA gene that correlate with CD, including 44 missense and nonsense mutations [3,4], numerous deletions and premature terminations [5]. In this study, we report a new hASPA gene mutation found in a family member of a CD-affected child homozygous for the known A731G mutation. The analysis of the genetic material of this family member indicated that the mutated hASPA gene causes an I226T amino acidic substitution in the protein sequence, potentially dangerous for the protein function. To clarify this issue, the hASPA encoded by this new mutated gene was expressed in Escherichia coli and showed no detectable catalytic activity. These results, as well as the peculiar family
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.01.021
612
V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
tree, indicating high frequency of both polymorphism and mutations of the hASPA gene, are discussed. Methods Clinical phenotypes of CD-affected child A 4-year old Caucasian child with suspected diagnosis of CD was hospitalized at the Policlinico Gemelli of the Catholic University “Sacro Cuore” of Rome. The patient was clinically characterized by mental retardation, sporadic epileptic episodes, megalocephaly, and uncontrolled head movements due to hypotonia of neck muscles. Magnetic resonance imaging evidenced spongy degeneration of the white matter in the brain and 1Hmagnetic resonance spectroscopy showed elevated brain NAA, thereby confirming the diagnosis of CD. The HPLC analysis of the urinary and serum concentration of this compound was used to further corroborate the diagnosis. To this purpose, 500 µL of urine were filtered through a 3 kD cut-off membrane-equipped tube (Pall Life Sciences, Ann Arbor, MI, USA) and properly diluted prior to the HPLC analysis. An isocratic, ion-pairing HPLC separation of NAA was performed according to a method suitable to screen for different inborn errors of metabolism (including CD), as described in detail elsewhere [6]. Molecular biological analysis Genomic DNA purification of the CD patient and his family members was performed on 100 μL of fresh whole blood using advanced silica-based membrane SpinClean™ genomic DNA purification kit (Mbiotech, Seul, Korea). The six exons of the ASPA gene were amplified by PCR using the appropriate ASPA primers designed according to the 0.2 version of the Primer3 Input software (primer3_www.cgi) developed by the Whitehead Institute for Biomedical Research (Cambridge, MA, USA). The sequence of hASPA published in NCBI (Gene Bank Accession Number NM_0000049.2) was used as template. Since no mutations were found in exons 1, 2, 3, 4 and 6 of any family members screened in this study, we described in detail only procedures and results referring to exon 5. To obtain the complete exon 5 sequence, we used flanking primers covering exon 5 and amplifying a fragment of 447 bp: forward 5′-CTCAGGTGATCCACCCAACT-3′ and reverse 5′-AGCGTGCAGGCCATACTTAC-3′. The resulting mixture after PCR was electrophoresed in a 1.5% agarose gel, at 10 V/cm for 60 min. The amplified DNA band was cut out, purified and sequenced using an ABI PRISM 310 automatic sequencer (Applied Biosystems, Foster City, CA, USA). Protein expression and purification The total RNA extracted from human fibroblast was used to clone the wild type mRNA of hASPA. Properly designed primers amplified the entire hASPA open reading frame (ORF), minus the start and stop codons: Fw 5′-ACTTCTTGTCACATTGCTG-3′ Rev 5′-ATGTAAACAGCAGCGAATAC-3′
The hASPA cDNA was cloned into the pBAD/ThioTOPO bacterial expression vector (Invitrogen, Carlsbad, CA, USA). An N-terminus thioredoxine/hASPA fusion protein, containing on the C-terminus a V5 epitope and a 6 × histidine tag, was the resulting vector product. hASPA expression was obtained by inoculating a selected single recombinant E. coli colony in 5 mL of ampicillinsupplemented (100 µg/mL) Luria–Bertani medium followed by overnight incubation at 37 °C under constant shaking. The culture medium (O.D.600 ÷ 1.0–2.0) was then diluted 10 times with fresh ampicillin-containing medium and incubated at 37 °C under shaking until having an O.D.600 ≅ 0.5. Addition of 0.2% arabinose to the medium and subsequent incubation for 4.5 h at 37 °C were used to induce recombinant hASPA expression. At the end of incubation, the medium was centrifuged and the pellet suspended in 5 mL of a buffer composed by 20 mM Na2HPO4, 500 mM NaCl, 1 mM β-mercaptoethanol, 0.1% of the non-ionic detergent IGEPAL CA630 (Sigma, St. Louis, MO, USA) and 1 tablet/ 50 mL of protease inhibitors (Boehringer