- Email: [email protected]
Identification and characterization of 5′ extension of mammalian agrin cDNA, the exons and the promoter sequences
Biochimica et Biophysica Acta 1494 (2000) 170^174
www.elsevier.com/locate/bba
Promoter paper
Identi¢cation and characterization of 5P extension of mammalian agrin cDNA, the exons and the promoter sequences Kazuhiro Shigemoto a; *, Sachiho Kubo b , Naoki Maruyama b , Seiichiro Yamada a , Keiko Obata a , Kazuo Kikuchi b , Ikuko Kondo a a
Department of Hygiene, School of Medicine, Ehime University, 514 Shitsukawa, Shigenobu-cho, Onsengun, Ehime 791-0295, Japan b Department of Molecular Pathology, Tokyo Metropolitan Institute for Gerontology, Tokyo 173, Japan Received 22 February 2000; received in revised form 10 August 2000 ; accepted 22 August 2000
Abstract Agrin, which is secreted from motor neurons, is essential for the formation and maintenance of the vertebrate neuromuscular junctions. Here we show the complete N-terminal sequence of the mammalian cDNA required for the expression and secretion as well as the intron/ exon structure and the 5P-flanking sequence required for basal promoter activity. The 5P-flanking region and the first exon are extremely GC rich and contain a CpG island. These features may account for hindrance in identification of the 5P end of the cDNA and the promoter region of the mammalian agrin gene. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Agrin; Neuromuscular junction; Synapse formation
Agrin, a large, multidomain heparan sulfate proteoglycan (HSPG), released from the nerve terminal has been shown to activate the MuSK receptor tyrosine kinase leading to the local accumulation of AchR in the postsynaptic membrane at the vertebrate neuromuscular junction [1^4]. Agrin may also have a complex role in the formation of synapses in central neurons [5]. The expression of agrin in neurons is regulated by synapse formation and by electric activity [6^8]. Agrin induces CREB (cAMP response element-binding protein) phosphorylation in hippocampal neurons in a Ca2 -dependent manner, and the early immediately gene c-fos in cortical neurons [9,10]. Recently, the accumulation of agrin in senile plaques and neuro¢brillary tangles of Alzheimer's disease brain as a major HSPG was shown and the contribution to the pathogenesis was suggested [11,12]. Agrin cDNAs have been cloned in rats, chicks and a marine ray [13,14]. Subsequent sequence analysis revealed that the agrin molecule contains several domains that are homologous to motifs found in other proteins of the extracellular matrix and that the molecular mass deduced from these cDNAs is more than 200 kDa. Chick agrin cDNA contains a signal sequence and a laminin-binding domain in the NH2 -terminal and
* Corresponding author. Fax: +81-89-960-5279; E-mail : [email protected]
can be secreted from transfected COS cells as endogenous agrin, however, the rat cDNA sequence lacks all of these properties [15]. Sequencing of cDNA from human agrin established that the N-terminal domain is highly conserved between humans and chicks, but human cDNA still lacks the signal sequence necessary for secretion [16]. In order to characterize the 5P-£anking region of the mouse agrin gene, we identi¢ed the previously unknown complete nucleotide sequence of the mouse agrin gene, the intron/exon structures of its N-terminal domain, and its promoter region required for the expression. To isolate mouse agrin cDNA containing the signal sequence and the ¢rst ATG codon, we generated an 872-bp mouse agrin cDNA probe reverse transcription-PCR using a set of primers and total RNA from C2C12 myotube cells. The primers were designed according to the results by Rupp et al. as follows : 5P-GTGCAGGGGAATGTTATGT-3P and 5P-CACACGGGCCCTGGTGCTTG-3P [17]. Using this probe, we isolated an agrin cDNA from the mouse C2C12 myotube cDNA library. The cDNA contained a 1.5-kb insert starting at position +79 (for the location, see Fig. 1). Since the cDNA clone isolated here still lacked a signal sequence that was found at the NH2 -terminus of soluble secretory molecules, additional cDNA clones were screened to isolate the region containing the ¢rst ATG codon. However, no such clones hybridized with the probe. To circumvent this problem, the next
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 1 4 - 1
BBAEXP 91475 31-10-00
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Fig. 1. 5P Sequence of mouse agrin mRNA and its immediate £anking region. An arrow indicates the major transcription start site. The ATG translation initiation site is in boldface type and numbered as +1. Consensus sequences for Sp1, c-Rel, Ik-3, MZF1 are underlined. An arrowhead shows the exon boundaries. The beginning of a 1.5-kb cDNA clone is shown by an asterisk. Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under accession no. AF190858 and Af190859.
