Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet potato plants

ARTICLE IN PRESS Journal of Plant Physiology 162 (2005) 1169—1176

www.elsevier.de/jplph

Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet potato plants Thomas Berbericha,b, Toshiyuki Takagia, Atsushi Miyazakia, Motoyasu Otanic, Takiko Shimadac, Tomonobu Kusanoa, a

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan Botanisches Institut, Goethe-Universita ¨t, Postfach 11 19 32, D-60054, Frankfurt am Main, Germany c Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, Ishikawa 921-8836, Japan b

Received 24 November 2004; accepted 4 January 2005

KEYWORDS Adiponectin; Agrobacteriummediated transformation; Diabetes; Ipomoea batatas; Transgenic plant

Summary Adiponectin is a 30 kDa protein exclusively produced and secreted from adipocytes and as a cytokine has been found to link obesity, insulin resistance, and type 2 diabetes. Production of biologically active adiponectin in large scale is desirable for pharmaceutical applications. Mouse adiponectin cDNA was used for developing transgenic sweet potato plants via Agrobacterium-mediated transformation. The presence of the transgene was verified by PCR and DNA gel blot analysis. Further investigated were five independent transgenic lines, all of which expressed high levels of adiponectin mRNA. Immuno blot analysis with a mouse adiponectin antiserum revealed that, in addition to a 29 kDa-protein which co-migrates with the adiponectin protein produced in Escherichia coli cells, a 31 kDa-protein was produced, indicative of a post-translational modification of the protein. The transgenic plants did not show obvious differences in growth rate and morphology in response to adiponectin production. & 2005 Elsevier GmbH. All rights reserved.

Introduction Plant-based expression of pharmaceutical proteins is a cost-effective alternative to traditional large-scale production systems such as microbial fermentation, insect and mammalian cell cultures, and transgenic animals. Plants are the most

economical makers of biomass using only sunlight, water, carbon dioxide and minerals. They offer a proper modification system for eukaryotic proteins, such as folding, post-translational modification, and sub-cellular targeting. Another advantage of using plant-based expression systems might be from a view point of safety, as it avoids animal

Corresponding author. Tel./fax: +81 22 217 5709.

E-mail address: [email protected] (T. Kusano). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.01.009

ARTICLE IN PRESS 1170 cell-culture contaminants such as mammalian pathogens and extraneous viral or bacterial components (Ma et al., 2003; Peterson and Arntzen, 2004). Since the human growth hormone was expressed in transgenic tobacco in 1986 (Barta et al., 1986), many more pharmaceutically relevant proteins have been successfully produced in plants comprising for example recombinant antibodies (Hiatt et al., 1989; Larrick et al., 2001) and vaccines (Mason et al., 1992; Streatfield et al., 2001). The proteins expressed in plants can be extracted from whole plant parts or organelles and used as a source for medicinal applications. Proteins that are expressed in edible plants could function directly in food. Roots and tuber crops such as sweet potato have a high production yield of biomass, thus they could have large impact as industrial material for application in biotechnology. Production of sweet potato ranks fifth after that of rice, wheat, maize and white potato, among the food crops of the world (Jansson and Raman, 1991). Establishing sweet potato as a production site for the delivery of recombinant proteins is worth striving for. Adiponectin or Acrp30 (adipocyte complement-related protein of 30 kDa) is a cytokine produced exclusively in and secreted from adipose tissue (Scherer et al., 1995) and is abundant in human plasma, with concentrations of 5–30 mg/mL, thus accounting for approximately 0.01% of total plasma proteins (Arita et al., 1999). Several studies showed that low levels of adiponectin can be assigned to human diseases which has recently been summarized by Dı´ez and Iglesias (2003). Reduced levels of adiponectin are associated with obesity, hypertension, or type 2 diabetes mellitus. Although the precise physiological role of adiponectin is not yet fully understood, its function as an anti-inflammatory and antiatherogenic compound is well established. Also, increased adiponectin levels are associated with increased insulin sensitivity and glucose tolerance. Thus, adiponectin might be useful for therapeutic application against diseases associated with insulin resistance such as type 2 diabetes mellitus and obesity. Because of its anti-inflammatory effect it

T. Berberich et al. might be useful as a protective factor for atherosclerosis development in cases where adiponectin levels in plasma are low. Furthermore, increasing plasma adiponectin might also be useful in preventing vascular restenosis after vascular intervention (Dı´ez and Iglesias, 2003). Testing these kinds of applications in a clinical setting, creation of a source for large amounts of functional recombinant protein is necessary. Here we report the expression of mouse adiponectin in sweet potato as a first step in establishing the production of adiponectin in an edible plant to meet future demands for this interesting protein.

