- Email: [email protected]
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
Agricultural Sciences in China
July 2009
2009, 8(7): 777-783
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L. WANG Sheng, ZHAO Guo-hong, JIA Yin-hua and DU Xiong-ming Cotton Germplasm Division, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang 455000, P.R.China
Abstract The aim of this study was to clone CAP (adenylyl cyclase-associated protein) gene from Gossypium arboreum L. and develop a platform for expressing and purifying CAP protein, which is a base for the construction and function researches of CAP. In this work, a CAP homolog from cotton (DPL971) ovule was identified and cloned. And the cDNA sequence consisted of an open reading frame of 1 416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa. To gain insight on the CAP role in cotton fiber development, the cloned CAP cDNA was expressed. A significant higher yield pure protein was obtained with the chromatographic method. Further experiments showed that the purified protein can bind with the actin in vitro indicating that the recombinant cotton CAP is functional. The procedure described here produced high yield pure protein through one chromatographic step, suitable for further structure-function studies. Key words: adenylyl cyclase-associated protein, CAP, cotton fiber, protein expression, protein purification, Gossypium arboreum L.
INTRODUCTION Cotton (Gossypium hirsutum L.) is a major source of textile fibers. Fiber cell initiation in the epidermal cells of cotton ovules represents a unique example of trichome development in higher plants (Ramsey and Berlin 1976). Many candidate genes that are expressed in fiber cells have been cloned and characterized (Lee et al. 2007). For example, the expression of some cytoskeleton genes is associated with the fiber development (Amor et al. 1995; Pear et al. 1996; Kawai et al. 1998; Babb and Haigler 2001; Ji et al. 2003; Ruan et al. 2005). Actin cytoskeleton is a key component of plant cytoskeleton which involves in the cell division and expansion (Kost et al. 2002). The reorganization of the actin cytoskeleton is thought to be mediated by actin binding proteins. The majority of actin binding proteins interReceived 21 July, 2008
act with filamentous (F) actin and serve to regulate assembly of the actin filaments and to link other proteins to the F-actin network. Actin sequestering factors can either enhance polymerization or cause disassembly of F-actin. Examples of these actin binding proteins include profiling (Sohn and GoldschmidtClermont 1994), β-thymosins (Carlier and Pantaloni 1994), Wiskott-Aldrich syndrome protein (WASp) (Zigmond 2000), and adenylyl cyclase-associated proteins (CAPs). To the date, CAP cDNA has been isolated from Saccharomyces cerevisiae (Field et al. 1990), Homo sapiens (Matviw et al. 1992), Rattus norvegicus (Zelicof et al. 1993), Mus musculus (Vojtek and Cooper 1993), hydra (Fenger et al. 1994), Dictyostelium discoideum (Gottwald et al. 1996), Lentinus edodes (Zhou et al. 1998), Drosophila (Baum et al. 2000), Arabidopsis thaliana (Barrero et al. 2002), and Gossypium hirsutum
Accepted 10 January, 2009
Correspondence DU Xiong-ming, Tel: +86-372-2525352, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60278-3
778
(Kawai et al. 1998). Deletion analysis has revealed that CAPs are multifunction proteins with several structural domains. It has been well understood that CAP contains four crucial functional domains: adenylyl cyclase domain, actin binding domain, Src homology 3 (SH3) binding domain (poly-proline amino acid domain), and multimerization domain (Hubberstey and Mottillo 2002). These domains seem to be characteristic of CAP and the related homologues because no other proteins present in databases share such motifs. These conservation, and the presence of the unique amino acid sequence (e.g., Arg, Leu, Glu repeats domain in N-terminal), suggest that CAP could play a similar role in all these organisms. Previously, the studies in A. thaliana and Nicotiana tabacum addressed the importance of CAP in actin cytoskeleton networking, which is significant for proper cell division and elongation (Barrero et al. 2002, 2003). In this paper, we report the identification, purification, and characterization of G. arboreum CAP, and describe a strategy for the production of high yield pure GaCAP for further estimating its roles in fiber cell development, and its relationship with other proteins of this important process.
MATERIALS AND METHODS Plant materials In this study, Gossypium arboreum DPL971 at nine successive selfed generations was used. The cotton, DPL971, was planted in a field (36°06´N lat., 114°21´E long.). Ovules from -1 to 9 DPA were harvested from individual plants of DPL971. Cotton fruit age was determined by tagging the petioles of a flower when it was fully opened. Leaves were also collected at anthesis. All selected materials were stored at -80°C until use.
Reagents, strains and plasmids The oligonucleotides (Table) were synthesized from Sangon (Shanghai, China). PCR components were from TaKaRa (Dalian, China). Restriction enzymes were from Fermentas (Burlington, ON, Canada). The reagents for protein purification and electrophoresis were purchased from USB (St. Louis, MO, USA) and Sigma (St. Louis, MO, USA). Actin protein (human platelet non-muscle, > 95% pure) was purchased from Cytosk-
WANG Sheng et al.
eleton (Denver, CO, USA). T-vector (pGEM-T easy) was purchased from Promega (Madison, WI). Plasmid pET-28a and Escherichia coli BL21 (DE3) pLysS (genotype: F- ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR) were from Novagen (Darmstadt, Germany). All other chemicals were of analytical grade.