Mannheim GmbH, Mannheim, Ge), pH 7.8. The suspension was sonicated, centrifuged at 20,680 ×g for 15 min at 4 °C and the resulting supernatant was used for further purification. To 5 g of a nickel-charged Sepharose affinity resin (Probond, Invitrogen, Carlsbad, CA, USA), previously equilibrated with 20 mM Na2HPO4, 500 mM NaCl, 1 mM β-mercaptoethanol, and pH 7.8, 5 mL of the bacterial-extracted supernatant was added and let to stir in a beaker for 20 min at 4 °C. Extensive washings were then performed with the equilibrating buffer. Release of recombinant hASPA from the resin was carried out by subsequent batchwise washings with equilibrating buffer supplemented with increasing imidazole concentrations (50, 100, 300 and 500 mM). Each washing was collected and assayed for ASPA activity. Site-directed mutagenesis Vector-inserted cDNA of hASPA was utilized to introduce the desired point mutation (indicated in bold), using the below reported primers and following the recommendations of the site-directed mutagenesis kit (Quick Change II, Stratagene, La Jolla, CA, USA): Fw 5′-GGTCTATAAAATTACAGAGAAAGTTGATTACCC-3′ Rev 5′-GGGTAATCAACTTTCTCTGTAATTTTATAGACC-3′ Mutant plasmid DNA was then isolated and sequenced to confirm the occurrence of the inserted mutation. Further expression and purification of the mutated protein were carried out as previously described for the recombinant hASPA wild type. hASPA enzyme assay The enzyme assay of the purified wild type and suspected mutated recombinant hASPA was performed in a mixture (total volume 400 µL) containing as substrate variable NAA concentrations (0.01–1 mM). Incubation of the mixtures at 37 °C lasted 15, 30, 60, 120 min and was terminated by adding 800 μL
V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
613
Table 1 Kinetic parameters of the E. coli expressed, purified hASPA of both wild type and I226T mutated proteins
hASPA wild type I226T-mutated hASPA
Vmax (μmol/min)
Km (μM)
Kcat (s− 1)
5.23 N.D.
163 N.D.
37.5 N.D.
Protocol for hASPA expression and conditions to evaluate hASPA activity are fully described in Methods section. Fig. 1. Nucleotide sequence of the exon 5 of the hASPA gene of the CD-affected patient carrying in homozygosis the A731G mutation.
of HPLC-grade acetonitrile. The precipitated proteins were removed by centrifugation at 21,680 ×g for 15 min at 4 °C, and supernatants were extracted three times with chloroform for organic solvent and lipid contamination removal. The aqueous phase was injected onto the HPLC column to evaluate the amount of residual NAA in the incubation mixture [6]. The enzymatic activity was evaluated by calculating the rate of NAA degradation and was expressed in U/mg protein (where 1 U = 1 μmol of NAA degraded/min). Each sample was assayed in triplicate. Results and discussion As it could be expected, concentration of NAA in urinary sample of the CD-affected child was abnormally elevated, being equal to 1.18 mmol/mmol creatinine (NAA in 20 age-matched controls = 3.41 ± 1.99 µmol/mmol creatinine), whilst NAA in serum was equal to 18.0 µmol/L plasma (NAA in 20 agematched controls = not detectable). As shown in Fig. 1, the analysis of the genetic material of the compound showed the known A731G mutation. This result prompted us to undertake the screening of the parents and, on request, of the consanguineous aunt and uncle-in-law of the compound. The parents of the compound were, as expected, carriers of the A731G mutation. Moreover, the father showed also to carry the known silent polymorphism C693T, which was not present in the CD patient. Surprisingly, the complete sequence of exon 5 of the patient's consanguineous aunt revealed either the presence of the C693T polymorphism or a nucleotide substitution T677C
in heterozygosis (Fig. 2). Consequently, the resulting primary sequence of the mutated hASPA should present an I226T amino acidic substitution. The possible dramatic modifications of the secondary, tertiary and quaternary structures of the mutated protein might produce a loss of its catalytic activity. This phenomenon might be caused either by the non-conservative I → T amino acidic substitution, or by the fact that the potential mutation would fall within a hot region of the protein (four mutations causing CD are reported to occur within the hASPA primary sequence comprised between the 214th and 231st residue). To verify the effect of this amino acidic substitution on the protein catalytic activity we expressed either the control or the mutated hASPA in E. coli. Results demonstrated that, whilst