Materials and Methods Plant material and growth conditions The Ipomoea batatas (L.) Lam. cultivar Kokei 14 was used as plant material. The plants were grown in soil in a growth chamber at 25 1C under a 16 h light/8 h dark photocycle. For some applications, plants were grown under sterile conditions in plastic containers on Murashige and Skoog (1962) medium supplemented with 3% sucrose and solidified with 0.8% agar.

Preparation of expression constructs for adiponectin in a plant expression vector A mouse adiponectin cDNA (accession number AK003138) was amplified by the polymerase chain reaction (PCR) using a mouse cDNA derived from liver organ, a gift from Drs. K. Mizuno and K. Ohashi (Graduate School of Life Sciences, Tohoku University), as the template and the primer pairs of AdiF (50 -gcggatccatgctactgttgcaagctctc-30 ) and AdiR (50 -gcgagctctcagttggtatcatggtagag-30 ). For cloning purpose, the restriction enzyme sites for BamHI and SacI were introduced in the above primer sequences (see underlined regions). The PCR products were ligated to the pGEM-T Easy vector (Promega, USA) and used to transform Escherichia coli strain

Figure 1. Schematic drawing of the plasmid pIG121Hm-AN used for transforming sweet potato. Mouse adiponectin fulllength cDNA has been placed under the control of 35S cauliflower mosaic virus promoter. The construct carries the left and right borders (LB, RB) of the transferred DNA that demarcates the sequences that are incorporated into the plant genomic DNA via Agrobacterium-mediated transformation. hpt, hygromycin phosphotransferase gene; NOS-p, nopaline synthase gene promoter, NOS-t, nopaline synthase gene terminator, nptII, neomycin phosphotransferase II gene; 35S pro, cauliflower mosaic virus 35S promoter.

ARTICLE IN PRESS Adiponectin production in sweet potato DH5a. After confirming the nucleotide sequence, the adiponectin cDNA fragment of 744 bp was excised by cutting with BamHI and SacI and subcloned into the corresponding sites of the plant expression vector pIG121-Hm (Ohta et al., 1990) which was under the control of the constitutive CaMV 35S promoter. The resulting construct pIG121-Hm-AN (Fig. 1) was introduced into A. tumefaciens strain EHA101 (Hood et al., 1986) by an electroporation method (Shen and Forde, 1989).

Transformation and regeneration Transgenic plants were produced by infecting embryogenic calli derived from shoot meristems of the sweet potato cultivar with Agrobacterium containing the recombinant binary vector plasmid as described by Otani et al. (1998). In brief, a single colony of Agrobacterium was grown on LB medium supplemented with 50 mg/L kanamycin, 50 mg/L hygromycin and 1.5% (w/v) agar were transferred to liquid LS medium supplemented with 10 mg/L acetosyringone and 1 mg/L 4FA, and shaken for 1 h at 29 1C. Embryogenic calli were soaked in the bacterial suspension for 2 min, blotted dry and transferred onto co-culture medium consisting of LS medium supplemented with 10 mg/L acetosyringone and 1 mg/L 4FA, 3% (w/v) sucrose and 0.32% (w/v) gellan gum. Following a period of three days of co-cultivation, infected calli were transferred onto selection medium that was LS medium containing 1 mg/L 4FA, 25 mg/L hygromycin, 500 mg/L carbenicillin, 3% (w/v) sucrose, and 0.32% (w/v) gellan gum. The cultures were propagated for 60 days at 26 1C in the dark with transfer to fresh selection medium every 2 weeks. Thereafter, the calli were transferred onto somatic embryo formation medium which was LS medium supplemented with 4 mg/L abscisic acid, 1 mg/L gibberellic acid, 25 mg/L hygromycin, 500 mg/L carbenicillin, 3% (w/ v) sucrose, and 0.32% (w/v) gellan gum. After 21 days, somatic embryos formed from hygromycinresistant calli were used for regeneration of plants by transfer onto LS medium supplemented with 0.05 mg/L gibberellic acid, 25 mg/L hygromycin, 500 mg/L carbenicillin, 3% (w/v) sucrose, and 0.32% (w/v) gellan gum. Regenerated plants were cultured on the same medium without gibberellic acid, and maintained at 26 1C under a 16 h photoperiod at 38 mmol/m2/s light from fluorescent tubes.