Nucleotide acid manipulation and sequence analysis Genomic DNA was obtained from leaves using a lysis buffer containing 2% (w/v) cetyltrimethylammonium bromide (CTAB), 100 mmol L-1 Tris-HCl, 10 mmol L-1 EDTA, 3 M NaCl and 2% (v/v) of 2-mercaptoethanol. Total RNA was isolated from ovules according to the method described previously (Wan and Wilkins 1994), and its cDNA was synthesized using 1 g of total RNA following the RNA PCR Kit (TaKaRa, Dalian, China). To amplify the full-length CAP from genomic DNA, two primers were designed from the GhCAP cDNA sequence (GenBank accession no. AB014884) as shown in Table. Touch-down PCR was performed with above DNA over 30 cycles (45 s at 94°C, 1 min at 58-52°C, -0.2°C/ cycle, and 3 min at 72°C) under standard reaction condition as recommended by the supplier (PTC-100, MJ). The PCR products were cloned and sequenced.
Expression and purification of the G. arboreum CAP gene All purification steps were carried out at 25°C. CAP was monitored by SDS-PAGE. Protein was detected by staining with silver stain. To express the CAP gene, two primers for the full length CAP cDNA were synthesized (Table) and amplified by PCR under the following condition: heating at 94°C for 5 min, followed by 30 cycles of denaturation at Table Primer sequences for cloning and expression Primer name 1) CCRI205F CCRI205R CCRI205FN CCRI205RE 1) 2)
Sequence (5´
3´) 2)
AAT GGA GGC GAA GCT GAT AG AGG CCT CAT TTA TGC TCC TG CTA GCT AGC ATG GAG GCG AAG CTG ATA GAG CCG GAA TTC CGG TGC TCC TGA GTG CGA CAC
Application Cloning Cloning Expression Expression
N, Nhe I; E, EcoR I. The enzyme sites are underlined.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
94°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 120 s. The 5´-primer (CCRI205FN) contained a unique Nhe I site and the 3´-primer (CCRI205RE) contained a common unique EcoR I site. CCRI205RE does not have a stop codon. The PCR product that contained a Nhe I site at the 5´ end and a EcoR I site at the 3´ end was subcloned into the pGEM-T easy vector and transformed into E. coli XL1-Blue competent cells. The recombinant plasmid was digested with Nhe I and EcoR I, purified by 0.8% agarose gel electrophoresis, and ligated to the pET-28a(+) expression vector, which contained the inducible T7 promoter, carried an N-terminal His-tag and plus an optional C-terminal His-tag sequence which was prepared by using the stop codon-excluded primer. The ligated plasmids were transformed into the expression host, E. coli BL21 (DE3) pLysS, which was confirmed by restriction enzyme analysis. Then, E. coli containing the gene were cultured to the mid-log phase (OD600 0.6-1) in 200 mL of LB broth containing 30 mg mL-1 of kanamycin and then induced with 1 mmol L -1 IPTG at 25°C over night. The cultured cells were centrifuged at 5 000 × g for 25 min at 4°C, resuspended in 7 mL of ice-cold 1× binding buffer [20 mmol L-1 sodium phosphate buffer (pH 7.4) containing 500 mmol L-1 NaCl and 20 mmol L-1 imidazole], sonicated at 4°C, and centrifuged at 10 000 × g for 30 min. The resulting supernatant was filtered through a 0.4 mm membrane to prevent clogging of resins, and applied to the HiTrap IMAC HP (GE Healthcare), charged with 4 column volumes of 1× charge buffer (0.1 mol L-1 NiSO4), and washed with 5 column volumes of 1 × elution buffer (20 mmol L-1 sodium phosphate buffer containing 0.5 mol L-1 NaCl and 0.5 mol L-1 imidazole) and 10 column volumes of 1× binding buffer. The homogeneity of CAP was tested by SDS-PAGE gel electrophoresis. Denaturing gel electrophoresis was performed with the Bio-Rad Mini-Protean II System using 15% polyacrylamide/bisacrylamide gels as described previously (Laemmli 1970).
Protein determination Protein concentration was measured by a Bradford assay based on a bovine serum albumin (Sigma, St. Louis, MO, USA) standard curve (Bradford 1976). Reagents for protein assays were purchased from Bio-Rad (Hercules, CA, USA).
779
Optimum temperature and stability CAPs have been shown to inhibit actin polymerisation in vitro, by sequestering monomeric actin (Amberg et al. 1995; Freeman et al. 1995; Gottwald et al. 1996). So, here, we used actin as the substrate and made the purified CAP protein bound with actin. Separation of actin from CAP/actin complex was performed as described previously (Gieselmann and Mann 1992). Actin concentration was determined photometrically with an absorption coefficient of 0.64 for 1 mg mL-1 at 290 nm (Lehrer and Kerwar 1972). Optimum temperature was determined by varying assay temperature in the reaction mixture and thermal stability was obtained after preincubation of CAP in the different temperatures (range from 5 to 75°C) for 30 min.
Optimum pH and stability Optimum pH was obtained by varying assay pH value in the reaction mixture. pH stability was investigated after preincubation of CAP in different pH buffers for 30 min, then rapidly recovered to the assay pH 7.0. In all cases, overlaps were obtained when buffers were changed so that correction could be made for spurious buffer effects.
Protein sequence analysis of CAP The amino acid sequence of CAP was analyzed using standard protein-protein BLAST. Multiple sequence alignment was generated by using the program Clustal X.
