Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase

125

Biochimica et Biophysica A cta, 1171 (1992) 125-128 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 90424

Short Sequence-Paper

Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase Yasukazu Yamada, Haruko Goto and Nobuaki Ogasawara Department of Genetics, Institute for DeL'elopmental Research, Aichi Prefectural Colony, Kasugai, Aichi (Japan) (Received 15 September 1992)

Key words: Erythrocyte AMP deaminase; cDNA; Cloning; Sequence

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.

AMP deaminase (EC 3.5.4.6) catalyzing the irreversible deamination of AMP to IMP is widely distributed in various mammalian cells and tissue-specific isozymes were found [1-3]. In human, there are three fundamental isoforms, isozyme M found in skeletal muscle, isozyme L in liver, and isozyme E1 existing as the major isozyme in erythrocyte, whereas the three parental isozymes in rat were A (musCle type), B (liver type) and C (heart type), respectively. The concentration of AMP deaminase in muscle is high compared to that in other tissues, cDNAs encoding rat and human muscle specific AMP deaminase (myoadenylate deaminase) consisted of 16 exons have been cloned [4,5], and two different genes for AMP deaminase have been identified [6]: AMPD-1, which is only expressed at high level in adult skeletal muscle and encodes muscle type isozyme; AMPD-2, which encodes liver type isozyme. Myoadenylate deaminase deficiency [7] was discovered in patients with muscle weakness and cramping after exercise. In contrast, erythrocyte AMP deaminase deficiency identified firstly by us [8,9], is clinically completely asymptomatic. The inheritance is autosomal recessive and the heterozygote frequency is estimated

Correspondence to: Y. Yamada, Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya-cho 713-8, Kasugai, Aichi 480-03, Japan. The sequence data reported in this paper have been submitted to the E M B L / G e n b a n k / D D B J databases under the accession number D12775.

at about 1/30. In this study, we determined a 3.7 kb cDNA sequence of the third gene, AMPD-3, encoding human erythrocyte AMP deaminase, prior to identify the gene mutation in the complete deficiency. The first screening from human spleen Agtl0 cDNA library [10], with a 327 bp SalI-EcoRI cDNA fragment of rat AMPD-1 as a probe, provided 16 positive phage clones and the second screening one positive clone. When the clone's DNA was digested by EcoRI, the electrophoresis of insert DNA (HS8) showed two fragments hybridizing with the rat AMPD-1 prove (Fig. 1A). The~e two EcoRI fragments (HS8L and HS8S) were subcloned into plasmid vector pUC18, and the nucleotide sequences were analyzed. The homology searches of HS8L and HS8S sequences to rat and human AMPD-1 [4,5] suggested that the HS8 was consisted of 5' region HS8S (1.2 kb) and 3' region HS8L (1.6 kb), connected by EcoRI sites. The HS8 cDNA contains an open reading frame of 1560 bp (520 amino acid) which showed 74% identity at the nucleotide level and 80% similarity at amino acid level with 3'-end of human AMPD-1 cDNA [5]. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The cDNA has a comparatively longer 3'-untranslated region (> 1.2 kb), whereas those were extremely short (13-17 bp) in both rat and human muscle genes previously reported [4,5]. Since a partial cDNA sequence of AMPD-2 corresponding to the exon 13 and 14 of AMPD-1 had been identified [6], in that region, the homologies between each two of the HS8 cDNA, rat

126 and human AMPD-1, and rat AMPD-2 were compared (Table I). The results suggest strongly that the HS8 is a portion of AMPD-3 cDNA encoding erythrocyte AMP deaminase. The HS8 contains about 2/3 of 3'-end of the coding region, but not 1/3 region at the 5'-end (Fig. 1A). The library was rescreened, but no positive clone larger than the original 2.8 kb HS8 clone was detected. Other cDNA libraries from human spleen, heart and Blymphoblasts in which AMPD-3 was expressed were also tested, but it was unsuccessful. Thus, we have tried to clone 5'-end of AMPD-3 cDNA by the method of 5'-RACE [11]. The reversetranscription of mRNA isolated from human Blymphoblasts was carried out using a specific RT primer E8B3 corresponding to 253-223 bp from the 5'-end of HS8. And then the PCR amplification was performed using 5' adaptor primer and a specific PCR primer E8B2 corresponding to 220-198 bp. A 816 bp clone (YAB202), having the same 220 bp sequence in 3'-end region as 5'-end of HS8, was obtained by cloning the PCR products into PstI digested pUC18 (Fig. 1B). Nucleotide sequence analysis indicated the YAB202 has an open reading frame. Further, by cloning the products between SalI and PstI sites, YAB207 clone was obtained, which has the SalI digested adapter