1171 transformants using the PlantGenElute Kit following the manufacturer0 s instructions (Sigma, USA). The adiponectin gene was detected by PCR analysis with the primer pair described above. As controls, the presence of b-amylase gene using the primers amyF (50 -cggtgttggagagatcaa-30 ) and amyR 0 (5 -cgttcttatttcttaggctc-30 ) and the hpt gene using the primers hptF (50 -cgtctgtcgagaagtttct-30 ) and hptR (50 -ggtgtcgtccatcacagttt-30 ) was determined. Genomic DNAs of transformed- and control plants were used as template in the PCR under the following conditions: 94 1C for 30 s, 55 1C for 30 s and 72 1C for 30 s for a total of 30 cycles. The amplified DNA fragments were size fractionated on a 1% agarose gel and visualized by staining with ethidium bromide. For genomic DNA gel blot hybridization, genomic DNA (10 mg each) digested with HindIII was electrophoresed on a 1% agarose gel. After staining with ethidium bromide, the DNA was transferred onto nylon membrane (Hybond-N+, Amersham, USA) by capillary transfer with 0.4 N NaOH. The adiponectin cDNA fragment was radioactively labeled with 32 P-dCTP (Amersham, USA) employing the Ladderman labeling kit (TaKaRa, Japan) and used as a probe for hybridizing the DNA gel blots. After hybridizing for overnight at 65 1C, the blots were washed twice in 2  SSC, 0.1% SDS at room temperature for 15 min, followed by a single wash in 1  SSC, 0.1% SDS at 65 1C for 20 min. The signals were detected by autoradiography on X-ray films (Hyperfilm MP, Amersham, USA).

RNA gel blot analysis Total RNA from leaves of wild-type and transformed plants was isolated by the ATA method (Nagy et al., 1988). RNA samples of 15 mg each were size-fractionated on formaldehyde-1% agarose gels and transferred to a nylon membrane (Hybond-N+, Amersham, USA). The blots were hybridized with a 32 P-labeled DNA-probe coding for adiponectin. After hybridization at 42 1C for 12 h, the blots were washed twice in 2  SSC, 0.1% SDS at room temperature for 15 min followed by a single wash in 1  SSC, 0.1% SDS at 65 1C for 20 min. The signals were detected by autoradiography on X-ray films (Hyperfilm MP, Amersham, USA).

Immunodetection of adiponectin protein in transgenic plants

PCR analysis and genomic DNA gel blot Genomic DNA was isolated from leaf tissue of non-transgenic plants and kanamycin resistant

The leaf tissue of wild-type and transgenic plants was homogenized and extracted on ice with 1.5 mL of 50 mM K-phosphate buffer (pH 7.4) per 1 g tissue.

ARTICLE IN PRESS 1172 Extracts were centrifuged for 10 min at 4 1C and 14,000 rpm in a microcentrifuge and the resulting supernatants were used for immunoblotting. Electrophoresis was performed in the presence of 0.4% (w/v) SDS on polyacrylamide slab gels consisting of a 12.5% (w/v) polyacrylamide resolving gel and a 4% (w/v) stacking gel in the buffer system of Laemmli (1970). Proteins were transferred electrophoretically to a nitrocellulose membrane (BA85, Schleicher and Schuell, Germany). A commercially available antibody (catalog number 107920, Calbiochem, USA) raised against amino acids 18–32 and 187–200 of mouse adiponectin Acrp30 was used to detect the recombinant adiponectin protein. Visualization of the antigen–antibody complex with a peroxidase-conjugated goat-anti-rabbit antiserum and the ECL system was performed as described by the manufacturer (Amersham, USA).

