Aminoacylase I Deficiency: a Novel Inborn Error of Metabolism

BBRC Biochemical and Biophysical Research Communications 338 (2005) 1322–1326 www.elsevier.com/locate/ybbrc

Aminoacylase I deﬁciency: A novel inborn error of metabolism

R.N. Van Coster a,*, E.A. Gerlo b, T.G. Giardina c, U.F. Engelke d, J.E. Smet a, C.M. De Praeter e, V.A. Meersschaut f, L.J. De Meirleir g, S.H. Seneca g, B. Devreese h, J.G. Leroy a, S. Herga c, J.P. Perrier c, R.A. Wevers d, W. Lissens g

a Department of Pediatrics, Division of Neurology and Metabolism, University Hospital, Ghent, Belgium b Department of Clinical Chemistry, University Hospital, Free University, Brussels, Belgium c Institut Me´diterrane´en de Recherche en Nutrition, INRA-UMR 1111 Service 342, Universite´ Paul Ce´zanne—Aix-Marseille III, Faculte´ des Sciences et Techniques, 13397 Marseille Cedex 20, France d Laboratory of Pediatrics and Neurology, University Medical Center Nijmegen, Nijmegen, The Netherlands e Department of Neonatal Intensive Care, University Hospital, Ghent, Belgium f Department of Radiology, Division of Pediatric Radiology, University Hospital, Ghent, Belgium g Department of Medical Genetics, University Hospital, Free University, Brussels, Belgium h Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Ghent, Belgium

Received 12 September 2005 Available online 2 November 2005

Abstract

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography–mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C > T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity. Ó 2005 Elsevier Inc. All rights reserved.

Keywords: Aminoacylase I; N-acetylated amino acids; N-acetylated proteins; Enzyme deﬁciency; Western blotting; Messenger RNA; Mutation analysis; Protein expression study; Newborn; Laminar cortical necrosis

Aminoacylase I (EC 3.5.1.14) is a homodimeric zinc- hydrolase (EC 3.4.19.1) with the release of the N-acetylated binding metalloenzyme located in the cytosol [1]. It catalyz- amino acid and a shorter free peptide. In the next step, ami- es the hydrolysis of N-linked acyl groups in L-amino acids, noacylase I hydrolyzes the N-acetylated amino acid to ace- including the N-acetylated derivatives of serine, glutamic tate and its free amino acid. acid, alanine, methionine, glycine, leucine, and valine. The detection of several N-acetylated amino acids in the Among the proteolytic systems known to be involved in urine of a young infant with an encephalopathy to be intracellular catabolism of intrinsic proteins, the intracellu- described in this report has led to the identification of a lar catabolism of N-acetylated proteins is mediated by the deficiency of aminoacylase I. ATP-ubiquitin-dependent proteasome [2]. This large com- plex degrades proteins into peptides 5–30 amino residues Materials and methods long. N-acetylated peptides are then cleaved by acylpeptide Patient description. The propositus was born at 39 weeks gestation * Corresponding author. Fax: +32 9 240 38 75. after an uneventful pregnancy as the first child of non-consanguineous E-mail address: [email protected] (R.N. Van Coster). parents. He had a birth weight of 2,565 g. Length and head circumference