RESULTS Cloning of the Gossypium arboreum CAP gene CAP cDNA sequence was obtained by RT-PCR using specific primers. After amplification, the products were cloned into pGEM-T easy vector and sequenced (GenBank accession no. EU106853). Sequence analysis showed that DPL971 cDNA encode 471 amino acid polypeptides which was structurally homologous to the GhCAP protein and other CAP homologs, therefore we designated the isolated genes as GaCAP. The CAP protein in G. arboreum (471 amino acid residues) and
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
780
A. thaliana (476 amino acid residues) were similar in size, but the former genomic sequence (~4 kb) was much larger than the later (~2.9 kb) indicating the sequence complexity of introns in G. arboreum. To establish the gene structure of CAP, the genomic PCR was performed. An amplified fragment about 4 kb was cloned and sequenced (GenBank accession no. EU106854). Comparison of cDNA and genomic sequences showed that the gene contained 10 exons and nine introns. The G. arboreum CAP shares high homology with other CAPs. The alignment of the amino acid sequences between G. arboreum CAP and the CAPs from other species is shown in Fig.1. The identity between the C-terminal domain of G. arboreum L. CAPs and those of A. thaliana and Oryza sativa CAP was 83%, while the Nterminal domains of G. arboreum L. CAP were 74 and 63% identical with the corresponding regions of A. thaliana and O. sativa CAP, respectively. So the C-terminal domain of G. arboreum L. CAP showed higher homology with plant CAPs than does the N-terminal domain. The CAP key motifs (e.g., Arg, Leu, Glu repeats motif, actin binding domain, SH3 motif, and multimerization domain) were consistently found in all CAPs.
Expression and purification of CAP To confirm whether the cloned gene was really adenylyl cyclase-associated protein, the cloned gene was inserted into the pET-28a(+) vector with a 6 His-tag, transformed into the expression host E. coli BL21 (DE3) pLysS, expressed by induction with IPTG, and collected by centrifugation. Excellent inducible expression of a CAP protein, identical to the native form (471 amino acid residues), was obtained from the recombinant pCAP plasmid. CAP protein was expressed in a soluble form in the cytosol. The expressed recombinant CAP was purified from the collected cells by sonication and His Bind column chromatography. The purified recombinant protein was homogenous by SDS-PAGE (Fig.2), and its molecular weight was approximately 50 kDa, which similar to native CAP.
Effects of temperature and pH The protein relative activity was kept a high level at a wide range temperature (from 30 to 55°C), then the activity was significantly decreased by the incubation
WANG Sheng et al.
temperature up to 75°C (Fig.3). And the binding capability of recombinant protein was stable (~90%) after incubated at 29 and 37°C for 2 h, respectively (Fig.4). Meanwhile, the optimum temperature was also obtained at 36°C and recombinant enzyme can remain nearly 75% activity when incubated at 53°C for 30 min. The optimum pH of CAP was 6.0, and the recombinant enzyme was stable at pH 4-7 (Fig.5).
DISCUSSION AND CONCLUSION This paper describes the cloning, expression, and characterization of an adenylyl cyclase-associated protein (CAP) from Gossypium arboretum. This is the first report describing the cloning and expression of CAP from Gossypium spp., although CAP has been cloned from G. hirsutum (Kawai et al. 1998). The CAP gene cloned from G. arboreum consisted of 1 416 bp, with 471 predicted amino acid residues. This CAP gene was homologus to other CAP genes, and the amino acid sequence showed high homology to dicot CAPs. In comparison with plant CAP DNA sequences, the GaCAP shows conservative exon-intron borders, except the number of introns. The average size of identified introns (~300 bp) is larger than other known organism CAPs. Multi-sequence alignment showed that CAP has a different similarity pattern in the N- and the C-terminal actin-binding domains tending to be well-conserved among different organisms. Nonetheless, a common feature of CAP proteins is the presence of a CAP motif in their N-terminal domains (amino acid 5-17 in GaCAP, Fig.1). The motif is characterized by RLE repeats (Arg, Leu, Glu), which have the potential to form an amphipathic helix that mediates protein-protein interactions (Cohen and Parry 1986; Bush and Sassone-Corsi 1990). Therefore, the N-terminal function also may be conserved among various organisms. The full length cDNA of GaCAP gene was cloned and expressed in E. coli. Using Ni 2+ -chelated chromatography, the recombinant protein was successfully obtained. Here, we used human platelet nonmuscle actin as substrate to test CAP binding activity under different temperatures and pH. The optimal pH of the recombinant CAP was around 6 and the optimal temperature range of the recombinant CAP was 3550°C, which was basically the same with that of the © 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
781
Fig. 1 Clustal X multiple amino acid sequence of the CAP from Gossypium arboreum with CAP from Arabidopsis thaliana (AtCAP1, accession no. NP_195175), Oryza sativa (OsCAP, accession no. NP_001051112), Vitis vinifera (VvCAP, accession no. CAN61863) and Homo sapiens (HsCAP, accession no. CAI11022). The asterisks below the aligned sequence indicate the same amino acids in all five sequences. The colons and points below the aligned sequence indicate the strongly conserved residues and the more weakly conserved residues, respectively. Gaps introduced to align the amino acid sequence optimally are indicated by dashes.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
782
WANG Sheng et al.
Fig. 2 SDS-PAGE analysis of His-tag-GaCAP expression in E. coli and purification by HiTrap chelating HP column. Lane 1, protein molecular weight markers (Sangon); lane 2, total cell lysate of uninduced culture; lane 3, supernatant of the cell lysate after induction; lane 4, preparation after chelating column.
Fig. 3 Effects of temperature on the activity of recombinant enzyme from 5 to 75°C.
Fig. 4 Thermal stability of the recombinant protein characterization of the protein at four incubation temperature levels using actin as substrate.