step was to screen a mouse genomic library with a DNA probe spanning nucleotides +123 to +491. Of the two positive clones yielded by using 1U106 phages, nucleotide sequence analysis revealed two putative exons containing the homology sequence of chicken cDNA. Moreover, the 5P exon, which was 2 kb distant from the second exon, encoded not only a reading frame further upstream from the isolated cDNA but also an in-frame ATG followed by 24 mainly hydrophobic amino acids that were a possible signal sequence. This ATG codon matched the Kozak consensus sequence for a translation initiation site [18]. The 36 exons of mouse agrin gene previously identi¢ed,
including the 3P untranslated region, did not include the exon that encodes the initiator methionine and the signal sequence [13]. The ¢rst exon in that report began at position +528 in the extension sequences as shown here in Fig. 1. Since we could not ¢nd the third exon in two genomic clones containing the ¢rst two exons, we screened the same phage genomic library with a DNA probe corresponding to position +438 to +553 from the extension sequence, resulting in four positive clones. Subsequent nucleotide sequence analysis showed an exon at the corresponding position +470 to +518 in the extension located 5.5 kb
Table 1 5P Exons structure of the mouse agrin gene Exon
Nucleotide numbera
Amino acid
Length (bp)
1 2 3
368^207 208^469 470^517
1^69 70^157 158^174
275 262 48
a
Splice acceptor
Splice donor
Intron size (kb)
tccatgtccgctcagGTTCG acttttcccccctagACAAA
GCAAGgtgggccccggccgg AGAAGgtgcgtggtgagagt TGATGgtaagtgtggtggtg
2 10 5.5
Position 1 refers to A in the ¢rst in-frame ATG in the cDNA sequence reported in this paper.
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Fig. 2. Amino acid sequence alignment of the 5P sequence of mouse, human, chicken and rat agrin. The alignment was generated using ClustalW 1.7 at the Baylor College of Medicine (BCM) Search Launcher site (http://dot.imagen.bcm.tmc.edu:9331/multi-align/multi-align.html) and shaded using Boxshade 3.21 (http://www.isrec.isb-sib.ch/software/BOX form.html).
upstream from the most 5P exon reported by Rupp et al. [13]. The distance between the second and the third exons was about 10 kb as characterized by a long-distance PCR method and restriction enzyme mapping using mouse genomic DNA (data not shown). All GT and AG dinucleotide consensus sequences for the splicing signals were conserved on these exons as shown in Table 1. Now we can identify the complete intron/exon structure of the extension sequence. When the deduced amino acid sequence of this extension was compared with the chicken and human sequence shown in Fig. 2, the mouse agrin sequence identi¢ed had 94% identity with human agrin and 77% identity with chicken agrin, but no homology with that of the rat
Fig. 3. Secretion of recombinant agrin from COS-7 cells. A 4.9-kb agrin cDNA containing the ¢rst initiation codon was ampli¢ed by PCR using a set of primers, 5P-CCGTTCCGGGCTGCGCCATGGTCC-3P and 5PCATCTGCACAAGTTGGGCCAA-AGCGGCCAGG-3P, cloned into pCDNA3.1/Myc-His vector. Two Wg of 4.9-kb agrin cDNA in the vector was transfected into 3U105 COS-7 cells in 35-mm dishes using FuGENE 6 transfection reagent (Roche). Then, 3 days later, the supernatant was harvested and separated by SDS^PAGE on a 6% gel, transferred to a nitrocellulose membrane and detected by anti-myc monoclonal antibody (9E10) with a ECL detection system (Amersham). A mock transfection (Mock) provided the control.