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Fig. 1. Identification of cDNA encoding human erythrocyte-specific AMP deaminase. The restriction sites are indicated: A, AccI; B, BamHI; D, DralII, E, EcoRI; Hc, HinclI; Hd, HindlII; Nc, NcoI; No, NotI; P, PstI; Sc, Sacl; SI, Sall; Sm, Smal; X, XhoI. Technically introduced sites are in parenthesis. The sense ( ~ ) and antisense ( ,-- ) primers are shown: AE2, 5'-GccATGgCGCGGCAGTTTCCCAA-3' (position: (-3)-20); AE7, 5'-GgATCCAAGTTCAGCCTTCATG-3' (844-865); A E l l , 5'-AaAgCTTCCTGCCCCTTTT C A A G G - 3 ' (1484-1507); BE9, 5 ' - G G T T G T A T c T c G A G T T G A A C T T G T C A - 3 ' (1183-1158); BE14, 5 ' - C C T T G T G T A G GAATTCCCTCA-3' (1963-1944); BE16, 5'-GAaGCtTCCTCTTC A C A G A G G A A A G - 3 ' (2411-2387); E8B2, 5'-GCtGCAGATGCT"ITTGGTTCATG-3' (990-969); E8B3, 5'-CAGGCTCCGTCTGGTATGTGTGCTTGATGAA-3' (1024-994); adaptor primer (ADT), 5'-GACTCGAGTCGACATCGA-3'. The base position numbers were counted by the A of the initiation codon ATG as base 1.

TABLE I

Comparison of four types of AMP deaminase cDNA The homologies between each two of rat and human AMPD-1, rat AMPD-2, and HS8 cDNA were compared in the region corresponding to the exons 13 and 14 of AMPD-1 (264 bp, 88 amino acid) at the nucleotide level (normal letter) and the deduced amino acid level (italic letter). Amino acid level

Nucleotide level rat AMPD-1 human AMPD-1 rat AMPD-2 human HS8

rat AMPD-1

human AMPD-I

rat AMPD-2

human HS8

81.5 69.3 72.7

98.9 66.3 67.8

72.7 71.6

80.7 79.5 79.5 -

72.0

primer sequence, poly(dT), and a 371 bp including a 174 bp open reading frame at the 3'-end. The amplification using AE2 in YAB207 and BE9 in HS8 resulted in a reasonable size of product (PC209), indicating the order, YAB207-YAB202-HS8, and the connection of YAB207 to YAB202 at PstI site. Further, by the analysis (direct sequencing or numbers of subcloning and sequencing) of the PC209 and two additional products (PC714, using primers AE7 and BE14; PCl116, using A E l l and BE16), the sequence of coding region of AMPD-3 cDNA was confirmed (Fig. 1C). Thus, the entire cDNA sequence of 3732 bp contained a 2301 bp open reading frame encoding 767 amino acids and resulted in 89 kDa protein (Fig. 2). Preliminarily, we constructed the expression plasmid by ligating the blunted entire coding region into pUC18 HincII site to produce lacZ'-AMP deaminase fusion protein. Escherichia coli does not exhibit detectable AMP deaminase activity without transformation with the expression plasmid. The deaminase activity was observed in the lysate from E. coli transformed with the plasmid in the sense orientation of coding region, but not in the opposite orientation. Western blot analyses were done on these extracts, and a band at approx. 85 kDa was probed with antisera specific for isozyme El, only in the deaminase active extracts. These expression results, as well as homologies in nucleotide and amino acid sequences with those of other isozymes (AMPD-1 and 2), indicate that the identified sequence is AMPD-3 cDNA encoding erythrocyte AMP deaminase. Identification of human AMPD-3 encoding erythrocyte AMP deaminase is indispensable to study gene mutation of the interesting enzyme deficiency [9]. In this study, we elucidated the 3.7 kb entire cDNA sequence of AMPD-3. The molecular analyses of gene mutation responsible for the deficiency are now in progress using RT-PCR technique coupled with direct sequencing.