Fig. 5 pH stability of the recombinant enzyme.
native CAP. The recombinant protein can preserve 90% of its activities in room temperature for 2 h and be stable in a wide range pH conditions. These protein enzyme properties suggested that it could be produced in a common condition and suitable for further biochemical or structural studies. In conclusion, this is the first report on the cloning and expression of CAP from Gossypium arboreum. Our results suggest that recombinant GaCAP-His6 is a functional protein and also could play an analogous physiological role as the other members of the family. The strategy described in the report allows us to generate enough quantities of GaCAP required to perform structural and functional studies. Understanding these protein helps examine the genetic characteristics and role of CAP in regulating fiber cell development.
(2006BAD13B04), and the National Basic Research Program of China (973 Program, 2004CB117301).
Acknowledgements This work was supported by the support of the National Key Technology R&D Program of China
References Amberg D C, Basart E, Botstein D. 1995. Defining protein interactions with yeast actin in vivo. Nature Structural Biology, 2, 28-35. Amor Y, Haigler C H, Johnson S, Wainscott M, Delmer D P. 1995. A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proceedings of the National Academy of Sciences of the USA, 92, 9353-9357. Babb V M, Haigler C H. 2001. Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems. Plant Physiology, 127, 12341242. Barrero R A, Umeda M, Yamamura S, Uchimiya H. 2002. Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division. The Plant Cell, 14, 149163. Barrero R A, Umeda M, Yamamura S, Uchimiya H. 2003. Over-
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants. Annals of Botany, 91, 599-603. Baum B, Li W, Perrimon N. 2000. A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. Current Biology, 10, 964-973. Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Bush S, Sassone-Corsi P. 1990. Dimers, leucine zippers and DNA-binding domains. Trends in Genetics, 6, 36-40. Carlier M F, Pantaloni D. 1994. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Seminars in Cell Biology, 5, 183-191. Cohen C, Parry D. 1986. Alpha-helical coiled-coils: A widespread motif in proteins. Trends in Biochemical Sciences, 11, 245-248. Fenger U, Hofmann M, Galliot B, Schaller H C. 1994. The role of the cAMP pathway in mediating the effect of head activator on nerve-cell determination and differentiation in hydra. Mechanisms of Development, 47, 115-125. Field J, Vojtek A, Ballester R, Bolger G, Colicelli J, Ferguson K, Gerst J, Kataoka T, Michaeli T, Powers S, et al. 1990. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell, 61, 319327. Freeman N L, Chen Z, Horenstein J, Weber A, Field J. 1995. An actin monomer binding activity localizes to the carboxylterminal half of the Saccharomyces cerevisiae cyclaseassociated protein. The Journal of Biological Chemistry, 270, 5680-5685. Gieselmann R, Mann K. 1992. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast. FEBS Letters, 298, 149-153. Gottwald U, Brokamp R, Karakesisoglou I, Schleicher M, Noegel A A. 1996. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin. Molecular Biology of the Cell, 7, 261-272. Hubberstey A V, Mottillo E P. 2002. Cyclase-associated proteins: CAP actity for linking signal transduction and actin polymerization. The FASEB Journal, 16, 487-499. Ji S J, Lu Y C, Feng J X, Wei G, Li J, Shi Y H, Fu Q, Liu D, Luo J C, Zhu Y X. 2003. Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucleic Acids Research, 31, 2534-2543. Kawai M, Aotsuka S, Uchimiya H. 1998. Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated
783
protein highly expressed during fiber elongation. Plant Cell Physiology, 39, 1380-1383. Kost B, BaoY Q, Chua N H. 2002. Cytoskeleton and plant organogenesis. Philosophical transactions of the Royal Society of London (Series B: Biological Sciences), 357, 777-789. Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685. Lee J J, Woodward A W, Chen Z J. 2007. Gene expression changes and early events in cotton fibre development. Annals of Botany, 100, 1391-1401. Lehrer S S, Kerwar G. 1972. Intrinsic fluorescence of actin. Biochemistry, 11, 1211-1217. Matviw H, Yu G, Young D. 1992. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. Molecular and Cellular Biology, 12, 5033-5040. Pear J R, Kawagoe Y, Schreckengost W E, Delmer D P, Stalker D M. 1996. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proceedings of the National Academy of Sciences of the USA, 93, 12637-12642. Ramsey J C, Berlin J D. 1976. Ultrastructure of early stages of cotton fiber differentiation. Botanical Gazette, 137, 11-19. Ruan Y L, Llewellyn D J, Furbank R T, Chourey P S. 2005. The delayed initiation and slow elongation of fuzz-like short fibre cells in relation to altered patterns of sucrose synthase expression and plasmodesmata gating in a lintless mutant of cotton. Journal of Experimental Botany, 56, 977-984. Sohn R H, Goldschmidt-Clermont P J. 1994. Profilin: at the crossroads of signal transduction and the actin cytoskeleton. Bioessays, 16, 465-472. Vojtek A B, Cooper J A. 1993. Identification and characterization of a cDNA encoding mouse CAP: A homolog of the yeast adenylyl cyclase associated protein. Journal of Cell Science, 105, 777-785. Wan C Y, Wilkins T A. 1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry, 223, 7-12. Zelicof A, Gatica J, Gerst J E. 1993. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae. The Journal of Biological Chemistry, 268, 13448-13453. Zhou G L, Miyazaki Y, Nakagawa T, Tanaka K, Shishido K, Matsuda H, Kawamukai M. 1998. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants. Microbiology, 144, 1085-1093. Zigmond S H. 2000. How WASP regulates actin polymerization. The Journal of Cell Science, 150, F117-120. (Edited by ZHANG Yi-min)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
July 2009
2009, 8(7): 777-783
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L. WANG Sheng, ZHAO Guo-hong, JIA Yin-hua and DU Xiong-ming Cotton Germplasm Division, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang 455000, P.R.China
Abstract The aim of this study was to clone CAP (adenylyl cyclase-associated protein) gene from Gossypium arboreum L. and develop a platform for expressing and purifying CAP protein, which is a base for the construction and function researches of CAP. In this work, a CAP homolog from cotton (DPL971) ovule was identified and cloned. And the cDNA sequence consisted of an open reading frame of 1 416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa. To gain insight on the CAP role in cotton fiber development, the cloned CAP cDNA was expressed. A significant higher yield pure protein was obtained with the chromatographic method. Further experiments showed that the purified protein can bind with the actin in vitro indicating that the recombinant cotton CAP is functional. The procedure described here produced high yield pure protein through one chromatographic step, suitable for further structure-function studies. Key words: adenylyl cyclase-associated protein, CAP, cotton fiber, protein expression, protein purification, Gossypium arboreum L.