Fig. 4. RNase protection assay for determination of the transcription initiation site. The 523-bp EcoRI^NotI fragment (from nucleotide residues 3461 to +62) was subcloned into pBluescript. This plasmid was digested by EcoRI and transcribed by T3 RNA polymerase to make an antisense probe, which was labeled with [K-32 P]UTP. The probe was incubated with 20 Wg of mouse C2C12 myotube RNA (lane 1) or tRNA (lane 2) and subjected to RNase digestion, as the manufacturer instructed (Ambion). The products were electrophoresed on a 6% polyacrylamide/7 M urea gel. The size of protected RNA fragment was determined using RNA size markers, as the relative mobility of RNA and DNA fragments in 7 M urea polyacrylamide gels di¡ers by approximately 5^10%. The template DNA fragments for RNA size markers were generated by PCR using reverse primer 5P-GGAAACAGCTATGACCATG-3P, 5P-TACCGGGCCCCCCCTCGAGG-3P, 5P-TTGGGTACCGGGCCCCCCCT3P and 5P-ACTATAGGGCGAATTGGGTA-3P with pBluescript plasmid DNA. The PCR fragments contained a T3 promoter derived from pBluescript and transcribed by T3 RNA polymerase with [K-32 P]UTP to generate 120-bp, 125-bp and 138-bp RNA.
BBAEXP 91475 31-10-00
K. Shigemoto et al. / Biochimica et Biophysica Acta 1494 (2000) 170^174
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Fig. 5. Plasmid construction and functional activity of mouse agrin promoter deletion mutants based on luciferase activity in transient transfected C2C12 cells. The deletion constructs were made from an HindIII/NotI 1.5-kb fragment cloned into pGVB basic vector (Promega). The ¢lled box denotes the luciferase reporter gene. Data are means þ S.E.M. of three independent experiments. The nucleotides are numbered from the transcription start site.
(Fig. 2). Moreover, Campanelli et al. performed expression analysis using the rat cDNA, however, the recombinant agrin located on the cell surface of transfected COS and CHO cells [19]. To prove that the isolated cDNA containing the extension was expressed as a secreted form, we cloned 4.9-kb agrin cDNA in an expression vector. This construct contained the putative ¢rst ATG codon and the signal sequence at the N-terminal region as well as the putative glycosaminoglycan attachment sites. The result of transiently transfecting the DNA into COS-7 cells was secretion of the recombinant protein with an apparent molecular mass between 400 and 600 kDa attached by a heparan sulfate glycosaminoglycan chain on SDS^PAGE in culture medium as expected (Fig. 3). We could express agrin as a soluble HSPG using isolated cDNA and these results solidly pave the way for further functional studies using whole molecules of mammalian agrin. Then we sought the transcription initiation site by ribonuclease protection assay. The results indicated that the transcription start site lay 68 bp upstream of the ATG codon (Fig. 4). The primer extension method was also performed to determine the start site, however, we could not obtain any signi¢cant bands probably by the secondary structure of agrin mRNA. Next we determined the promoter region for the basal expression in mouse C2C12 myoblast cells by a luciferase reporter gene assay (Fig. 5). Approximately 1.5 kb of the 5P-£anking region showed promoter activity in C2C12 cells. The deletion analysis indicated the basal promoter activity was remained in the 3461-bp upstream region. This region contains 11 GC boxes (GGGGCGGGGC) that might serve as binding sites for Sp1 but had no canonical TATA or CAAT box. Other transcription regulatory sites including c-Rel, Ik-3, MZF1 were also identi¢ed. The genomic region including the 11 Sp1-binding sites and the ¢rst exon extending from 3353 to +111 accommodated more than 80% G and C nucleotides and formed a CpG island. This feature may hinder the isolation of the 5P end of the mam-
malian cDNAs, and the 5P-£anking region. The promoter activity was also observed between 31.5 kb and 3461 bp of the 5P-£anking in C2C12 cells (Fig. 5). Both of the truncated constructs showed stronger transcriptional activity than the full-length construct. This ¢nding suggests the location of a repressor element between these truncated constructs. Agrin is expressed as tissue-speci¢c manners in adult tissues, spatiotemporal manners in developmental embryo and an activity-dependent manner in adult brain, therefore further studies will be needed to determine these regulatory mechanisms [20].
