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A Single Nucleotide Change in the Prolidase Gene in Fibroblasts from Two Patients with Polypeptide Positive Prolidase Deficiency

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A Single Nucleotide Change in the Prolidase Gene in Fibroblasts from Two Patients with Polypeptide Positive Prolidase Deficiency A single nucleotide change in the prolidase gene in fibroblasts from two patients with polypeptide positive prolidase deficiency. Expression of the mutant enzyme in NIH 3T3 cells. A Tanoue, … , A Kitano, I Matsuda J Clin Invest. 1990;86(1):351-355. https://doi.org/10.1172/JCI114708. Research Article Prolidase deficiency is an autosomal recessive disorder characterized by mental retardation and various skin lesions. Cultured skin fibroblasts were obtained from two independent patients with abnormal prolidase. Using the polymerase chain reaction, we amplified the entire coding region of human prolidase mRNA derived from patients' fibroblasts. Nucleotide sequence analysis of amplified cDNA products revealed a G to A substitution at position 826 in exon 12, where aspartic acid was replaced by asparagine at the amino acid residue 276, in cells from both patients. An analysis of the DNA showed that the substitution was homozygous. An expression plasmid clone containing a normal human prolidase cDNA (pEPD-W) or mutant prolidase cDNA (pEPD-M) was prepared, transfected, and tested for expression in NIH 3T3 cells. Incorporation of pEPD-W and pEPD-M resulted in the synthesis of an immunological polypeptide that corresponded to human prolidase. Active human enzyme was detected in cells transfected with pEPD-W, but not in those transfected with pEPD-M. These results were compatible with our observation of fibroblasts and confirmed that the substitution was responsible for the enzyme deficiency. As active prolidase was recovered in prolidase-deficient fibroblasts transfected with pEPD-W, this restoration of prolidase activity after transfection means that gene replacement therapy for individuals with this human disorder can be given due consideration. Find the latest version: https://jci.me/114708/pdf Rapid Publication A Single Nucleotide Change in the Prolidase Gene in Fibroblasts from Two Patients with Polypeptide Positive Prolidase Deficiency Expression of the Mutant Enzyme in NIH 3T3 Cells Akito Tanoue, Fumio Endo, Akito Kitano, and Ichiro Matsuda Department ofPediatrics, Kumamoto University Medical School, Kumamoto 860, Japan Abstract seems to play an important role in the recycling of proline. Prolidase deficiency is an autosomal recessive disorder with a Prolidase deficiency is an autosomal recessive disorder charac- variable clinical phenotype and a metabolic phenotype, nota- terized by mental retardation and various skin lesions. Cul- bly massive imidodipeptiduria (1). Among patients with clini- tured skin fibroblasts were obtained from two independent pa- cal symptoms of this disorder, skin ulcers, mental retardation, tients with abnormal prolidase. Using the polymerase chain and abnormalities in bones occur. Data on 28 patients with reaction, we amplified the entire coding region of human proli- prolidase deficiency have been documented in the past 20 dase mRNA derived from patients' fibroblasts. Nucleotide se- years (1), and we (2, 3) and others (4) have shown that there quence analysis of amplified cDNA products revealed a G to A were at least three types of mutations: immunologically substitution at position 826 in exon 12, where aspartic acid was crossreacting material (CRM)' negative, normal-size CRM, replaced by asparagine at the amino acid residue 276, in cells and enlarged CRM. from both patients. An analysis of the DNA showed that the We identified a cDNA clone corresponding to the human substitution was homozygous. prolidase (5), and determined the nucleotide sequence and the An expression plasmid clone containing a normal human primary structure of the enzyme (6). The prolidase gene is prolidase cDNA (pEPD-W) or mutant prolidase cDNA located on chromosome 19 (6) and consists of 15 exons and 14 (pEPD-M) was prepared, transfected, and tested for expres- introns, the span being > 130 kb (2a). We have now examined sion in NIH 3T3 cells. Incorporation of pEPD-W and pEPD- the molecular events in two patients with polypeptide positive M resulted in the synthesis of an immunological polypeptide prolidase deficiency. The nucleotide sequences of cDNAs that corresponded to human prolidase. Active human enzyme from the patients were analyzed by making comparisons with was detected in cells transfected with pEPD-W, but not in the sequence of cDNA and genomic DNA from a normal those transfected with pEPD-M. These results were compati- individual. We obtained evidence for the same amino acid ble with our observation of fibroblasts and confirmed that the substitution in the mutant enzymes, thereby confirming that substitution was responsible for the enzyme deficiency. this substitution is responsible for defects in the activity. As active prolidase was recovered in prolidase-deficient fi- broblasts transfected with pEPD-W, this restoration of proli- Methods dase activity after transfection means that gene replacement therapy for individuals with this human disorder can be given Materials. Purified human prolidase, a monoclonal antibody (EP2) due consideration. (J. Clin. Invest. 