the wild type protein had kinetic parameters even better than those reported in literature [8], the purified mutated hASPA did not show catalytic activity (Table 1) at any substrate concentration tested (Fig. 3). The recent results reporting the crystal structure of recombinant hASPA at 2.8 Å unequivocally evidenced that, notwithstanding previous erroneous indications [7], the protein is a homodimer in which each subunit covalently links a Zn2+ atom, essential for the catalytic activity [2,9]. The homology of the site of catalysis and the proposed mechanism of action render hASPA similar to the zinc-dependent hydrolase carboxypeptidase A. Secondary structure of hASPA monomers is characterized by nine α-helix and thirteen β-sheets organized into two main domains, the N-terminal and the C-terminal [2,9]. The one third of the sequence representing the C-terminal domain has a globular structure with a two-stranded anti-parallel β-sheet linker (β-sheets 8 and 12) wrapping around the N-terminal region. In particular, a close interaction occurs between the β-sheet 5 of the C-terminal domain and the β-sheet 8 of the
Fig. 2. Nucleotide sequence of the exon 5 of the hASPA gene of the consanguineous aunt of the CD-affected patient carrying in heterozygosis both the T677C mutation and the silent C693T polymorphism.
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V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
Fig. 3. Initial rate (V0) of the reaction of NAA hydrolysis catalyzed by the wild type and I226T-mutated hASPA, as a function of increasing substrate concentration. No residual activity of the I226T-mutated hASPA was detectable at any NAA tested. Sigmoidal curve of the wild type hASPA indicates the dimeric structure of the mature protein (Hill's coefficient n = 1.8, data not shown).
N-terminal domain, where a G123 of the β-sheet 5 is hydropobically in contact with a I225 and I226 of the β-sheet 8 [2]. In the deducted primary structure of our mutated protein the amino acidic substitution I → T at the position 226 should deeply modify the interaction among the two β-sheets 5 and 8 by dramatically decreasing the hydrophobicity of the region and, therefore, leading to decrease in the overall strength of interaction between the two domains. Since this interaction is fundamental to preserve the hydrophobic core of the protein, where the link with substrate occurs, it is conceivable to hypothesize that the mutated protein we found in the consanguineous aunt of the CD patient is inactive because of profound
tridimensional changes, ultimately altering the structure of the catalytic site. At present, it is not possible to speculate whether NAA can enter or not the site of catalysis of this mutated protein. The unusual pedigree summarizing the mutations found in the hASPA gene of the family members screened in this study, is illustrated in Fig. 4. In addition to the new T677C mutation, it is worth underlining that, notwithstanding the very low probability, the C693T polymorphism was present in the father, in the consanguineous aunt and in the uncle-in-law of the compound, even though no parental relationship occurred among any of the three carriers. In conclusion, we have presented a new mutation in the hASPA gene encoding for an inactive protein with an I → T substitution at the position 226 in its primary structure. On the basis of the available structural data [2,9], this amino acidic substitution presumably causes profound changes in the mutated hASPA tridimensional structure. Such changes are in turn responsible for the complete loss of the protein catalytic activity. Therefore, it may be affirmed that this mutation in homozygosis would lead to the phenotype characteristic of CD. Acknowledgments This work has been supported in part by research funds of University of Rome “Sacro Cuore” and University of Catania.
Fig. 4. The familial tree of the CD-affected patient showing the new mutation T677C in the consanguineous aunt and the anomalous frequency of the silent C693T polymorphism in three non-consanguineous subjects. Black and white square and circle = father (I, 1; A731G/WT, C693T/WT; 35 years old) and mother (I, 2; A731G/WT; 32 years old) of the CD patient; dotted and white circle = consanguineous aunt (I, 3; T677C/WT, C693T/WT; 28 years old) of the CD patient; white square = uncle-in-law (I, 4; C693T/WT; 33 years old) of the CD patient. Black square = CD-affected patient (II, 1; A731G/A731G; 4 years old). Asterisks indicate heterozygotic carriers of the C693T silent polymorphism.