References [1] G. Tsen, W. Halfter, S. Kroger, G.J. Cole, J. Biol. Chem. 270 (1995) 3392^3399. [2] D.M. Valenzuela, T.N. Sitt, P.S. DiStefano, E. Rojas, K. Mattsson, D.L. Compton, L. Nunez, J.S. Park, J.L. Stark, D.R. Gies, S. Thomas, M.M. Le Beau, A.A. Fernald, N.G. Copeland, N.A. Jenkins, S.J. Burden, D.J. Glass, G.D. Yancopoulos, Neuron 15 (1995) 573^ 584. [3] T.M. DeChiara, D.C. Bowen, D.M. Valenzuela, M.V. Simmons, W.T. Poueymirou, S. Thomas, E. Kinetz, D.L. Compton, E. Rojas, J.S. Park, C. Smith, P.S. DiStefano, D.J. Glass, S.J. Burden, G.D. Yancopoulos, Cell 83 (1995) 312^322. [4] D.J. Glass, D.C. Bowen, T.N. Stitt, C. Radziejewski, J. Bruno, T.E. Ryan, D.R. Gies, S. Shah, K. Mattsson, S.J. Burden, P.S. DiStefano, D.M. Valenzuela, T.M. DeChiara, G.D. Yancopoulos, Cell 85 (1996) 513^523. [5] A. Ferreira, J. Cell. Sci. 112 (1999) 4729^4738. [6] L.T. O'Connor, J.C. Lauterborn, C.M. Gall, M.A. Smith, J. Neurosci. 14 (1994) 1141^1152. [7] L.T. O'Connor, J.C. Lauterborn, M.A. Smith, C.M. Gall, Brain Res. Mol. Brain Res. 33 (1995) 277^287. [8] N.A. Cohen, W.E. Kaufmann, P.F. Worley, F. Rupp, Neuroscience 76 (1997) 581^596. [9] R.R. Ji, C.M. Bose, C. Lesuisse, D. Qiu, J.C. Huang, Q. Zhang, F. Rupp, J. Neurosci. 18 (1998) 9695^9702. [10] L.G.W. Hilgenberg, C.L. Hoover, M.A. Smith, J. Neurosci. 19 (1999) 7384^7393. [11] J.E. Donahue, T.M. Berzin, M.S. Ra¢i, D.J. Glass, G.D. Yancopou-
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[12]
[13] [14] [15]
K. Shigemoto et al. / Biochimica et Biophysica Acta 1494 (2000) 170^174 los, J.R. Fallon, E.G. Stopa, Proc. Natl. Acad. Sci. USA 96 (1999) 6468^6472. M.M. Verbeek, I. Otte-Holler, J. van den Born, L.P. van den Heubel, G. David, P. Xesseling, R.M. de Waal, Am. J. Pathol. 155 (1999) 2115^2125. F. Rupp, D.G. Payan, C. Magill-Solc, D.M. Cowan, R.H. Scheller, Neuron 6 (1991) 811^823. K.W. Tsim, M.A. Ruegg, G. Escher, S. Kroger, U.J. McMahan, Neuron 8 (1992) 677^689. A.J. Denzer, M. Gesemann, B. Schumacher, M.A. Ruegg, J. Cell. Biol. 131 (1995) 1547^1560.
[16] A.J. Gro¡en, C.A. Buskens, T.H. van Kuppevelt, J.H. Veerkamp, L.A. Monnens, L.P. van den Heuvel, Eur. J. Biochem. 254 (1998) 123^128. [17] F. Rupp, T. Ozcelik, M. Linial, K. Peterson, U. Francke, R. Scheller, J. Neurosci. 12 (1992) 3535^3544. [18] M. Kozak, Nucleic Acids Res. 12 (1984) 857^872. [19] J.T. Campanelli, S.L. Roberds, K.P. Campbell, R.H. Scheller, Cell 77 (1994) 663^674. [20] W. Hoch, M. Ferns, J.T. Campanelli, Z.W. Hall, R.H. Scheller, Neuron 11 (1993) 479^490.
