Characterization of the human MANB gene encoding lysosomal α-d -mannosidase

Gene 198 (1997) 351–357

Characterization of the human MANB gene encoding lysosomal a--mannosidase Nobuaki Wakamatsu *, Yasuo Gotoda, Shiro Saito, Hisaomi Kawai First Department of Internal Medicine, School of Medicine, The University of Tokushima, 3 Kuramoto-cho, Tokushima 770, Japan Received 25 March 1997; accepted 12 May 1997; Received by T. Sekiya

Abstract Genomic clones of human MANB gene encoding the lysosomal enzyme, a-mannosidase, have been isolated, sequenced and analyzed. The human MANB gene spans approximately 22 kb and consists of 24 exons. The 5∞ flanking region of the gene shows a high G+C content and has two Sp1 and three AP-2 sites. Promoter analysis using deletion constructs of the 5∞ flanking region fused to the bacterial CAT gene showed that 150 bp of 5∞ sequence could drive the expression of MANB in COS 7 cells. Determination of the sequence of the 5∞ end of the a-mannosidase mRNA by 5∞ RACE protocol showed that transcription is initiated from a cluster of sites centered −28 and −20 bp from the first in-frame ATG. These data demonstrate that, like other lysosomal enzyme genes such as those for b-glucuronidase or b-hexosaminidase, the human MANB gene is controlled by a short 5∞ flanking sequence located near the initiation codon. © 1997 Elsevier Science B.V. Keywords: a-Mannosidase; Gene structure; Promoter; CAT assay

1. Introduction a--mannosidase (EC 3.2.1.24) is a lysosomal hydrolase that catalyses the hydrolysis of terminal a-mannoside residues of N-linked oligosaccharides of glycoproteins. Lysosomal a-mannosidase occurs in two forms, a-mannosidase A and B (Cheng et al., 1986). Tsuji and Suzuki (1987) have purified a-mannosidase from placental tissues using a specific antibody for the enzyme and demonstrated that the a-mannosidase contains only 65 and 27 kDa polypeptides. This result and the fact that the two forms of the enzyme are indistinguishable immunologically (Cheng et al., 1986) suggest that they are derived from a common precursor polypeptide chain. The enzyme has been found in all mammalian tissues (Conchie et al., 1959). Mannosidosis caused by a defect of this enzyme leads to accumulation of mannose-rich oligosaccharides in lysosomes ( Thomas and * Corresponding author. Tel.: +81 886 313111, ext. 2302; Fax: +81 886 337121; e-mail:[email protected] Abbreviations: bp, base pair(s); kb, kilobase(s); CAT, chloramphenicol acetyltransferase; HSV, herpes simplex virus; TK, thymidine kinase; ELISA, enzyme-linked immunosorbent assay; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; TPA, 12-O-tetradecanoyl-phorbol 13-acetate. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 37 - 5

Beaudet, 1995). We examined and reported two siblings with a-mannosidosis presenting with coarse face, hearing loss, and spastic paraplegia ( Kawai et al., 1985). Recently, human MANB cDNA was isolated from retina/muscle cDNA libraries with PCR-mediated cloning methods (Nebes and Schmidt, 1994) and from human spleen and fibroblast cDNA sources by RT-PCR (Liao et al., 1996). The partial amino acid sequences of purified enzyme matched the deduced amino acid sequence of the cDNA ( Emiliani et al., 1995), and transfection experiments with the MANB cDNA gave increased lysosomal a-mannosidase enzyme activity in murine fibroblasts ( Wang et al., 1996) and Pichia pastoris (Liao et al., 1996). These data confirmed that the cloned cDNAs encode human lysosomal a-mannosidase. With the aim of determining the organization of the gene and analyzing molecular defects of a-mannosidosis, we have isolated and investigated a genomic DNA clone encoding the entire human MANB gene.