0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.126 R.N. Van Coster et al. / Biochemical and Biophysical Research Communications 338 (2005) 1322–1326 1323 were 48 and 33 cm, respectively. Delivery was by elective caesarian section TTGCTGATCCACATCTGC-30, and 50-GCTAAACTCCCACACAGA due to breech presentation. On the third day of life, the newborn no longer TGTAGTGCCTGTCTTC-30, and 50-AGGTTCATTTCCTTGTGGGC drank effectively, started vomiting and became cyanotic. Seizures and CCCGCTGAAAGC-30, respectively. The expected sizes of RT-PCR apnoeic spells were noticed. In addition to daily bouts of convulsions, products were 838 bp for the b-actin and 747 bp for the ACY1. Each cycle generalized hypotonia was most prominent. During the following days, of PCR included 30 s of denaturation at 94 °C. The annealing step was episodes of bradycardia compounded the hypotonia. Cerebral MRI carried out at appropriate temperature using 0.4 lmol of each couple of showed abnormal signals in the cortico-subcortical zones of the fronto- primers for 2 min, whereas the extension step was performed at 72 °C for parietal and temporo-occipital areas consistent with cortical laminar 3 min using an overall volume of 50 ll and a total number of 40 cycles for necrosis. A sensorineural hearing deficit (left 70 dB, right 60 dB) was PCR in a Mastercycler gradient 5331 thermocycler (Eppendorf). After detected by auditory brain stem response. The convulsions ceased around appropriate cycles, 10 ll of the RT-PCR products was separated on a 1.2% the age of two weeks, when also the feeding problems started improving. TAE (Tris acetate EDTA)–agarose gel, stained with ethidium bromide, and At six weeks, the feeding difficulties had subsided and convulsions had not photographed under UV light. recurred. Clinical neurological examination revealed no abnormalities. For Southern blot analyses, RT-PCR products were separated by However, auditory brain stem response confirmed significant hearing electrophoresis, the gel was denatured (0.4 M NaOH, 1.5 M NaCl) for deficit (left 65 dB, right 100 dB). The MRI revealed mild signs of cerebral 30 min, rinsed twice with distilled water, neutralized with 0.5 M Tris–HCl, cortical atrophy. Clinical examination at six months of age was normal. At 1.5 M NaCl, pH 7.5 buffer, 30 min, and transferred onto a nylon mem- nine months, he had reached normal milestones and abnormal clinical brane (Hybond, Amersham Pharmacia Biotech). After blotting, the neurological signs were not detected. membranes were dried and cross-linked by ultraviolet light. Organic acids. Organic acids were extracted by a standard ethyl ace- Oligonucleotides with internal sequences to our ACY1 sequence were tate-diethyl ether procedure, converted to their trimethylsilyl (TMS) used for synthesis of the labelled-PCR probe (50-GCCCAGGACA derivatives, and analyzed on a Hewlett Packard 5973 gas chromatogra- TGAAGAGTGTCAGCATCCAGTACC-30 and 50-TCCCGCTTCCT phy–mass spectrometry (GC–MS) system [3]. For a detailed description, GGTGCCAGCTCTGCAGC-30). The ACY1-PCR DNA probe was see Gerlo et al. (in preparation). random primed labelled by using the DIG-high prime DNA labelling kit Proton NMR spectroscopy. Proton NMR spectroscopy of urine (1- according to the manufacturerÕs instructions (Roche Diagnostics Corpo- dimensional and COSY) was performed on a Bruker 500 MHz spec- ration). Finally, the RT-PCR products were probed at 45 °C in the trometer as described before [4]. manufactured hybridization conditions and detected by using the DIG Enzyme activities. Cultured lymphoblast cell lines were washed in cold detection starter kit II (Roche). phosphate-buffered saline, pelleted, and resuspended in 2 mL of 10 mM Cloning of human aminoacylase I. The clone (#IRAUp969D1218D) Hepes buffer at pH 7.2, containing 5 mM glucose. After cells were lysed containing the complete open reading frame of hACY1 was obtained from with an Ultraturax for 10 s in ice-cold Hepes buffer, the resulting RZPD German Resource Center for Genome Research (Berlin, Germa- homogenate was centrifuged at 100,000g for 30 min. The supernatant was ny). hACY1 nucleotide sequence, fused to an upstream GST sequence, either directly used for measurement of acylpeptide hydrolase activity, or was cloned using the Gateway system (Invitrogen). hACY1 cDNA was for acylase I activity, as previously described [5]. Protein concentrations first synthesized, in a PCR mixture, using the primers, 50-G GGG ACA were determined using BradfordÕs method [6]. AGT TTG TAC AAA AAA GCA GGC TTG