References [1] Matalon R, Michals-Matalon K, Sebesta M, Deanching M, Gashokoff P, Casanova J. Aspartoacylase deficiency and N-acetylaspartic aciduria in patient with Canavan disease. Am J Med Genet 1988;29:463–71. [2] Bitto E, Bingman CA, Wesenberg GE, McCoy JG, Phillips Jr GN. Structure of aspartoacylase, the brain enzyme impaired in Canavan disease. Proceedings of the Nat Acad Sci 2007;104:456–61.
V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615 [3] Stenson PD, Ball EV, Mort M, et al. Human Gene Mutation Database (HGMD). Hum Mutat 2003;21:577–81. [4] Zeng BJ, Pastores GM, Leone P, et al. Mutation analysis of the aspartoacylase gene in non-Jewish patients with Canavan disease. Adv Exp Med Biol 2006;576:165–73. [5] Zeng BJ, Wang ZH, Ribeiro LA, et al. Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease. J Inherit Metab Dis 2002;25:557–70. [6] Tavazzi B, Lazzarino G, Leone P, et al. Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical
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diagnosis of inborn errors of metabolism. Clin Biochem 2005;38: 997–1008. [7] Hershfield JR, Madhavarao CN, Moffett JR, Benjamins JA, Garbern JY, Namboodiri A. Aspartoacylase is a regulated nuclear–cytoplasmic enzyme. FASEB J 2006;20:2139–41. [8] Le Coq J, An HJ, Lebrilla C, Viola RE. Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease. Biochemistry 2006;45:5878–84. [9] Hershfield JR, Pattabiraman N, Madhavarao CN, Namboodiri MA. Mutational analysis of aspartoacylase: implications for Canavan disease. Brain Res 2007;1148:1–14.
Clinical Biochemistry 41 (2008) 611 – 615
A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity Valentina Di Pietro a , Alessandra Gambacurta b , Angela Maria Amorini c , Antonino Finocchiaro a , Serena D'Urso c , Lia Ceccarelli a , Barbara Tavazzi a , Bruno Giardina a , Giuseppe Lazzarino c,⁎ a Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome “Sacro Cuore”, Rome, Italy Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, Rome, Italy Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b
c
Received 26 September 2007; received in revised form 17 January 2008; accepted 22 January 2008 Available online 7 February 2008
Abstract Objective: To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. Design and methods: Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. Results: Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. Conclusion: Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Canavan disease; N-acetylaspartate; N-acetylaspartoacylase; T677C mutation; Site-directed mutagenesis
Introduction Human aspartoacylase (hASPA; EC 3.5.1.15) deficiency is responsible for Canavan disease (CD, MIM # 271900), an autosomal recessive form of leukodystrophy characterized by spongy degeneration of the white matter in the brain, for which there is currently no effective treatment [1]. The salient clinical features of Canavan disease include atonia of neck muscles, hypotonia, hyperextension of legs and flexion of arms, blindness, severe mental defect, and megalocephaly and are manifest from early infancy. The hASPA gene, located in 17pterp13, is made up of 6 exons and encodes a 313-amino acid polypeptide chain with a molecular mass of 36 kD. The mature form of hASPA has recently been fully characterized [2] and consists of two identical subunits, each carrying a covalentlybound zinc atom essential for its catalytic activity. hASPA has evident similarities to carboxypeptidase A in both the tridimen-
⁎ Corresponding author. Fax: +39 095337036. E-mail address: [email protected] (G. Lazzarino).