T. Berberich et al. fragments derived from the hpt and adiponectin genes, respectively, were only detected in the transformants, the 378-bp fragment of the bamylase gene was detected in all plants also including untransformed control plants (Fig. 2). Based on these experiments, the integration of the pIG121-Hm-AN construct in all five selected lines could be confirmed. In order to compare the integration of T-DNA in the transformants, DNA gel blot hybridization of genomic DNA digested with HindIII having one recognition site in the 50 edge of the CaMV 35S promoter of the transformation vector (Fig. 1) was performed using the adiponectin cDNA as a probe. The hybridization patterns were different in all five lines (Fig. 3), showing that T-DNA insertion

Results Selection of transgenic sweet potato plants In previous experiments the production of transgenic sweet potato from embryogenic callus via A. tumefaciens infection has been demonstrated (Otani et al., 1998). In our study the transformation of sweet potato with A. tumefaciens carrying the binary vector construct pIG121-Hm-AN comprising the DNA coding for mouse adiponection has been successful as examined by the growth of plants in the presence of 25 mg/L hygromycin. Thus, the stably inserted genes were transcribed under the control of the CaMV 35S promoter and translated, providing the synthesis of the hpt gene product enabling growth on medium containing hygromycin. As hygromycin-resistant transformants, we obtained five lines and used them for further examination. Untransformed sweet potato plants served as controls in the analysis of the mouse adiponectin gene integration and its expression on mRNA and protein levels.

Figure 2. Detection of adiponectin, hpt and b-amylase genes in control- and transgenic sweet potato leaves by PCR.

Analysis of adiponectin DNA in transgenic plants As the first step confirming the presence of the recombinant DNA in the regenerated plants, PCR analysis was performed on their genomic DNA. The hpt and adiponectin genes present on the pIG121Hm-AN plasmid used for transformation and the sweet potato b-amylase gene as a control were amplified using the gene-specific primer pairs described. While the 482-bp and 744-bp DNA

Figure 3. Genomic DNA gel blot analysis of control- and the five transgenic sweet potato lines. The blot was hybridized with adiponectin cDNA as a probe: wt, cultivar Kokei14 wild type; lane 1, transgenic line 1; lane 2, line 4; lane 3, line 5, lane 4, line 6; lane 5, line 13.

ARTICLE IN PRESS Adiponectin production in sweet potato

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occurred at different sites in the sweet potato genome. From the hybridization pattern, we can further speculate that line 1 contains only one copy of the mouse adiponectin gene whereas lines 4, 5, 6 and 13 seem to contain at least two but not more than three copies each. No adiponectin-coding DNA was detected in the control wild-type plants.

Detection of the adiponectin transcripts The expression of the adiponectin gene on mRNA level in the selected sweet potato transformant lines 1, 4, 5, 6 and 13 was examined by RNA gel blot analysis using the mouse adiponectin cDNA as a probe. As shown in Fig. 4, all transformants contained relatively high levels of adiponectin transcripts which could be easily detected after hybridization. Furthermore, the amount of adiponectin mRNA in the individual lines was almost invariant when compared with the stained rRNA as loading control. These results show that the integrated adiponectin genes were efficiently transcribed under the control of the 35S promoter in sweet potato plants.

Expression of the adiponectin protein in transgenic plants From the data of RNA gel blot analysis, we supposed that the detected adiponectin transcripts should be sufficient for translation of a detectable amount of adiponectin protein. To examine whether the adiponectin protein was synthesized in plants and whether the protein has the expected size, we performed immuno blot analysis using crude leaf extracts and a commercially available antiserum against mouse adiponectin. In Fig. 5, the data of protein blot analysis are shown. As expected, no adiponectin protein was detected in the untransformed wild-type plants. Also in ex-

Figure 4. RNA gel blot analysis of wild-type and transgenic sweet potato leaves with adiponectin cDNA as a probe. Before hybridization, the blot was stained with 0.04% methylene blue solution to verify the equal loading of the RNA samples: wt, Kokei 14; lane 1, transgenic line 1; lane 2, line 4; lane 3, line 5, lane 4, line 6; lane 5, line 13.

Figure 5. Immuno blot analysis of wild-type and transgenic sweet potato leaves with anti-adiponectin antibody. wt, Kokei 14; lane 1, transgenic line 1; lane 2, line 4; lane 3, line 5, lane 4, line 6; lane 5, line 13.

tracts of the transgenic line 13 no signal could be detected, suggesting that the existing transcript is not translated in this line. In the extracts from lines 1, 4, 5 and 6, the antibody recognized two proteins with the sizes of approximately 29 and 31 kDa, respectively. While the theoretical size of the polypeptide derived from the cDNA sequence is 26.8 kDa, the 29 kDa-protein co-migrates with the adiponectin produced in E. coli cells in the gelsystem (data not shown). The higher amount of protein could be detected in lines 4 and 5, whereas lines 1 and 6 contained significantly less adiponectin protein. It should be also referred to our observation that aged leaves of those transformants contained the higher molecular mass protein which immunoreacts with the anti-adiponection antibody (data not shown). We have roughly estimated that one gram of the transformant lines 4 and 5 leaf tissue contained a few hundred nanograms or less of adiponectin protein.