INTRODUCTION Cotton (Gossypium hirsutum L.) is a major source of textile fibers. Fiber cell initiation in the epidermal cells of cotton ovules represents a unique example of trichome development in higher plants (Ramsey and Berlin 1976). Many candidate genes that are expressed in fiber cells have been cloned and characterized (Lee et al. 2007). For example, the expression of some cytoskeleton genes is associated with the fiber development (Amor et al. 1995; Pear et al. 1996; Kawai et al. 1998; Babb and Haigler 2001; Ji et al. 2003; Ruan et al. 2005). Actin cytoskeleton is a key component of plant cytoskeleton which involves in the cell division and expansion (Kost et al. 2002). The reorganization of the actin cytoskeleton is thought to be mediated by actin binding proteins. The majority of actin binding proteins interReceived 21 July, 2008
act with filamentous (F) actin and serve to regulate assembly of the actin filaments and to link other proteins to the F-actin network. Actin sequestering factors can either enhance polymerization or cause disassembly of F-actin. Examples of these actin binding proteins include profiling (Sohn and GoldschmidtClermont 1994), β-thymosins (Carlier and Pantaloni 1994), Wiskott-Aldrich syndrome protein (WASp) (Zigmond 2000), and adenylyl cyclase-associated proteins (CAPs). To the date, CAP cDNA has been isolated from Saccharomyces cerevisiae (Field et al. 1990), Homo sapiens (Matviw et al. 1992), Rattus norvegicus (Zelicof et al. 1993), Mus musculus (Vojtek and Cooper 1993), hydra (Fenger et al. 1994), Dictyostelium discoideum (Gottwald et al. 1996), Lentinus edodes (Zhou et al. 1998), Drosophila (Baum et al. 2000), Arabidopsis thaliana (Barrero et al. 2002), and Gossypium hirsutum
Accepted 10 January, 2009
Correspondence DU Xiong-ming, Tel: +86-372-2525352, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60278-3
778
(Kawai et al. 1998). Deletion analysis has revealed that CAPs are multifunction proteins with several structural domains. It has been well understood that CAP contains four crucial functional domains: adenylyl cyclase domain, actin binding domain, Src homology 3 (SH3) binding domain (poly-proline amino acid domain), and multimerization domain (Hubberstey and Mottillo 2002). These domains seem to be characteristic of CAP and the related homologues because no other proteins present in databases share such motifs. These conservation, and the presence of the unique amino acid sequence (e.g., Arg, Leu, Glu repeats domain in N-terminal), suggest that CAP could play a similar role in all these organisms. Previously, the studies in A. thaliana and Nicotiana tabacum addressed the importance of CAP in actin cytoskeleton networking, which is significant for proper cell division and elongation (Barrero et al. 2002, 2003). In this paper, we report the identification, purification, and characterization of G. arboreum CAP, and describe a strategy for the production of high yield pure GaCAP for further estimating its roles in fiber cell development, and its relationship with other proteins of this important process.
MATERIALS AND METHODS Plant materials In this study, Gossypium arboreum DPL971 at nine successive selfed generations was used. The cotton, DPL971, was planted in a field (36°06´N lat., 114°21´E long.). Ovules from -1 to 9 DPA were harvested from individual plants of DPL971. Cotton fruit age was determined by tagging the petioles of a flower when it was fully opened. Leaves were also collected at anthesis. All selected materials were stored at -80°C until use.
Reagents, strains and plasmids The oligonucleotides (Table) were synthesized from Sangon (Shanghai, China). PCR components were from TaKaRa (Dalian, China). Restriction enzymes were from Fermentas (Burlington, ON, Canada). The reagents for protein purification and electrophoresis were purchased from USB (St. Louis, MO, USA) and Sigma (St. Louis, MO, USA). Actin protein (human platelet non-muscle, > 95% pure) was purchased from Cytosk-
WANG Sheng et al.
eleton (Denver, CO, USA). T-vector (pGEM-T easy) was purchased from Promega (Madison, WI). Plasmid pET-28a and Escherichia coli BL21 (DE3) pLysS (genotype: F- ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR) were from Novagen (Darmstadt, Germany). All other chemicals were of analytical grade.