sional structure of the catalytic site and the mechanism of catalysis [2]. In mammals, ASPA plays a critical role in brain metabolism, catalyzing in oligodendrocytes the deacetylation of the major Nacetylated amino acid, N-acetylaspartate (NAA) and generating free acetate and aspartate. Since abnormally high NAA values are detectable in CD-affected patients [1], determination of NAA levels in the brain, cerebrospinal fluid and urine is generally used as a clinical biochemical index to diagnose CD. Currently, there are at least 56 known mutations in the hASPA gene that correlate with CD, including 44 missense and nonsense mutations [3,4], numerous deletions and premature terminations [5]. In this study, we report a new hASPA gene mutation found in a family member of a CD-affected child homozygous for the known A731G mutation. The analysis of the genetic material of this family member indicated that the mutated hASPA gene causes an I226T amino acidic substitution in the protein sequence, potentially dangerous for the protein function. To clarify this issue, the hASPA encoded by this new mutated gene was expressed in Escherichia coli and showed no detectable catalytic activity. These results, as well as the peculiar family
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.01.021
612
V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
tree, indicating high frequency of both polymorphism and mutations of the hASPA gene, are discussed. Methods Clinical phenotypes of CD-affected child A 4-year old Caucasian child with suspected diagnosis of CD was hospitalized at the Policlinico Gemelli of the Catholic University “Sacro Cuore” of Rome. The patient was clinically characterized by mental retardation, sporadic epileptic episodes, megalocephaly, and uncontrolled head movements due to hypotonia of neck muscles. Magnetic resonance imaging evidenced spongy degeneration of the white matter in the brain and 1Hmagnetic resonance spectroscopy showed elevated brain NAA, thereby confirming the diagnosis of CD. The HPLC analysis of the urinary and serum concentration of this compound was used to further corroborate the diagnosis. To this purpose, 500 µL of urine were filtered through a 3 kD cut-off membrane-equipped tube (Pall Life Sciences, Ann Arbor, MI, USA) and properly diluted prior to the HPLC analysis. An isocratic, ion-pairing HPLC separation of NAA was performed according to a method suitable to screen for different inborn errors of metabolism (including CD), as described in detail elsewhere [6]. Molecular biological analysis Genomic DNA purification of the CD patient and his family members was performed on 100 μL of fresh whole blood using advanced silica-based membrane SpinClean™ genomic DNA purification kit (Mbiotech, Seul, Korea). The six exons of the ASPA gene were amplified by PCR using the appropriate ASPA primers designed according to the 0.2 version of the Primer3 Input software (primer3_www.cgi) developed by the Whitehead Institute for Biomedical Research (Cambridge, MA, USA). The sequence of hASPA published in NCBI (Gene Bank Accession Number NM_0000049.2) was used as template. Since no mutations were found in exons 1, 2, 3, 4 and 6 of any family members screened in this study, we described in detail only procedures and results referring to exon 5. To obtain the complete exon 5 sequence, we used flanking primers covering exon 5 and amplifying a fragment of 447 bp: forward 5′-CTCAGGTGATCCACCCAACT-3′ and reverse 5′-AGCGTGCAGGCCATACTTAC-3′. The resulting mixture after PCR was electrophoresed in a 1.5% agarose gel, at 10 V/cm for 60 min. The amplified DNA band was cut out, purified and sequenced using an ABI PRISM 310 automatic sequencer (Applied Biosystems, Foster City, CA, USA). Protein expression and purification The total RNA extracted from human fibroblast was used to clone the wild type mRNA of hASPA. Properly designed primers amplified the entire hASPA open reading frame (ORF), minus the start and stop codons: Fw 5′-ACTTCTTGTCACATTGCTG-3′ Rev 5′-ATGTAAACAGCAGCGAATAC-3′
The hASPA cDNA was cloned into the pBAD/ThioTOPO bacterial expression vector (Invitrogen, Carlsbad, CA, USA). An N-terminus thioredoxine/hASPA fusion protein, containing on the C-terminus a V5 epitope and a 6 × histidine tag, was the resulting vector product. hASPA expression was obtained by inoculating a selected single recombinant E. coli colony in 5 mL of ampicillinsupplemented (100 µg/mL) Luria–Bertani medium followed by overnight incubation at 37 °C under constant shaking. The culture medium (O.D.600 ÷ 1.0–2.0) was then diluted 10 times with fresh ampicillin-containing medium and incubated at 37 °C under shaking until having an O.D.600 ≅ 0.5. Addition of 0.2% arabinose to the medium and subsequent incubation