BBAEXP 91475 31-10-00
www.elsevier.com/locate/bba
Promoter paper
Identi¢cation and characterization of 5P extension of mammalian agrin cDNA, the exons and the promoter sequences Kazuhiro Shigemoto a; *, Sachiho Kubo b , Naoki Maruyama b , Seiichiro Yamada a , Keiko Obata a , Kazuo Kikuchi b , Ikuko Kondo a a
Department of Hygiene, School of Medicine, Ehime University, 514 Shitsukawa, Shigenobu-cho, Onsengun, Ehime 791-0295, Japan b Department of Molecular Pathology, Tokyo Metropolitan Institute for Gerontology, Tokyo 173, Japan Received 22 February 2000; received in revised form 10 August 2000 ; accepted 22 August 2000
Abstract Agrin, which is secreted from motor neurons, is essential for the formation and maintenance of the vertebrate neuromuscular junctions. Here we show the complete N-terminal sequence of the mammalian cDNA required for the expression and secretion as well as the intron/ exon structure and the 5P-flanking sequence required for basal promoter activity. The 5P-flanking region and the first exon are extremely GC rich and contain a CpG island. These features may account for hindrance in identification of the 5P end of the cDNA and the promoter region of the mammalian agrin gene. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Agrin; Neuromuscular junction; Synapse formation
Agrin, a large, multidomain heparan sulfate proteoglycan (HSPG), released from the nerve terminal has been shown to activate the MuSK receptor tyrosine kinase leading to the local accumulation of AchR in the postsynaptic membrane at the vertebrate neuromuscular junction [1^4]. Agrin may also have a complex role in the formation of synapses in central neurons [5]. The expression of agrin in neurons is regulated by synapse formation and by electric activity [6^8]. Agrin induces CREB (cAMP response element-binding protein) phosphorylation in hippocampal neurons in a Ca2 -dependent manner, and the early immediately gene c-fos in cortical neurons [9,10]. Recently, the accumulation of agrin in senile plaques and neuro¢brillary tangles of Alzheimer's disease brain as a major HSPG was shown and the contribution to the pathogenesis was suggested [11,12]. Agrin cDNAs have been cloned in rats, chicks and a marine ray [13,14]. Subsequent sequence analysis revealed that the agrin molecule contains several domains that are homologous to motifs found in other proteins of the extracellular matrix and that the molecular mass deduced from these cDNAs is more than 200 kDa. Chick agrin cDNA contains a signal sequence and a laminin-binding domain in the NH2 -terminal and
* Corresponding author. Fax: +81-89-960-5279; E-mail : [email protected]
can be secreted from transfected COS cells as endogenous agrin, however, the rat cDNA sequence lacks all of these properties [15]. Sequencing of cDNA from human agrin established that the N-terminal domain is highly conserved between humans and chicks, but human cDNA still lacks the signal sequence necessary for secretion [16]. In order to characterize the 5P-£anking region of the mouse agrin gene, we identi¢ed the previously unknown complete nucleotide sequence of the mouse agrin gene, the intron/exon structures of its N-terminal domain, and its promoter region required for the expression. To isolate mouse agrin cDNA containing the signal sequence and the ¢rst ATG codon, we generated an 872-bp mouse agrin cDNA probe reverse transcription-PCR using a set of primers and total RNA from C2C12 myotube cells. The primers were designed according to the results by Rupp et al. as follows : 5P-GTGCAGGGGAATGTTATGT-3P and 5P-CACACGGGCCCTGGTGCTTG-3P [17]. Using this probe, we isolated an agrin cDNA from the mouse C2C12 myotube cDNA library. The cDNA contained a 1.5-kb insert starting at position +79 (for the location, see Fig. 1). Since the cDNA clone isolated here still lacked a signal sequence that was found at the NH2 -terminus of soluble secretory molecules, additional cDNA clones were screened to isolate the region containing the ¢rst ATG codon. However, no such clones hybridized with the probe. To circumvent this problem, the next
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 1 4 - 1
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Fig. 1. 5P Sequence of mouse agrin mRNA and its immediate £anking region. An arrow indicates the major transcription start site. The ATG translation initiation site is in boldface type and numbered as +1. Consensus sequences for Sp1, c-Rel, Ik-3, MZF1 are underlined. An arrowhead shows the exon boundaries. The beginning of a 1.5-kb cDNA clone is shown by an asterisk. Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under accession no. AF190858 and Af190859.