127 GACTCTCCTAAAGGGCAGATGAAGATCAGAGCTTTGCACCCTGTGATGCCATTTTAATCAACCCTGCTTGG -121 TTTTAGAGGATTGCTCCcGTGGGTCACTTGAGGCAGGCTCCACCTTCCCCAGGA~AGTGGCAGAGTcCAGccAGcG~TCGGAGcTGGAGGccCACGTGGGAGCAGTGAGcGGCTCTGAG ATGCCGCGGCAGTTTCCCAAGCTGAACATC~CTGAAGT~GATGAGCAAGTCCGG~T~CTGGCG~AGM~GGTGTTTGCTAAAGTGCTCCGAGAAGAGGACAGcAM~GATGCCC~GT CCCTG M P R 0 F P g L N I S E V fl f 0 V R L L A E K V F A K V L R E g D $ K D A L S L

-I

120 40

TTCACTGTCCCAGAGGACTGCCCCATcGGGCkA.~.GG.~GCCAAGGAGAGGGAGCrGCAGAA~AGCTGGCAGAGCAG~GTCTGT~AGACCGC~G~G~GTTTc~GATG 240 F T V P E D C P I G 0 K E A K E R E L 0 K E L A E 0 I[ S V E T A K R K I[ S F I( M 80 ATTCGGTCCCAGTCCCTGTCTcTG¢AAATGCCGCCACAGCAAGATTGGAAGGGCcCCCCGGCAGCCAGTCCGGCCATGTCTCcCACAACCcCTGTGGTCACTGGAGCCACTTCCCTGCCC I R S Q S L S L Q M P P 0 0 D W K G P P A A S P A M S P T T P V V T G A T S L P

360 120

ACGCCAGCACCCTATGCCATGCCTGAGTTCCAGCGGGTCACCATCAGCGGAGATTACTGTGCCGGGATCACTTTGGAG~ACTATGAGCAGGCAGCCAAGAGTCTGGCCAAGGCCCTAATG 480 T P A P Y A M P E F 0 R V T I S G D Y C A G I T L E D Y E 0 A A • $ L A K A L M 180 AT C CGGGAGAAGTATGCGCGG CT C GC CTA C CACCGCTT C CCGCGGAT CA CAT C CCAGTACCTGGGTCAT C CGCGGGCGGATA CTGCA C CT C CGGAAGAGGGCCT T C CA GA C TT C CA C C CT I R E K Y A R L A Y H R F P R I T S 0 Y L G H P R A D T A P P E E G L P D F H P

600 200

CCTCCACTGCCCCAGGAAGACCCCTACTG~CTGGATGATGCACCCCCCAACCTGGATTACTTGGTC~ACATG~AGGGGGGCATCCT~TTTGTGTATGATAACAAGAAGATGCTGGAGCAC 720 P P L P 0 E D P Y C L D D A P P If L D Y L V H M Q G G I L F V Y D N K K M L E H 240 CAGGAGCCGCACAGCCTACCCTACCCCGACCTGGAGACCTACACGGTGGACATGAGCCACATCCTGGCTCTCATCACCGATGGCCCCACGAAAAC•TATTGTCACCGGCGACTGAACTTT840 0 E P H S L P Y P D L E T Y T V D M S H I L A L I T D G P T K T Y C H R R L N F 280 CTGGAATCCAAGTTCAGCCTTCATGAGATGTTAAACGAAATGTCCGAGTTCAA.~GAGTTGAAGAGTAACCCCCACCGGGA~TCTAT~CGTGAG~GGTGGACACACACATCCATGCG 960 L E S K F S L H E M L N E M S E F K E L K S N P H R D F Y N V R K V D T H I H A 320 GCCGCCTGCATG~A~AAAAGCATCTGCTGCGCTTCATCAAG~ACACATACCAGACGGAGCCTGACAGGACTGTGGCAGAGAAGCGGGGCCGGAAGATCA~CTGCGGCAGGTGTTTGAC A A C M N 0 K H L L R F I K H T Y 0 T E P D R T V A E g R G R K I T L R 0 V F D