for 4.5 h at 37 °C were used to induce recombinant hASPA expression. At the end of incubation, the medium was centrifuged and the pellet suspended in 5 mL of a buffer composed by 20 mM Na2HPO4, 500 mM NaCl, 1 mM β-mercaptoethanol, 0.1% of the non-ionic detergent IGEPAL CA630 (Sigma, St. Louis, MO, USA) and 1 tablet/ 50 mL of protease inhibitors (Boehringer Mannheim GmbH, Mannheim, Ge), pH 7.8. The suspension was sonicated, centrifuged at 20,680 ×g for 15 min at 4 °C and the resulting supernatant was used for further purification. To 5 g of a nickel-charged Sepharose affinity resin (Probond, Invitrogen, Carlsbad, CA, USA), previously equilibrated with 20 mM Na2HPO4, 500 mM NaCl, 1 mM β-mercaptoethanol, and pH 7.8, 5 mL of the bacterial-extracted supernatant was added and let to stir in a beaker for 20 min at 4 °C. Extensive washings were then performed with the equilibrating buffer. Release of recombinant hASPA from the resin was carried out by subsequent batchwise washings with equilibrating buffer supplemented with increasing imidazole concentrations (50, 100, 300 and 500 mM). Each washing was collected and assayed for ASPA activity. Site-directed mutagenesis Vector-inserted cDNA of hASPA was utilized to introduce the desired point mutation (indicated in bold), using the below reported primers and following the recommendations of the site-directed mutagenesis kit (Quick Change II, Stratagene, La Jolla, CA, USA): Fw 5′-GGTCTATAAAATTACAGAGAAAGTTGATTACCC-3′ Rev 5′-GGGTAATCAACTTTCTCTGTAATTTTATAGACC-3′ Mutant plasmid DNA was then isolated and sequenced to confirm the occurrence of the inserted mutation. Further expression and purification of the mutated protein were carried out as previously described for the recombinant hASPA wild type. hASPA enzyme assay The enzyme assay of the purified wild type and suspected mutated recombinant hASPA was performed in a mixture (total volume 400 µL) containing as substrate variable NAA concentrations (0.01–1 mM). Incubation of the mixtures at 37 °C lasted 15, 30, 60, 120 min and was terminated by adding 800 μL
V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
613
Table 1 Kinetic parameters of the E. coli expressed, purified hASPA of both wild type and I226T mutated proteins
hASPA wild type I226T-mutated hASPA
Vmax (μmol/min)
Km (μM)
Kcat (s− 1)
5.23 N.D.
163 N.D.
37.5 N.D.
Protocol for hASPA expression and conditions to evaluate hASPA activity are fully described in Methods section. Fig. 1. Nucleotide sequence of the exon 5 of the hASPA gene of the CD-affected patient carrying in homozygosis the A731G mutation.
of HPLC-grade acetonitrile. The precipitated proteins were removed by centrifugation at 21,680 ×g for 15 min at 4 °C, and supernatants were extracted three times with chloroform for organic solvent and lipid contamination removal. The aqueous phase was injected onto the HPLC column to evaluate the amount of residual NAA in the incubation mixture [6]. The enzymatic activity was evaluated by calculating the rate of NAA degradation and was expressed in U/mg protein (where 1 U = 1 μmol of NAA degraded/min). Each sample was assayed in triplicate. Results and discussion As it could be expected, concentration of NAA in urinary sample of the CD-affected child was abnormally elevated, being equal to 1.18 mmol/mmol creatinine (NAA in 20 age-matched controls = 3.41 ± 1.99 µmol/mmol creatinine), whilst NAA in serum was equal to 18.0 µmol/L plasma (NAA in 20 agematched controls = not detectable). As shown in Fig. 1, the analysis of the genetic material of the compound showed the known A731G mutation. This result prompted us to undertake the screening of the parents and, on request, of the consanguineous aunt and uncle-in-law of the compound. The parents of the compound were, as expected, carriers of the A731G mutation. Moreover, the father showed also to carry the known silent polymorphism C693T, which was not present in the CD patient. Surprisingly, the complete sequence of exon 5 of the patient's consanguineous aunt revealed either the presence of the C693T polymorphism or a nucleotide substitution T677C