step was to screen a mouse genomic library with a DNA probe spanning nucleotides +123 to +491. Of the two positive clones yielded by using 1U106 phages, nucleotide sequence analysis revealed two putative exons containing the homology sequence of chicken cDNA. Moreover, the 5P exon, which was 2 kb distant from the second exon, encoded not only a reading frame further upstream from the isolated cDNA but also an in-frame ATG followed by 24 mainly hydrophobic amino acids that were a possible signal sequence. This ATG codon matched the Kozak consensus sequence for a translation initiation site [18]. The 36 exons of mouse agrin gene previously identi¢ed,
including the 3P untranslated region, did not include the exon that encodes the initiator methionine and the signal sequence [13]. The ¢rst exon in that report began at position +528 in the extension sequences as shown here in Fig. 1. Since we could not ¢nd the third exon in two genomic clones containing the ¢rst two exons, we screened the same phage genomic library with a DNA probe corresponding to position +438 to +553 from the extension sequence, resulting in four positive clones. Subsequent nucleotide sequence analysis showed an exon at the corresponding position +470 to +518 in the extension located 5.5 kb
Table 1 5P Exons structure of the mouse agrin gene Exon
Nucleotide numbera
Amino acid
Length (bp)
1 2 3
368^207 208^469 470^517
1^69 70^157 158^174
275 262 48
a
Splice acceptor
Splice donor
Intron size (kb)
tccatgtccgctcagGTTCG acttttcccccctagACAAA
GCAAGgtgggccccggccgg AGAAGgtgcgtggtgagagt TGATGgtaagtgtggtggtg
2 10 5.5
Position 1 refers to A in the ¢rst in-frame ATG in the cDNA sequence reported in this paper.
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172
K. Shigemoto et al. / Biochimica et Biophysica Acta 1494 (2000) 170^174
Fig. 2. Amino acid sequence alignment of the 5P sequence of mouse, human, chicken and rat agrin. The alignment was generated using ClustalW 1.7 at the Baylor College of Medicine (BCM) Search Launcher site (http://dot.imagen.bcm.tmc.edu:9331/multi-align/multi-align.html) and shaded using Boxshade 3.21 (http://www.isrec.isb-sib.ch/software/BOX form.html).
upstream from the most 5P exon reported by Rupp et al. [13]. The distance between the second and the third exons was about 10 kb as characterized by a long-distance PCR method and restriction enzyme mapping using mouse genomic DNA (data not shown). All GT and AG dinucleotide consensus sequences for the splicing signals were conserved on these exons as shown in Table 1. Now we can identify the complete intron/exon structure of the extension sequence. When the deduced amino acid sequence of this extension was compared with the chicken and human sequence shown in Fig. 2, the mouse agrin sequence identi¢ed had 94% identity with human agrin and 77% identity with chicken agrin, but no homology with that of the rat
Fig. 3. Secretion of recombinant agrin from COS-7 cells. A 4.9-kb agrin cDNA containing the ¢rst initiation codon was ampli¢ed by PCR using a set of primers, 5P-CCGTTCCGGGCTGCGCCATGGTCC-3P and 5PCATCTGCACAAGTTGGGCCAA-AGCGGCCAGG-3P, cloned into pCDNA3.1/Myc-His vector. Two Wg of 4.9-kb agrin cDNA in the vector was transfected into 3U105 COS-7 cells in 35-mm dishes using FuGENE 6 transfection reagent (Roche). Then, 3 days later, the supernatant was harvested and separated by SDS^PAGE on a 6% gel, transferred to a nitrocellulose membrane and detected by anti-myc monoclonal antibody (9E10) with a ECL detection system (Amersham). A mock transfection (Mock) provided the control.