1080 360

GGCCTGCACATGGACCC•TACGACCTCACTGTGGACTCACTGGATGTCCACGCGGGCCGGCAGACATT•CA•CGCTTTGACAAGTT•AACTC•AAATACAACCCTGTGGGGGCCAGTGAG 1200 G L H M D P Y D L T V D S L D V H A G R 0 T F H R F D K F N S K Y N P V G A S E 400 CTGCGTGACCTGTATTTGAAAACTGAkAACTATCTGGGAGGA~AGTACTTTGCTCG~ATGGTCAAGGAGGTT~CC~GGGAG~TG~A~GAGAG~AAGTACCAGTACTCAGAGCCACGGCTC L R D L Y L K T E N Y L G G E Y F A R M V K g V h R E L E E S K Y 0 Y S E P R L

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TCCATCTACG~CCGCAGTCCT~AGGAGTGGCCCAACCTGGCCTACTGGTTCAT~A~CACAAG~TCTACTCT~CCAACATGCG~TGGAT~ATC~AGGTGCC~GGATTTATGA~ATATTT S I Y G R S P E E W P N L A Y W F I 0 H K V Y S P N M R W I I 0 V P R I Y D I F

1440 480

AGGTCAAAG~AGCTGCTGCCAAACTTTGGGAAC-ATGCTGGAGAACATCTTCCTGCCCCTTTTC~GGCCACTATCAACCCCCAAGATCATCGAGAGCTTCACCTCTTCCTTAAATATGTG 1560 R S K K L L P N F G K M L E N I F L P L F K A T 'I N P 0 D H R E L H L F £ K Y V 520 ACGGGGTTTGACAGCGTGGATGATGAGTCCAAGCACAGCGACCACATGTTTT~CGACAAGAGCCCAAACCCGGACGTCTGGACCAGTGAGCAGAACCCACCCTACAGCTA~TACCTGTAC 1680 T G F D S V D D E S K H S D H M F S D K S P N P D V W T S E 0 N P P Y S Y Y L Y 560

TACATGTATGCCAACATCATGGTGCTCAACAACCTCCGCAGGGAGCGCGGCCTGAGCACGTTCCTGTTCCGGCCGCACTGTGGGGAAGCCGGCTCCATCACCCACCTGGTGTCTGCCTTC Y

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ATTGCAGCTCAAGTGTGGAAGCTGAGCACCTGCGACCTGTGTGAGATCGCCAGGAACAGCGTGCTGCAGAGCGGCCTCTCG~AT~AGGA~AAGCAAAAGTTTCTGGGACA~AATTATTAT I A A Q V W K L S T C D L C E I A R N S V L 0 S G L S H 0 E K 0 K F

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CTCGAATATTCCAAGAACCCTCTGAGGG~ /TTCCTACACAAGGGACTGCATGTTTCTCTTTCCACCGATGACCCCATG~AGTTCCACTACACGAAGGAAGCACTTATC G ' AAG~TATGCC L

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2040 680

2160 720

AAAGAAGGACCTGAAGGAAATGATATTCGAAAGACA.A~ATGTC'~CTCAGATCCGGATGGCATTCCGATATGAGACCTTATGCAATGAGCTCAGCTTCCTGTCTGATGCTATG~TCAGAA 2280 K E G P E G N D I R K T N V A 0 I R M A F £ Y E T L C N E L $ F L S D A M K S E 760 GAGATCACCGCCTTGACCAACTAGGTCCAGCATTTGACATGCATTTTAACTTTTTGGTTCAATTTCAAGTCTGCTGTGGCTAATAGTGGTCAAGATTCCGAACTAGGACTTTCCTCTGTG 2400 E I T A L T N * 767