GTT CCG CGT GGA Immunoblotting. Cultured lymphoblast cell lines were washed with PBS TTC ACC AGC AAG GGT CCC GAG GAG-30 and 50-GGG GAC buffer. The resulting pellets were homogenized in buffer containing 10 mM CAC TTT GTA CAA GAA AGC TGG GTA AAG GTT TAG GAG sodium phosphate, 150 mM sodium chloride, and 2 mM EDTA (pH 7.4), TTC CAG GGC TCA GC-30, the underlined sequence corresponds to the and centrifuged at 16,000g. Protein concentration in the supernatant was thrombin cleavage site. Each primer (1 lM) was annealed to matrix determined according to Lowry et al. [7]. Sixty micrograms of protein was (50 ng) and both strands of the plasmid amplified using 1 U Platinium loaded per lane. Proteins were separated in a 16.5% tricine SDS–PAGE gel HIFI DNA polymerase (Invitrogen) in a linear extension reaction carried as described by Laemmli [8]. Electroblotting was performed according to out by PCR under the following conditions: 2 min denaturation at 95 °C, Towbin [9]. As primary antibody an anti-aminoacylase I polyclonal serum and 12 cycles of 30 s denaturation at 95 °C, 1 min 30 annealing at 55 °C, was used (Abnova, Taiwan). To demonstrate equal loading, the mem- 3 min at 68 °C, and 5 min extension at 68 °C. PCR cycling was performed brane was reprobed with a b-actin antibody (Abcam, UK). in a Mastercycler gradient 5331 system (Eppendorf). Full length PCR Gene analysis. PCR primers were manually designed to cover the 14 product was purified by electrophoresis in a 1% agarose gel using a exons and parts of the flanking intronic regions of the ACY1 gene. QIAquick Gel Extraction kit (Qiagen) and was inserted into a pDONR/ Genomic and cDNA sequences of the gene were from GenBank (Acces- Zeo entry vector using a PCR Cloning System with Gateway Technology sion Nos. AC115284 and L07548, respectively). The 14 exons were PCR kit (Invitrogen). Starting with this entry clone, hACY1 DNA construct amplified in 12 fragments (exons 5 and 6, and exons 8 and 9 were each was cloned into a pDEST15 expression vector to create the DNA for the amplified as one fragment) and sequenced using the PCR primers on an GST fusion protein. The expression vector was used to transform BL21 ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). DE3 cells (Invitrogen). Transformants were selected on LB medium plates PCR primer sequences and PCR conditions are electronically available containing 50 lg/mL ampicillin and the sequence was confirmed by DNA from W. Lissens. sequencing which was carried out using the dideoxychain-termination Total RNA isolation, ACY1 mRNA expression by RT-PCR, and procedure by Genome Express (Grenoble, France). Southern blot analysis. After the cells were washed once with cold PBS, Site-directed mutagenesis. The mutation was introduced into the 200 ll of RNAwiz isolation solution (Ambion, Austin, USA) was added to pGEX1kT-ACY1 plasmid using the QuickChange XL site-directed each sample. Total RNA was immediately extracted according to the mutagenesis kit (Stratagene Europe, Amsterdam Zuidoost, The Nether- instructions of the manufacturer and stored at 20 °C until used. The lands) with two overlapping complementary oligonucleotides containing quality of total RNA was systematically checked under non-denaturating the corresponding nucleotide changes 50-AACCGCTATATCTGC agarose gel. One microgram of total RNA was used for RT and the cDNA GCGGTGGGGGTCCCA-30 and 50-TGGGACCCCCACCGCGCAGA synthesis reaction was performed according to the instructions from TATAGCGGTT-30. Mutations were confirmed by DNA sequencing Invitrogen using their SuperScript III/Platinium Taq polymerase One-step which was carried out using the dideoxychain-termination procedure by RT-PCR System (Invitrogen Life Technologies) at 45 °C for 20 min, and Genome Express (Grenoble, France). Lastly, the plasmids were inserted terminated by heating at 95 °C for 5 min, for RT enzyme inactivation. PCR into E. coli BL21 DE3 competent cells to induce the expression of the primers used in this study were designed based on the EMBL/GenBank proteins required. (Accession #: Rattus b actin, NM_031144; ACY1, NM_001005383), and Protein expression and purification. Expression and purification of the the b-actin and ACY1 PCR primer sequences were 50-ATCTGGCACC wild-type and mutant proteins were performed as follows. Bacterial lysis ACACCTTCTACAATGAGCTGCG-30 and 50-CGTCATACTCCTGC was carried out in a 10 mM Tris–HCl, pH 8.0, buffer containing 1 mM 1324 R.N. Van Coster et al. / Biochemical and Biophysical Research Communications 338 (2005) 1322–1326