Nucleotide acid manipulation and sequence analysis Genomic DNA was obtained from leaves using a lysis buffer containing 2% (w/v) cetyltrimethylammonium bromide (CTAB), 100 mmol L-1 Tris-HCl, 10 mmol L-1 EDTA, 3 M NaCl and 2% (v/v) of 2-mercaptoethanol. Total RNA was isolated from ovules according to the method described previously (Wan and Wilkins 1994), and its cDNA was synthesized using 1 g of total RNA following the RNA PCR Kit (TaKaRa, Dalian, China). To amplify the full-length CAP from genomic DNA, two primers were designed from the GhCAP cDNA sequence (GenBank accession no. AB014884) as shown in Table. Touch-down PCR was performed with above DNA over 30 cycles (45 s at 94°C, 1 min at 58-52°C, -0.2°C/ cycle, and 3 min at 72°C) under standard reaction condition as recommended by the supplier (PTC-100, MJ). The PCR products were cloned and sequenced.
Expression and purification of the G. arboreum CAP gene All purification steps were carried out at 25°C. CAP was monitored by SDS-PAGE. Protein was detected by staining with silver stain. To express the CAP gene, two primers for the full length CAP cDNA were synthesized (Table) and amplified by PCR under the following condition: heating at 94°C for 5 min, followed by 30 cycles of denaturation at Table Primer sequences for cloning and expression Primer name 1) CCRI205F CCRI205R CCRI205FN CCRI205RE 1) 2)
Sequence (5´
3´) 2)
AAT GGA GGC GAA GCT GAT AG AGG CCT CAT TTA TGC TCC TG CTA GCT AGC ATG GAG GCG AAG CTG ATA GAG CCG GAA TTC CGG TGC TCC TGA GTG CGA CAC
Application Cloning Cloning Expression Expression
N, Nhe I; E, EcoR I. The enzyme sites are underlined.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
94°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 120 s. The 5´-primer (CCRI205FN) contained a unique Nhe I site and the 3´-primer (CCRI205RE) contained a common unique EcoR I site. CCRI205RE does not have a stop codon. The PCR product that contained a Nhe I site at the 5´ end and a EcoR I site at the 3´ end was subcloned into the pGEM-T easy vector and transformed into E. coli XL1-Blue competent cells. The recombinant plasmid was digested with Nhe I and EcoR I, purified by 0.8% agarose gel electrophoresis, and ligated to the pET-28a(+) expression vector, which contained the inducible T7 promoter, carried an N-terminal His-tag and plus an optional C-terminal His-tag sequence which was prepared by using the stop codon-excluded primer. The ligated plasmids were transformed into the expression host, E. coli BL21 (DE3) pLysS, which was confirmed by restriction enzyme analysis. Then, E. coli containing the gene were cultured to the mid-log phase (OD600 0.6-1) in 200 mL of LB broth containing 30 mg mL-1 of kanamycin and then induced with 1 mmol L -1 IPTG at 25°C over night. The cultured cells were centrifuged at 5 000 × g for 25 min at 4°C, resuspended in 7 mL of ice-cold 1× binding buffer [20 mmol L-1 sodium phosphate buffer (pH 7.4) containing 500 mmol L-1 NaCl and 20 mmol L-1 imidazole], sonicated at 4°C, and centrifuged at 10 000 × g for 30 min. The resulting supernatant was filtered through a 0.4 mm membrane to prevent clogging of resins, and applied to the HiTrap IMAC HP (GE Healthcare), charged with 4 column volumes of 1× charge buffer (0.1 mol L-1 NiSO4), and washed with 5 column volumes of 1 × elution buffer (20 mmol L-1 sodium phosphate buffer containing 0.5 mol L-1 NaCl and 0.5 mol L-1 imidazole) and 10 column volumes of 1× binding buffer. The homogeneity of CAP was tested by SDS-PAGE gel electrophoresis. Denaturing gel electrophoresis was performed with the Bio-Rad Mini-Protean II System using 15% polyacrylamide/bisacrylamide gels as described previously (Laemmli 1970).
Protein determination Protein concentration was measured by a Bradford assay based on a bovine serum albumin (Sigma, St. Louis, MO, USA) standard curve (Bradford 1976). Reagents for protein assays were purchased from Bio-Rad (Hercules, CA, USA).
779
Optimum temperature and stability CAPs have been shown to inhibit actin polymerisation in vitro, by sequestering monomeric actin (Amberg et al. 1995; Freeman et al. 1995; Gottwald et al. 1996). So, here, we used actin as the substrate and made the purified CAP protein bound with actin. Separation of actin from CAP/actin complex was performed as described previously (Gieselmann and Mann 1992). Actin concentration was determined photometrically with an absorption coefficient of 0.64 for 1 mg mL-1 at 290 nm (Lehrer and Kerwar 1972). Optimum temperature was determined by varying assay temperature in the reaction mixture and thermal stability was obtained after preincubation of CAP in the different temperatures (range from 5 to 75°C) for 30 min.
Optimum pH and stability Optimum pH was obtained by varying assay pH value in the reaction mixture. pH stability was investigated after preincubation of CAP in different pH buffers for 30 min, then rapidly recovered to the assay pH 7.0. In all cases, overlaps were obtained when buffers were changed so that correction could be made for spurious buffer effects.
Protein sequence analysis of CAP The amino acid sequence of CAP was analyzed using standard protein-protein BLAST. Multiple sequence alignment was generated by using the program Clustal X.