2. Materials and methods 2.1. Materials Cosmid libraries containing partial Sau3A digests of human genomic DNA were purchased from Stratagene

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(La Jolla, CA, USA). CAT ELISA kit was from 5 Prime3 Prime (Boulder, CO, USA). Human MANB cDNA clones, pHAM20, pHAM18 and pHAM31 were kindly provided by Dr. Vicki L. Nebes (Allegheny–Singer Research Institute, Pittsburgh, PA, USA). Human liver Marathon-Ready@ cDNA and Advantage@ cDNA PCR kit were from Clontech Laboratories (Palo Alto, CA, USA). 2.2. Isolation and sequencing of genomic clones One million clones of the cosmid libralies were screened using a 3.1 kb fragment of human MANB cDNA (Nebes and Schmidt, 1994) as a probe. Exoncontaining DNA fragments of the cosmid isolates were identified by Southern blot analysis using the same probe after digestion of the clones with EcoRI or BamHI. These fragments were subcloned into plasmid KS(+) and fine mapping of each genomic fragment was accomplished. DNA sequencing of clones was performed with an automated DNA Sequencer (SQ-5500, Hitachi, Tokyo, Japan) using a dye-primer cycle sequencing kit (Delta Taq, Amersham, Arlington Heights, IL, USA). 2.3. Construction of reporter genes used for promoter assays The vector B1.5, containing 1.5 kb of BamHI fragment (Fig. 1), was used to create the following reporter constructs: (1) The 370-bp NcoI fragment of a normal and a reverse orientation sequence was inserted into the BamHI site of pBLCAT3 (=pCAT3.5N or pCAT3.5NR). (2) The 150-bp SmaI–NcoI fragment was inserted into the BamHI site of pBLCAT3 (= pCAT1.5S ).

2.4. Cell culture, DNA transfection and promoter assays COS 7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum. Cells (5×105) were seeded the day before transfection at 70–80% confluence in 25 cm2 culture dishes. The COS 7 cells were transfected with 10 mg of test plasmid and 0.5 mg of pCMVb control plasmid, which is an expression vector containing the E. coli b-galactosidase gene driven by the human cytomegalovirus promoter/enhancer. Transfection was accomplished by the calcium phosphate coprecipitation method ( Wigler et al., 1978). Ten hours later, the medium was replaced with the fresh medium. After an additional 48 h culture, COS 7 cells were harvested and cell extracts were assayed for CAT activity, b-galactosidase activity (An et al., 1982), and protein concentration (Bradford, 1976). The CAT activity was calculated by determining the amount of CAT protein with a CAT ELISA kit (Neumann et al., 1987). 2.5. 5∞ RACE To determine the MANB transcription start sites, human liver Marathon-Ready@ cDNA was amplified with M1A antisense primer (5∞-CGAAGCCCATCTGCGCAAACAGCG-3∞, in exon 4 and 5 of MANB cDNA) and adaptor primer 1 (AP1:5∞-CCATCCTAATACGACTCACTATAGGGC-3∞) with KlenTaq polymerase. Amplification was carried out for 25 cycles: each cycle consisted of a 30-s denaturation at 94°C, 3 min annealing and extension at 68°C. The PCR product was cloned and sequenced using an antisense primer (M5A:5∞-CATGGTCCAGGGGCCTGCTGAGTC-3∞) in exon 1 of MANB gene. The PCR product and isolated

Fig. 1. Restriction map of the human MANB gene. The overall structure of the gene is presented on the top line. Exons are shown as solid boxes and numbered 1–24. Restriction maps using EcoRI and BamHI appear below the gene. Below the restriction maps are isolated cosmid clones, pCOS5.1, pCOS5.4, pCOS3.5 and pCOS3.4. The broken lines indicate extension of the clones further 5∞ or 3∞. Genomic fragments subcloned into plasmid KS(+) for exon localization and sequencing are shown below the cosmid clones.

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clones were reamplified with M5A and nested adaptor primer 2 (AP2:5∞-ACTCACTATAGGGCTCGAGCGGC-3∞) in the presence of [a-32P]dCTP and were electrophoresed in a 7% polyacrylamide gel.

putative CAT box. Two 25 bp direct repeats are at −292 and −261.

3. Results

To define the promoter of MANB gene, three deletion clones of the 5∞ region were constructed in CAT expression vectors and cotransfected with pCMVb in COS 7 cells. The constructs are presented in Fig. 4A. CAT activities were determined as CAT content in COS 7 cells using a CAT ELISA kit and are normalized to the activity obtained for pCAT3.5N. pCAT3.5N, containing 350 bp of an NcoI fragment, showed approximately 50% of the CAT activity of pBLCAT2, expressing the HSV TK promoter ( Fig. 4B, lanes 2 and 5). The construct containing only 150 bp of 5∞ sequence also showed 50% of the promoter activity of pBLCAT2 ( Fig. 4B, lanes 3 and 5). It is noteworthy that the promoter expressed in reverse orientation (pCAT3.5NR) showed higher activity than pCAT3.5N ( Fig. 4B, lane 4). These data demonstrate that as little as 150 bp of 5∞ flanking sequence was sufficient to promote the expression of CAT activity in COS 7 cells.