Fig. 4. RNase protection assay for determination of the transcription initiation site. The 523-bp EcoRI^NotI fragment (from nucleotide residues 3461 to +62) was subcloned into pBluescript. This plasmid was digested by EcoRI and transcribed by T3 RNA polymerase to make an antisense probe, which was labeled with [K-32 P]UTP. The probe was incubated with 20 Wg of mouse C2C12 myotube RNA (lane 1) or tRNA (lane 2) and subjected to RNase digestion, as the manufacturer instructed (Ambion). The products were electrophoresed on a 6% polyacrylamide/7 M urea gel. The size of protected RNA fragment was determined using RNA size markers, as the relative mobility of RNA and DNA fragments in 7 M urea polyacrylamide gels di¡ers by approximately 5^10%. The template DNA fragments for RNA size markers were generated by PCR using reverse primer 5P-GGAAACAGCTATGACCATG-3P, 5P-TACCGGGCCCCCCCTCGAGG-3P, 5P-TTGGGTACCGGGCCCCCCCT3P and 5P-ACTATAGGGCGAATTGGGTA-3P with pBluescript plasmid DNA. The PCR fragments contained a T3 promoter derived from pBluescript and transcribed by T3 RNA polymerase with [K-32 P]UTP to generate 120-bp, 125-bp and 138-bp RNA.
BBAEXP 91475 31-10-00
K. Shigemoto et al. / Biochimica et Biophysica Acta 1494 (2000) 170^174
173
Fig. 5. Plasmid construction and functional activity of mouse agrin promoter deletion mutants based on luciferase activity in transient transfected C2C12 cells. The deletion constructs were made from an HindIII/NotI 1.5-kb fragment cloned into pGVB basic vector (Promega). The ¢lled box denotes the luciferase reporter gene. Data are means þ S.E.M. of three independent experiments. The nucleotides are numbered from the transcription start site.
(Fig. 2). Moreover, Campanelli et al. performed expression analysis using the rat cDNA, however, the recombinant agrin located on the cell surface of transfected COS and CHO cells [19]. To prove that the isolated cDNA containing the extension was expressed as a secreted form, we cloned 4.9-kb agrin cDNA in an expression vector. This construct contained the putative ¢rst ATG codon and the signal sequence at the N-terminal region as well as the putative glycosaminoglycan attachment sites. The result of transiently transfecting the DNA into COS-7 cells was secretion of the recombinant protein with an apparent molecular mass between 400 and 600 kDa attached by a heparan sulfate glycosaminoglycan chain on SDS^PAGE in culture medium as expected (Fig. 3). We could express agrin as a soluble HSPG using isolated cDNA and these results solidly pave the way for further functional studies using whole molecules of mammalian agrin. Then we sought the transcription initiation site by ribonuclease protection assay. The results indicated that the transcription start site lay 68 bp upstream of the ATG codon (Fig. 4). The primer extension method was also performed to determine the start site, however, we could not obtain any signi¢cant bands probably by the secondary structure of agrin mRNA. Next we determined the promoter region for the basal expression in mouse C2C12 myoblast cells by a luciferase reporter gene assay (Fig. 5). Approximately 1.5 kb of the 5P-£anking region showed promoter activity in C2C12 cells. The deletion analysis indicated the basal promoter activity was remained in the 3461-bp upstream region. This region contains 11 GC boxes (GGGGCGGGGC) that might serve as binding sites for Sp1 but had no canonical TATA or CAAT box. Other transcription regulatory sites including c-Rel, Ik-3, MZF1 were also identi¢ed. The genomic region including the 11 Sp1-binding sites and the ¢rst exon extending from 3353 to +111 accommodated more than 80% G and C nucleotides and formed a CpG island. This feature may hinder the isolation of the 5P end of the mam-
malian cDNAs, and the 5P-£anking region. The promoter activity was also observed between 31.5 kb and 3461 bp of the 5P-£anking in C2C12 cells (Fig. 5). Both of the truncated constructs showed stronger transcriptional activity than the full-length construct. This ¢nding suggests the location of a repressor element between these truncated constructs. Agrin is expressed as tissue-speci¢c manners in adult tissues, spatiotemporal manners in developmental embryo and an activity-dependent manner in adult brain, therefore further studies will be needed to determine these regulatory mechanisms [20].
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