in heterozygosis (Fig. 2). Consequently, the resulting primary sequence of the mutated hASPA should present an I226T amino acidic substitution. The possible dramatic modifications of the secondary, tertiary and quaternary structures of the mutated protein might produce a loss of its catalytic activity. This phenomenon might be caused either by the non-conservative I → T amino acidic substitution, or by the fact that the potential mutation would fall within a hot region of the protein (four mutations causing CD are reported to occur within the hASPA primary sequence comprised between the 214th and 231st residue). To verify the effect of this amino acidic substitution on the protein catalytic activity we expressed either the control or the mutated hASPA in E. coli. Results demonstrated that, whilst the wild type protein had kinetic parameters even better than those reported in literature [8], the purified mutated hASPA did not show catalytic activity (Table 1) at any substrate concentration tested (Fig. 3). The recent results reporting the crystal structure of recombinant hASPA at 2.8 Å unequivocally evidenced that, notwithstanding previous erroneous indications [7], the protein is a homodimer in which each subunit covalently links a Zn2+ atom, essential for the catalytic activity [2,9]. The homology of the site of catalysis and the proposed mechanism of action render hASPA similar to the zinc-dependent hydrolase carboxypeptidase A. Secondary structure of hASPA monomers is characterized by nine α-helix and thirteen β-sheets organized into two main domains, the N-terminal and the C-terminal [2,9]. The one third of the sequence representing the C-terminal domain has a globular structure with a two-stranded anti-parallel β-sheet linker (β-sheets 8 and 12) wrapping around the N-terminal region. In particular, a close interaction occurs between the β-sheet 5 of the C-terminal domain and the β-sheet 8 of the
Fig. 2. Nucleotide sequence of the exon 5 of the hASPA gene of the consanguineous aunt of the CD-affected patient carrying in heterozygosis both the T677C mutation and the silent C693T polymorphism.
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V. Di Pietro et al. / Clinical Biochemistry 41 (2008) 611–615
Fig. 3. Initial rate (V0) of the reaction of NAA hydrolysis catalyzed by the wild type and I226T-mutated hASPA, as a function of increasing substrate concentration. No residual activity of the I226T-mutated hASPA was detectable at any NAA tested. Sigmoidal curve of the wild type hASPA indicates the dimeric structure of the mature protein (Hill's coefficient n = 1.8, data not shown).
N-terminal domain, where a G123 of the β-sheet 5 is hydropobically in contact with a I225 and I226 of the β-sheet 8 [2]. In the deducted primary structure of our mutated protein the amino acidic substitution I → T at the position 226 should deeply modify the interaction among the two β-sheets 5 and 8 by dramatically decreasing the hydrophobicity of the region and, therefore, leading to decrease in the overall strength of interaction between the two domains. Since this interaction is fundamental to preserve the hydrophobic core of the protein, where the link with substrate occurs, it is conceivable to hypothesize that the mutated protein we found in the consanguineous aunt of the CD patient is inactive because of profound
tridimensional changes, ultimately altering the structure of the catalytic site. At present, it is not possible to speculate whether NAA can enter or not the site of catalysis of this mutated protein. The unusual pedigree summarizing the mutations found in the hASPA gene of the family members screened in this study, is illustrated in Fig. 4. In addition to the new T677C mutation, it is worth underlining that, notwithstanding the very low probability, the C693T polymorphism was present in the father, in the consanguineous aunt and in the uncle-in-law of the compound, even though no parental relationship occurred among any of the three carriers. In conclusion, we have presented a new mutation in the hASPA gene encoding for an inactive protein with an I → T substitution at the position 226 in its primary structure. On the basis of the available structural data [2,9], this amino acidic substitution presumably causes profound changes in the mutated hASPA tridimensional structure. Such changes are in turn responsible for the complete loss of the protein catalytic activity. Therefore, it may be affirmed that this mutation in homozygosis would lead to the phenotype characteristic of CD. Acknowledgments This work has been supported in part by research funds of University of Rome “Sacro Cuore” and University of Catania.
Fig. 4. The familial tree of the CD-affected patient showing the new mutation T677C in the consanguineous aunt and the anomalous frequency of the silent C693T polymorphism in three non-consanguineous subjects. Black and white square and circle = father (I, 1; A731G/WT, C693T/WT; 35 years old) and mother (I, 2; A731G/WT; 32 years old) of the CD patient; dotted and white circle = consanguineous aunt (I, 3; T677C/WT, C693T/WT; 28 years old) of the CD patient; white square = uncle-in-law (I, 4; C693T/WT; 33 years old) of the CD patient. Black square = CD-affected patient (II, 1; A731G/A731G; 4 years old). Asterisks indicate heterozygotic carriers of the C693T silent polymorphism.
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