3.1. Isolation of the human MANB gene One million clones of human superCos cosmid libraries were screened using the human MANB cDNA (Nebes and Schmidt, 1994) as probe. Four overlapping clones, pCOS5.1, pCOS5.4, pCOS3.5 and pCOS3.4, were obtained. One of these clones, pCOS5.4, was found to contain the complete MANB gene and its structure was investigated in detail. The human MANB gene is approximately 22 kb long and consists of 24 exons (Fig. 1). 3.2. Nucleotide sequence of the MANB gene Six exon-containing EcoRI DNA fragments of 13.5, 6.0, 3.5, 3.5, 1.6 and 0.9 kb, of pCOS5.4 were subcloned into EcoRI site of plasmid KS(+). The nucleotide sequence of all exons, the exon/intron boundaries, and part of intron sequences were determined (Fig. 2). The sizes and sequences of the exons were identical to those of the cloned cDNAs except that the first in-frame ATG codon was found 69 bp upstream of the second ATG, which had been reported as a translation initiation site (Nebes and Schmidt, 1994; Liao et al., 1996). Also, three discrepancies in the sequence were identified, C/T935 ( Thr312Ile) and G/A1008 (Arg337Gln) in exon 7 (Nebes and Schmidt, 1994) and T/C1151 (His384Pro) in exon 9 (Liao et al., 1996). Other differences from the sequence of the MANB cDNA reported by Nebes and Schmidt have proved to be due to sequencing errors ( Wakamatsu, Gotoda, Kawai, unpublished data). The exons range in size from 79 bp (exon 10) to 228 bp (exon 21) and the introns range from 82 bp (intron 9) to 4.1 kb (intron 14). A putative polyadenylation signal, ATTAAA was found 84 bp downstream from the stop codon, TGA. 3.3. Nucleotide sequence of the MANB 5∞ region The nucleotide sequence of 0.8 kb of the proximal 5∞ flanking region and a part of exon 1 of the gene is shown in Fig. 3. The first 200 bp of 5∞ sequence shows a high G+C content (76%) with two Sp1 sites, in the form of a consensus GGGCGG or its inverse (Dynan and Tjian, 1985) at positions −96, −61, and three AP-2 sites, consensus CCC(A/C )N(G/C ) (G/C ) (G/C ) at −157, −145 and −91, respectively (Faisst and Meyer, 1992). Further upstream at position −460, there is one

3.4. Promoter activity of 5∞ flanking region of the MANB gene

3.5. Identification of transcription initiation site The 5∞ end of the coding sequence of a-mannosidase was determined using the RACE protocol. Human liver Marathon-Ready@ cDNA was amplified with a specific adaptor (AP1) and MANB antisense (M1A) primer. The size of the major PCR product was approximately 700 bp and this was the longest DNA fragment detected. The nucleotide sequence of this DNA fragment demonstrated that transcription initiation sites occurred at −28 and −20 bp from the first ATG codon in the MANB gene ( Fig. 5).

4. Discussion Our studies have shown that the MANB gene coding human lysosomal a-mannosidase contains 24 exons within a span of 22 kb. The three amino acid differences identified by comparison with the cloned cDNA, two in exon 7 and other in exon 9, were also in control DNA samples and most likely correspond to amino acid polymorphisms. The structure and nucleotide sequence of the human MANB gene presented here will be useful for the analysis of molecular defects of patients with amannosidosis. Two in-frame ATG codons were identified at the 5∞ end of the gene. The nucleotide sequence surrounding both ATG fit the Kozak consensus sequence (A/GCCATGG) for translation initiation ( Kozak, 1987), suggesting that the first is most likely the initia-

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Fig. 2. Nucleotide sequence of the 24 exons, a part of 23 introns and the 3∞ flanking region of the human MANB gene. Exon sequences are in boldface with the nucleotides in uppercase and the deduced amino acid sequence above the nucleotide sequence. The numbers correspond to the position of amino acid residues. The TGA translation termination codon is indicated by three dots, and the polyadenylation signal is underlined.