1990. 86:351-355.) Key directed to human prolidase, and antiserum raised against human words: prolidase- prolidase deficiency * polymerase chain reac- prolidase were prepared as described (2). [a-32P]dCTP (3,000 Ci/ Nitro- tion * point mutation -- transfection mmol) was purchased from ICN Radiochemicals (Irvine, CA). cellulose membrane was obtained from Schleicher and Schuell (Dassel, FRG). Restriction enzymes, T4 DNA ligase, and Taq DNA polymer- Introduction ase were purchased from Takara Shuzo Co. (Kyoto, Japan) and were used essentially as recommended by the supplier. Avian myeloblastosis Prolidase is a homodimeric enzyme catalyzing the hydrolysis virus reverse transcriptase and the reagent lipofectin were purchased of dipeptides with carboxy-terminal proline. This enzyme from Bethesda Research Laboratories (Gaithersburg, MD). A 7-deaza sequencing kit was obtained from Toyobo Co. (Osaka, Japan). Oligo- nucleotide were synthesized using a DNA synthesizer (model was in at the Annual Meeting of primers Part of this work presented Japanese 381 A; Applied Biosystems Inc., Foster City, CA). the Japanese Society for Inborn Errors of Metabolism, Fukui, Japan, Polymerase chain reaction of mRNA. Total cellular RNAs from October 1989. controls' and patients' fibroblasts were prepared using the guanidium Please address reprint requests to Dr. Fumio Endo, Department of Five were Medical School, Ku- thiocyanate procedure (7). oligonucleotide primers synthe- Pediatrics, Kumanmto University Honjo 1-1-1, sized for polymerase chain reaction (PCR) of mRNA, an oligonucleo- mamoto 860, Japan. and two sets of A2, B1, B2) 14 1990 and in revised 9 tide primer C, oligonucleotide primers (A1, Received for publication February form to the normal se- April 1990. consisting of 20 bases complementary published quence of human prolidase (6) (Fig. 1). J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/90/07/035i/05 $2.00 1. Abbreviations used in this paper: CRM, crossreacting material; PCR, Volume 86, July 1990, 351-355 polymerase chain reaction. A Single Nucleotide Change in Prolidase (PEPD) Gene from Prolidase Deficiency 351 A1* 933 bp *A2 Figure 1. Strategy for richia coli HBlBO were purified by two cycles of cesium chloride- +114~~ PCR amplification and ethidium bromide centrifugation. 3' 5' AUGGA AAAA the DNA sequence of Cells and DNA transfection. Cultured NIH 3T3 cells and fibroblasts Bi+ 975 bp *B2 oligonucleotide primers. with prolidase deficiency were used for transfection. Transfection of 4-C The PCR primers Al, DNA was performed using lipofection (13), according to instructions Primer Set A A2, B , and B2 were given by the supplier. Nearly confluent cells (80%) in 75-cm dishes Al;5'CCGGTGCCGGGCGAACATGG3' A2, B1 and B re- A2;5'CACCAGTCACCTGGCTTCAT3' used to amplify proli- were washed twice with 6 ml ofRPMI 1640 medium (Gibco Laborato- Primer Set B dase sequences from ries, Grand Island, NY), and 9 ml of RPMI 1640 medium containing Bl;5'TTGGAAAGCCTCTTCGAGCA3' specific primer C 10 jg of DNA and 30 ,ig of lipofection was added. To normalize for B2;5'CCCGGGAAACAGCACTGTTT3' primed DNA:RNA hy- Primer C by- any variability in the transfection efficiency, plasmid pCHl 10 which 5'GTGGTGCCTGCAAAAGGGTA3' primdsbrids. TheDNA:rNlarelative contained the E. coli lac Z gene sequence under the control of Simian priming sites and direc- virus 40 enhancer-promoter (14), was cotransfected as an internal tion of extension for each primer (arrows) are indicated. The bottom standard. The transfected cells were incubated for 6 h in a 7% CO2 panel shows the DNA sequence of PCR oligonucleotide primers; Al atmosphere at 370C, after which 9 ml of RPMI 1640 medium with (-16 to +4), A2 (958 to 977), B1 (664 to 683), B2 (1,620 to 1,639), 20% FCS (Gibco Laboratories) was added. After incubation of this and C (1,659 to 1,678) are complementary to the sequence of proli- preparation for 12 h, the medium was replaced with 18 ml of fresh dase (6). Of these, A2, B2, and C represent reverse or antisense se- medium with 10% FCS and the cells were harvested 2 d later. quences. Two segments, 933- and 975-bp long, were amplified with Cell extracts were prepared as described (3) and used for protein primer sets of A and B, respectively. The base number for the A of assay, immune precipitation, and immune blot analyses. Part of the the ATG start codon and the terminal G of the UAG stop codon are cell extracts were assayed for fl-galactosidase activity by incubation indicated. with O-nitrophenyl-B-D-galactopyranoside as described (15). The amount of protein was measured by a dye-binding method (kit from Bio-Rad Laboratories, Richmond, CA). A specific first-strand cDNA copy of human prolidase RNA was Enzyme assay, immune precipitation, and immune blot. Prolidase made from 10 tg of total cellular RNA using the oligonucleotide activities in the crude cell extracts were measured as described (16). primer C complementary to the RNA strand that serves as a primer for Immune precipitation ofhuman prolidase with the mouse monoclonal reverse transcriptase (8). Amplification of the first strand of cDNA was antibody (EP2) was carried out as described (2).
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