TCTGGGAGCTGAGCACTTGTCTATACTTGTTCCTAATTTTCCAAGTATTTCTCTTGM~CTGCCAGTGCCTGAACTGTT~GGGCCAG~ATTTTCCCTGGTCAGATGCCAAGTAACATGTG

2520 2640

GTTTTCTGCCATACTTTTCTCCAG~GCCAGGAG~CT/~TTGGTA~TTGTTCATTTCAGCCTCTGGATGGCTGGCTGCCTTAAACACAATC~TTTC~GCTCCATTTCAT~GGGGCT

2760

AAGAGGATGCCTCTGAAGAAATTTTAAACTGGTGATTTTGGTTGcACTGCTCACTTTAAGAGTTAACATGCTCACTTGTTAGTATTTCTGAGTAACAAGATGGTGACTTCTCCTTGGGGA

AcTTTG/u~.GGAGTTAAGATGG/L~GACTTCCTTCTTGACA~ATTGTGTTTTTAGTTGAATTTcTT/U~CGTTTTATTTAGCC~CCTTCCCTCTTTCTAGTTGG~GCC~TGTACTCA 2880 TGAA~CAGCCACTCCTATTCTGAGTCTTGGTTTCTTCACCTAG/U~GTGA~TTTGGACTAGATGAGTGGCTTTCAGGGTGTT~GTG~TCTCCTCATG~TACTTTAGGGTGGGGG 3000

AGGGAGTGAGTGATGCTCAGGGGCTGTCAAAGTGACTGCGTTCATCAGTTTACACTGGGGCTGCTATAT~TATTTTCATTTG~CG~G~CTTC~GCACAGGACTAGATGATCT CTGTTCCTTTTGGCTCTAATATGCTACAACTGTAGGCCAATTATCACTTTACCAATTAAGAGTTAGGCCAGATAA@TGAAATTTAACTTAAGG~CACACAGCTAAT~GT~TAGGCCTA

3120 3240 AACTGGATTTCCTTATTCCA.4ATC~TGTCTTTTCCCCACTATTCCATTAGACCCCACA~tTGTTAGTTGTGTGTGTGTGTGTGTGTTTTT~TCACTGT~CCGGGTGCATTTTTTT~G 3360 GCAAAATTTCTCCCTTATCTACTGTGATGACTTCAGAAGATACAATGGTCCCAGGG@CCAAGTAGAAAGCATTTTT~GATT~TCTG~TT~GCTTTATCAGTGTACTCTTTATCTG 3480 TGTTACTAGTGCCTGGTATGTAGTAGGTGCTCAATAAATGCATATTGAATAACCGGAATTC 3541

Fig. 2. Nucleotide and deduced amino acid sequence for human erythroeyteAMP deaminase eDNA. A 3732 bp eDNA contains an open reading frame of 2301 bp which encodes 767 amino acid chain resulting in a 89 kDa protein. The polyadenylation consensus signal is underlined.

We are grateful to Dr. R.L. Sabina, Medical College of Wisconsin, for providing the rat AMPD-1 cDNA done, and Dr. S. Ohno, Yokohama City University School of Medicine, for the gift of the Agtl0 human

spleen cDNA library. This work was supported by a Gout Research Foundation grant and an Intractable Disease grant from Ministry of Health and Welfare of Japan.

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6 Morisaki, T., Sabina, R.L. and Holmes, E.W. (1990) J. Biol. Chem. 265, 11482-11486. 7 Fishbein, W.N., Arumblustmacher, V.W. and Griffin, J.L. (1978) Science 200, 545-548. 8 Ogasawara, N., Goto, H., Yamada, Y., Nishigaki, 1., Itoh, T. and Hasegawa, I. (1984) Biochem. Biophys. Res. Commun. 122, 13441349. 9 Ogasawara, N., Goto, H., Yamada, Y., Nishigaki, I., Itoh, T., Hasegawa, I. and Park, K.S. (1987) Human Genet. 75, 15-18. 10 Ohno, S., Emori, Y., Sugihara, H., Imajoh, S. and Suzuki, K. (1987) Methods Enzymol. 139, 363-379. 11 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002.