RESULTS Cloning of the Gossypium arboreum CAP gene CAP cDNA sequence was obtained by RT-PCR using specific primers. After amplification, the products were cloned into pGEM-T easy vector and sequenced (GenBank accession no. EU106853). Sequence analysis showed that DPL971 cDNA encode 471 amino acid polypeptides which was structurally homologous to the GhCAP protein and other CAP homologs, therefore we designated the isolated genes as GaCAP. The CAP protein in G. arboreum (471 amino acid residues) and
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
780
A. thaliana (476 amino acid residues) were similar in size, but the former genomic sequence (~4 kb) was much larger than the later (~2.9 kb) indicating the sequence complexity of introns in G. arboreum. To establish the gene structure of CAP, the genomic PCR was performed. An amplified fragment about 4 kb was cloned and sequenced (GenBank accession no. EU106854). Comparison of cDNA and genomic sequences showed that the gene contained 10 exons and nine introns. The G. arboreum CAP shares high homology with other CAPs. The alignment of the amino acid sequences between G. arboreum CAP and the CAPs from other species is shown in Fig.1. The identity between the C-terminal domain of G. arboreum L. CAPs and those of A. thaliana and Oryza sativa CAP was 83%, while the Nterminal domains of G. arboreum L. CAP were 74 and 63% identical with the corresponding regions of A. thaliana and O. sativa CAP, respectively. So the C-terminal domain of G. arboreum L. CAP showed higher homology with plant CAPs than does the N-terminal domain. The CAP key motifs (e.g., Arg, Leu, Glu repeats motif, actin binding domain, SH3 motif, and multimerization domain) were consistently found in all CAPs.
Expression and purification of CAP To confirm whether the cloned gene was really adenylyl cyclase-associated protein, the cloned gene was inserted into the pET-28a(+) vector with a 6 His-tag, transformed into the expression host E. coli BL21 (DE3) pLysS, expressed by induction with IPTG, and collected by centrifugation. Excellent inducible expression of a CAP protein, identical to the native form (471 amino acid residues), was obtained from the recombinant pCAP plasmid. CAP protein was expressed in a soluble form in the cytosol. The expressed recombinant CAP was purified from the collected cells by sonication and His Bind column chromatography. The purified recombinant protein was homogenous by SDS-PAGE (Fig.2), and its molecular weight was approximately 50 kDa, which similar to native CAP.
Effects of temperature and pH The protein relative activity was kept a high level at a wide range temperature (from 30 to 55°C), then the activity was significantly decreased by the incubation
WANG Sheng et al.
temperature up to 75°C (Fig.3). And the binding capability of recombinant protein was stable (~90%) after incubated at 29 and 37°C for 2 h, respectively (Fig.4). Meanwhile, the optimum temperature was also obtained at 36°C and recombinant enzyme can remain nearly 75% activity when incubated at 53°C for 30 min. The optimum pH of CAP was 6.0, and the recombinant enzyme was stable at pH 4-7 (Fig.5).
DISCUSSION AND CONCLUSION This paper describes the cloning, expression, and characterization of an adenylyl cyclase-associated protein (CAP) from Gossypium arboretum. This is the first report describing the cloning and expression of CAP from Gossypium spp., although CAP has been cloned from G. hirsutum (Kawai et al. 1998). The CAP gene cloned from G. arboreum consisted of 1 416 bp, with 471 predicted amino acid residues. This CAP gene was homologus to other CAP genes, and the amino acid sequence showed high homology to dicot CAPs. In comparison with plant CAP DNA sequences, the GaCAP shows conservative exon-intron borders, except the number of introns. The average size of identified introns (~300 bp) is larger than other known organism CAPs. Multi-sequence alignment showed that CAP has a different similarity pattern in the N- and the C-terminal actin-binding domains tending to be well-conserved among different organisms. Nonetheless, a common feature of CAP proteins is the presence of a CAP motif in their N-terminal domains (amino acid 5-17 in GaCAP, Fig.1). The motif is characterized by RLE repeats (Arg, Leu, Glu), which have the potential to form an amphipathic helix that mediates protein-protein interactions (Cohen and Parry 1986; Bush and Sassone-Corsi 1990). Therefore, the N-terminal function also may be conserved among various organisms. The full length cDNA of GaCAP gene was cloned and expressed in E. coli. Using Ni 2+ -chelated chromatography, the recombinant protein was successfully obtained. Here, we used human platelet nonmuscle actin as substrate to test CAP binding activity under different temperatures and pH. The optimal pH of the recombinant CAP was around 6 and the optimal temperature range of the recombinant CAP was 3550°C, which was basically the same with that of the © 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
781
Fig. 1 Clustal X multiple amino acid sequence of the CAP from Gossypium arboreum with CAP from Arabidopsis thaliana (AtCAP1, accession no. NP_195175), Oryza sativa (OsCAP, accession no. NP_001051112), Vitis vinifera (VvCAP, accession no. CAN61863) and Homo sapiens (HsCAP, accession no. CAI11022). The asterisks below the aligned sequence indicate the same amino acids in all five sequences. The colons and points below the aligned sequence indicate the strongly conserved residues and the more weakly conserved residues, respectively. Gaps introduced to align the amino acid sequence optimally are indicated by dashes.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
782
WANG Sheng et al.
Fig. 2 SDS-PAGE analysis of His-tag-GaCAP expression in E. coli and purification by HiTrap chelating HP column. Lane 1, protein molecular weight markers (Sangon); lane 2, total cell lysate of uninduced culture; lane 3, supernatant of the cell lysate after induction; lane 4, preparation after chelating column.
Fig. 3 Effects of temperature on the activity of recombinant enzyme from 5 to 75°C.
Fig. 4 Thermal stability of the recombinant protein characterization of the protein at four incubation temperature levels using actin as substrate.