EDTA, 150 mM NaCl, and 10 mg/ml lysozyme (STE buffer). The diges- and smaller amounts of isoleucine, leucine, threonine, and tion was carried out at 4 °C for 15 min. Cells were then sonicated for 1 min valine were consistently found in four urine samples col- in STE containing 1 M DL-dithiothreitol and 10% Sarkosyl. After a lected over a two-month period (Table 1). Other organic 16,000 rpm centrifugation run, the supernatant was supplemented with 10% Triton X-100 and incubated for 30 min at room temperature. The acids including N-acetylaspartic acid were within reference resulting soluble fraction was mixed with GST-agarose beads pre-equili- limits. Proton nuclear magnetic resonance spectroscopy of brated with PBS. The fusion protein was first cleaved from GST by a patientÕs urine sample confirmed the presence of these incubating the gel overnight with thrombin (20 U/ml gel bed volume) at compounds and also revealed the presence of N-acetylated room temperature, and the resulting beads were subsequently washed with glutamine, asparagine, and an unidentified N-acetylated PBS buffer. The soluble fraction was used for characterization, while the remaining bound GST was released from the column into a 20 mM derivative (Table 1). N-Acetylaspartic acid was present in reduced glutathione solution containing 50 mM Tris–HCl buffer, pH 8.0. the patientÕs urine but was not significantly increased. Polyacrylamide gel electrophoresis and protein determination. SDS– The same held for the dipeptide N-acetylaspartylglutamate. PAGE was performed in 12% (w/v) polyacrylamide gel as described by N-acetylated forms of the aromatic amino acids tyrosine, Laemmli [8] to check the protein purity and determine the molecular mass. phenylalanine, and tryptophan were not found, and N- Molecular-mass markers and ACY1 samples were heated to 100 °C in the presence of 2% SDS under reducing conditions prior to performing elec- acetyllysine was also undetectable. trophoresis and Coomassie brilliant blue staining. Protein concentration was determined using BradfordÕs method with bovine serum albumin as Enzyme activities the standard [6]. Aminoacylase I activity was significantly decreased in Results the patient derived cell strains (Table 2). The activity of acylpeptide hydrolase was not significantly different in Metabolites in urine EBV transformed patient and control lymphoblast cell strains. The acylpeptide hydrolase/aminoacylase I activity GC–MS analysis of organic acids extracted from the ratio was 272 in the patient versus 23 in control cells. patientÕs urine was performed. The profile was character- ized by multiple unusual peaks identified as the TMS-deriv- Immunoblotting atives of N-acetylated amino acids by matching the retention times and the mass spectra of TMS-derivatives Western blotting showed that aminoacylase I CRM was prepared from model compounds. N-acetylated derivatives significantly decreased in the lymphoblast cells of the of alanine, glutamic acid, glycine, methionine, and, serine, patient (Fig. 1). Immunoblotting with b-actin antibody