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Fig. 2. (continued )

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tion codon. Other lysosomal hydrolases, such as human glucocerebrosidase (Sorge et al., 1987) and b subunit of human b-hexosaminidase (Neote et al., 1990), also have in-frame two or three AUG codons in the upstream region of the mRNA, respectively. In both cases, all of these ATG can act as the translation initiation codon in expression experiments. However, in the latter case, only the first in-frame ATG is used exclusively in vivo when all three ATG are present (Neote et al., 1990). The CAT assays showed that only 150 bp of 5∞ flanking sequence from the first ATG of the MANB gene was required for promoter activity. Similar findings have been made for the promoters of other lysosomal enzymes, such as mouse b-glucuronidase (Shipley et al., 1991), the mouse Hexa gene ( Yamanaka et al., 1994; Wakamatsu et al., 1994), and human N-acetylgalactosamine-6-sulfatase gene (Nakashima et al., 1994), where promoter activity was obtained with as little as 100 bp of 5∞ flanking region. The occurrence of Sp1 and Ap-2 sequences within the first 150 bp of the 5∞ region suggests that these elements are probably the main regulatory elements controlling expression of human a-mannosidase mRNA in COS 7 cells. The high activity of the reverse orientation of pCAT3.5NR was unexpected (Fig. 4B, lane 4) and may reflect the activity of reverse orientation of the Sp1 site. In some housekeeping genes, it has been reported that the reverse orientated promoter has activity in transfection experiments (Johnson and Friedmann, 1990; Sitzler

Fig. 3. Nucleotide sequence of 0.8 kb of the 5∞ flanking region of the MANB gene. Nucleotide residue +1 denotes the A of the first ATG in exon 1, and the nucleotides preceding it are indicated by negative numbers. Possible control elements are underlined or are in boldface, and are identified below the indicated sequence. The restriction enzyme sites used for the CAT expression constructs are underlined.

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Fig. 4. Human MANB promoter activity in COS 7 cells. (A) The MANB promoter sequences are represented by lines flanked by single letters corresponding to the identity of the restriction enzymes used in their construction. N1 and N2=NcoI, S=SmaI. (B) Each CAT construct (10 mg) was cotransfected with pCMVb (0.5 mg) into COS 7 cells, and 58 h later CAT activities were measured. The transfection efficiency for each CAT construct was normalized to the b-galactosidase activity. The activities were normalized to that of pCAT3.5N ( lane 2). A histogram shows the relative CAT activity±S.E.M. of three independent experiments. Numbers correspond to the designation in (A).

Fig. 5. Two major transcription initiation sites of a-mannosidase mRNA in liver tissue. (A) The PCR product of anchor-ligated cDNA with M1A and AP1 primer (see Materials and Methods) and isolated PCR products (M1 and M2) were reamplified with M5A antisense primer and nested adaptor primer 2 (AP2), and were electrophoresed through 7% acrylamide gels. Lane 1, 5∞-RACE (RT product plus anchor oligonucleotide); lane 2, PCR product (M1); lane 3, PCR product (M2); lane 4, water blank (no anchor-ligated template). (B) Nucleotide sequence of PCR products M1 and M2. The bracket to the left of each panel shows a portion of the anchor sequence. (C ) Nucleotide sequence surrounding the transcription initiation sites in the MANB gene. The sites identified as M1 and M2 are indicated by arrows.

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et al., 1991; Nakashima et al., 1994). Indeed, the promoter of the lysosomal, N-acetylgalactosamine6-sulfatase gene (Nakashima et al., 1994) has strong activity even in reverse orientation and the Sp1 site is considered to be a regulatory element. This result is very similar to the case of MANB gene, but more detailed promoter analysis, treatment with cAMP and TPA, will be required to clarify the regulation of expression of the human MANB gene.

Acknowledgement These studies were supported by the special research funds of the president (to N.M.) and The Research Grant (8A-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare (to H.K.). We thank Dr. Tomoichi Enomoto and Emiko Takeda (Nissei-Co., Atsuki, Japan) for invaluable sequencing assistance. We are grateful to Dr. Toshio Matsumoto ( Tokushima University, Tokushima) for helpful discussions. We are also grateful to Dr. Roy A. Gravel (McGill University, Montreal Children’s Hospital Research Institute) for critical reading and helpful discussion of the manuscript.

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