Fig. 5 pH stability of the recombinant enzyme.
native CAP. The recombinant protein can preserve 90% of its activities in room temperature for 2 h and be stable in a wide range pH conditions. These protein enzyme properties suggested that it could be produced in a common condition and suitable for further biochemical or structural studies. In conclusion, this is the first report on the cloning and expression of CAP from Gossypium arboreum. Our results suggest that recombinant GaCAP-His6 is a functional protein and also could play an analogous physiological role as the other members of the family. The strategy described in the report allows us to generate enough quantities of GaCAP required to perform structural and functional studies. Understanding these protein helps examine the genetic characteristics and role of CAP in regulating fiber cell development.
(2006BAD13B04), and the National Basic Research Program of China (973 Program, 2004CB117301).
Acknowledgements This work was supported by the support of the National Key Technology R&D Program of China
References Amberg D C, Basart E, Botstein D. 1995. Defining protein interactions with yeast actin in vivo. Nature Structural Biology, 2, 28-35. Amor Y, Haigler C H, Johnson S, Wainscott M, Delmer D P. 1995. A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proceedings of the National Academy of Sciences of the USA, 92, 9353-9357. Babb V M, Haigler C H. 2001. Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems. Plant Physiology, 127, 12341242. Barrero R A, Umeda M, Yamamura S, Uchimiya H. 2002. Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division. The Plant Cell, 14, 149163. Barrero R A, Umeda M, Yamamura S, Uchimiya H. 2003. Over-
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.
expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants. Annals of Botany, 91, 599-603. Baum B, Li W, Perrimon N. 2000. A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. Current Biology, 10, 964-973. Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Bush S, Sassone-Corsi P. 1990. Dimers, leucine zippers and DNA-binding domains. Trends in Genetics, 6, 36-40. Carlier M F, Pantaloni D. 1994. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Seminars in Cell Biology, 5, 183-191. Cohen C, Parry D. 1986. Alpha-helical coiled-coils: A widespread motif in proteins. Trends in Biochemical Sciences, 11, 245-248. Fenger U, Hofmann M, Galliot B, Schaller H C. 1994. The role of the cAMP pathway in mediating the effect of head activator on nerve-cell determination and differentiation in hydra. Mechanisms of Development, 47, 115-125. Field J, Vojtek A, Ballester R, Bolger G, Colicelli J, Ferguson K, Gerst J, Kataoka T, Michaeli T, Powers S, et al. 1990. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell, 61, 319327. Freeman N L, Chen Z, Horenstein J, Weber A, Field J. 1995. An actin monomer binding activity localizes to the carboxylterminal half of the Saccharomyces cerevisiae cyclaseassociated protein. The Journal of Biological Chemistry, 270, 5680-5685. Gieselmann R, Mann K. 1992. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast. FEBS Letters, 298, 149-153. Gottwald U, Brokamp R, Karakesisoglou I, Schleicher M, Noegel A A. 1996. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin. Molecular Biology of the Cell, 7, 261-272. Hubberstey A V, Mottillo E P. 2002. Cyclase-associated proteins: CAP actity for linking signal transduction and actin polymerization. The FASEB Journal, 16, 487-499. Ji S J, Lu Y C, Feng J X, Wei G, Li J, Shi Y H, Fu Q, Liu D, Luo J C, Zhu Y X. 2003. Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucleic Acids Research, 31, 2534-2543. Kawai M, Aotsuka S, Uchimiya H. 1998. Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated
783
protein highly expressed during fiber elongation. Plant Cell Physiology, 39, 1380-1383. Kost B, BaoY Q, Chua N H. 2002. Cytoskeleton and plant organogenesis. Philosophical transactions of the Royal Society of London (Series B: Biological Sciences), 357, 777-789. Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685. Lee J J, Woodward A W, Chen Z J. 2007. Gene expression changes and early events in cotton fibre development. Annals of Botany, 100, 1391-1401. Lehrer S S, Kerwar G. 1972. Intrinsic fluorescence of actin. Biochemistry, 11, 1211-1217. Matviw H, Yu G, Young D. 1992. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. Molecular and Cellular Biology, 12, 5033-5040. Pear J R, Kawagoe Y, Schreckengost W E, Delmer D P, Stalker D M. 1996. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proceedings of the National Academy of Sciences of the USA, 93, 12637-12642. Ramsey J C, Berlin J D. 1976. Ultrastructure of early stages of cotton fiber differentiation. Botanical Gazette, 137, 11-19. Ruan Y L, Llewellyn D J, Furbank R T, Chourey P S. 2005. The delayed initiation and slow elongation of fuzz-like short fibre cells in relation to altered patterns of sucrose synthase expression and plasmodesmata gating in a lintless mutant of cotton. Journal of Experimental Botany, 56, 977-984. Sohn R H, Goldschmidt-Clermont P J. 1994. Profilin: at the crossroads of signal transduction and the actin cytoskeleton. Bioessays, 16, 465-472. Vojtek A B, Cooper J A. 1993. Identification and characterization of a cDNA encoding mouse CAP: A homolog of the yeast adenylyl cyclase associated protein. Journal of Cell Science, 105, 777-785. Wan C Y, Wilkins T A. 1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry, 223, 7-12. Zelicof A, Gatica J, Gerst J E. 1993. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae. The Journal of Biological Chemistry, 268, 13448-13453. Zhou G L, Miyazaki Y, Nakagawa T, Tanaka K, Shishido K, Matsuda H, Kawamukai M. 1998. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants. Microbiology, 144, 1085-1093. Zigmond S H. 2000. How WASP regulates actin polymerization. The Journal of Cell Science, 150, F117-120. (Edited by ZHANG Yi-min)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.