Table 1 Urinary excretion (mmol/mol creatinine) of N-acetylated amino acids, as determined by gas chromatography mass–spectrometry (GC–MS) and proton magnetic resonance spectrometry (H1 NMR) Metabolite Identiﬁcations in diﬀerent (#) samples GC–MS H1 NMR #1 D21* #2 D51* #3 D65* #4 D112* Controls #5 D112* N-acetyl-serine 230 240 330 210 ND 326 N-acetyl-glutamic acid 310 390 560 330 ND 510 N-acetyl-glutamine ND ND ND ND ND 392 N-acetyl-alanine 200 210 250 150 ND 200 N-acetyl-methionine 110 30 80 110 ND 115 N-acetyl-glycine 160 180 190 150 ND 232 N-acetyl-aspartic acid 16 17 16 11 <31 ND N-acetyl-asparagine ND ND ND ND ND 337 N-acetyl-leucine 18 17 23 17 ND ± 40 N-acetyl-isoleucine 5 3 6 4 ND ± 40 N-acetyl-valine 11 12 17 12 ND ND N-acetyl-threonine 60 50 80 50 ND 44 N-acetyl-X ND ND ND ND ND 40 ND, not detectable. * Age of the patient (days).

Table 2 Specific activities of aminoacylase I (ACY1) and acylpeptide hydrolase (APH) in lymphoblasts from the patient and controls (U/mg protein) Samples Specific activity APH Specific activity ACY1 Ratio specific activity APH/ACY1 Mean controls (n = 3) 82 ± 22 3.6 ± 0.23 23 ± 6 Patient 49 0.18 272 R.N. Van Coster et al. / Biochemical and Biophysical Research Communications 338 (2005) 1322–1326 1325

Genomic analysis

Genomic sequencing of all 14 exons/introns in the patient and his parents showed that the patient was homozygous for a c.1057C > T change in exon 13, while both of his parents were heterozygous for this mutation. This nucleotide transition predicts the p.Arg353Cys. Five out of 161 controls were also heterozygous for this transition.

Site-directed mutagenesis and purification of wild-type and mutants ACY1 Fig. 1. Tricine SDS–PAGE was used for separation of proteins in lymphoblasts from the patient (P) and two controls (C1) and (C2). Immunoblotting with an anti-aminoacylase I polyclonal antibody revealed The wild-type and mutated ACY1 genes were expressed a severe decrease of the band corresponding to aminoacylase I in and purified to homogeneity by affinity chromatography. lymphoblasts from the patient as compared to controls. Reprobing with b The ACY1 protein size was checked by SDS–PAGE after actin antibody after stripping of the aminoacylase I antibody demon- removal of the GST tag by thrombin proteolysis. Each strated equal loading of the samples. enzyme produced a single band of an appropriate size (45 kDa) (data not shown). yielded comparable results in the patient and two control Role of R353C mutation in ACY1 cell lines. To investigate the possible importance of the arginine RT-PCR and Southern blot analyses residue at position 353 for human ACY1 activity, arginine was mutated into a cysteine. The R353C mutant resulted in RT-PCR product analyses were performed to compare complete absence of enzyme activity. the expression of aminoacylase I mRNA between the patient and two controls. A strong band for aminoacylase Discussion I mRNA was detected in the controls and only a weak band in the patient (Fig. 2A). The b-actin cDNA amplified Acetylation of the N-terminal amino acid (a-NH2 acet- as internal marker was present in all control and patient ylation) is a common protein modification in eukaryotes cells at a similar level (Fig. 2A). Using the RT-PCR prod- but is rarely encountered in prokaryotes. In mammalians, ucts, Southern blotting was performed with a DIG-ACY1 80–90% of the cytosolic proteins are subjected to an irre- PCR fragment confirming the above results (Fig. 2B). versible, cotranslational amino acid acetylation at their Moreover, a nested PCR with an aliquot of the RT-PCR N-terminus [10]. Many structural proteins such as actin from the patient was performed which allowed to conclude and tropomyosin are N-acetylated. This holds as well for that the weak band observed was indeed aminoacylase I several cytosolic enzymes, calcium and metal-binding mRNA (data not shown). proteins, transfer proteins, and peptide hormones [2]. N-acetylated proteins are catabolized in the cytosol by the ATP-ubiquitin-dependent proteasome pathway [2]. Functional aminoacylase I is crucial in the last step in this degradation as it catalyzes the hydrolysis of N-acetylated amino acids into acetate and the free amino acid. In this paper, a patient is described with a severe deficiency of the aminoacylase I. The propositus presented neonatally with an acute encephalopathy with onset on the third day of life and duration of about two weeks. This is the first report of a patient with a congenital defect in the cytoplas- mic protein degradation pathway of N-acetylated proteins. Detection of several N-acetylated amino acids in the patientÕs urine pointed to an as yet undescribed inborn error. The pattern of N-acetylated derivatives in the urine of the patient matched the substrate specificity of aminoacylase I. Fig. 2. RT-PCR products of b-actin (Act) and aminoacylase I (ACY1) in The enzyme is known to have a preference for neutral and ali- cultured lymphoblast cell lines from the patient (P) and two controls (C1 phatic N-acetyl-amino acids [11]. The patientÕs urine did not and C2). mRNA levels were determined by semi-quantitative RT-PCR as described in Materials and methods. In the upper panel are shown the contain N-acetylated forms of the basic and aromatic amino results after ethidium bromide staining (A), in the lower panel the acids. N-acetylaspartic acid was also not increased. These Southern blot after probing with the DIG-ACY1 PCR product (B). findings were consistent with the normal function of separate 1326 R.N. Van Coster et al. / Biochemical and Biophysical Research Communications 338 (2005) 1322–1326 aminoacylases deacetylating N-acetylaspartic acid (aspar- As this is a report of a single patient, there is no direct toacylase) and N-acetylated aromatic amino acids (aryl acy- evidence that aminoacylase I deficiency has caused the clin- lamidase) [11]. Aspartoacylase, deficient in Canavan disease ical findings observed. The clinical phenotype associated [12], is specific for N-acetylaspartic acid. The clinical and with aminoacylase I deficiency and the long-term clinical neuroradiologic features in our patient were different from implications of this hitherto unreported inborn error of the ones characterizing Canavan disease. metabolism must await further follow-up of the propositus The suspected deficiency of aminoacylase I was confirmed presented and the reporting of more patients with the same enzymatically in the EBV transformed lymphocytes of the enzyme defect. propositus. SDS–PAGE and immunoblotting showed that the loss of catalytic activity was due to severely decreased References amounts of aminoacylase I protein. The amount of aminoacylase I mRNA was also significantly decreased, probably [1] T. Giardina, A. Biagini, D. Massey-Harroche, A. Puigserver, Distri- due to inefficient transcriptional processing or to mRNA bution and subcellar localization of acylpeptide hydrolase and acylase instability. In the DNA extracted from the peripheral leuco- I along the hog gastro-intestinal tract, Biochimie 81 (1999) 1049– cytes, the homozygous point mutation (R353C) due to the 1055. [2] J. Perrier, A. Durand, T. Giardina, A. Puigserver, Catabolism of c.1057 C > T transition was detected in exon 13 of the intracellular N-terminal acetylated proteins: involvement of acylpep- ACY1 gene. Both parents, who denied consanguinity, were tide hydrolase and acylase, Biochimie 87 (2005) 673–685. heterozygous for this mutation. Based on a porcine ACY1 [3] K. Tanaka, D.G. Hine, A. West-Dull, T.B. Lynn, Gas-chromato- model [13] each ACY1 monomer consists of two domains: graphic method of analysis for urinary organic acids. I. Retention a globular catalytic subunit (residues 1–188 and 311–399) indices of 155 metabolically important compounds, Clin. Chem. 26 (1980) 1839–1846. consisting of a b-sheet sandwiched between a-helices and a [4] U.H.F. Engelke, M.L.F. 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Bradford, A rapid and sensitive method for the quantitation of genetic polymorphism, protein expression studies were per- microgram quantities of protein utilizing the principle of protein-dye formed which showed that the mutant protein had lost all binding, Anal. Biochem. 72 (1976) 248–254. [7] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein catalytic activity. Therefore, the metabolic condition measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) described might not be exceptionally rare. From the results 265–275. of the study presented it is expected that the abnormal [8] U.K. Laemmli, Cleavage of structural proteins during the assembly of compounds would be detected in 1/4,147 individuals. How- the head of bacteriophage T4, Nature 227 (1970) 680–685. ever, as organic acid analysis has become part of routine [9] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure testing for any encephalopathic patient at most major med- and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350– ical centers, one would predict that other patients with this 4354. unusual organic acid profile would have been identified in [10] B. Polevoda, F. Sherman, N-terminal acetyltransferases and sequence the past. N-Acetylated amino acids have poor extraction requirements for N-terminal acetylation of eukaryotic proteins, J. efficiency which can vary greatly from laboratory to labo- Mol. Biol. 325 (2003) 595–622. [11] M.W. Anders, W. Dekant, Aminoacylases, Adv. Pharmacol. 27 ratory. Therefore, it is not excluded that the abnormal pro- (1994) 431–448. file has been missed in other patients. Another theoretical [12] R. Matalon, K. Michals, D. Sebesta, et al., Aspartoacylase deficiency possibility is that not all patients with this enzyme deficien- and N-acetylaspartic aciduria in patients with Canavan disease, Am. cy excrete the abnormal compounds in the urine because of J. Med. Genet. 29 (1988) 463–471. the existence of an alternative pathway for degradation of [13] C. DÕAmbrosio, F. Talamo, R.M. Vitale, P. Amodeo, G. Tell, L. Ferrara, A. Scaloni, Probing the dimeric structure of porcine N-acetylated amino acids. Until now, however, such an aminoacylase I by mass spectrometric and modeling procedures, alternative pathway has not been demonstrated. Biochemistry 42 (2003) 4430–4443.