Analysis of morphological appearance of the transgenic plants All transgenic sweet potato lines were grown further to maturation stages for comparison with untransformed control plants for phenotypic alteration. Here we only showed two transgenic lines (Fig. 6). Line 4 represents a transgenic plant in which the adiponectin protein is synthesized as evaluated by immuno blot analysis. In the other transgenic plant, line 13, adiponectin transcripts are as abundant as in line 4, but no protein was detected. Despite this difference in adiponectin expression, both lines show no difference in leaf and shoot morphology compared with wild-type control plants. The only difference is displayed by the morphology of tubers. While tubers of line 4

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Figure 6. Phenotypic comparison of the wild-type and the transgenic sweet potatoes. Non-transgenic (wt) and transgenic plants (lines 4 and 13) were compared phenotypically. 1, young seedlings in plastic boxes; 2, mature leaves; 3, storage tubers. White bar represents 1 cm in length.

had approximately the same size and shape as tubers of wild-type plants, line 13 produced a single large tuber.

Discussion In our present study, we have shown that the expression of the mouse adiponectin protein in sweet potato plants was successful. The application of bioengineering to plants for production of pharmaceutical products for human use has expanded in recent years. Sweet potato, because of its high production yield of biomass could have large impact as industrial material for application in biotechnology. With the use of Agrobacteriummediated transformation, we could stably insert the DNA coding for mouse adiponectin into the sweet potato genome and could regenerate plants from the transfected calli. In all five investigated transgenic lines, low copy number (one to three) of integrated T-DNA was observed which is an advantage of Agrobacterium-mediated transformation over other methods like electroporation or particle bombardment which often lead to complex integration of multiple copies (Otani et al., 1998). The gene-copy number, the position of transgene integration and the structure of the transgenic locus are factors which lead to variable transgene expression

T. Berberich et al. and cannot be precisely controlled through construct design. At least with respect to transcription of the adiponectin-encoding DNA, no positional effect could be observed. In all five lines, the adiponectin gene was transcribed to approximately the same levels of stable mRNA. However, the evenly occurring transcript levels were not reflected by equally high levels of adiponectin protein in the different lines. In one line (line 13), no protein could be detected, and even though adiponectin protein was detected in the other four transgenic lines, the levels differed in contrast to the corresponding mRNA levels. So far, most plant-derived proteins have been produced in and directly harvested from leaves of transgenic tobacco with a typical yield of only 0.1% of the total soluble protein (Ma et al., 2003). Higher levels of up to 4.1% could be obtained when the proteins were expressed in tobacco chloroplasts (Daniell et al., 2001). A low level of protein production could be the result of instability or miss-folding of the foreign proteins within the plant cell. Another and the most probable reason could be a bias in codon usage between mouse and sweet potato plant, which would result in a low efficiency of translation of the mRNAs coding for the foreign protein (Mason et al., 1980). In the present stage of our work it cannot be ruled out that adjustment of the adiponectin coding DNA to plant codon usage would enhance the production of adiponectin protein from the high levels of transcripts that are disposable in the transgenic sweet potato plants. In total soluble protein extracts of the transgenic sweet potato lines, two clear protein bands of 29 and 31 kDa in sizes were detected by the antibody in immuno blot analysis. The expected size of mouse adiponectin protein derived from the nucleotide sequence of the cDNA we used for expression in sweet potato is 26.8 kDa. In fact, the authentic protein produced in bacterial cells migrates to the position similar to that of the 29 kDaprotein (not shown). The cDNA encodes for all features that are necessary for producing a functional adiponectin protein. As shown by Wang et al. (2002) the four lysine residues in the collagenous domain of adiponectin secreted by adipocytes are modified by hydroxylation and glycosylation. These post-translational modifications could not be detected in recombinant adiponectin produced by E. coli. The data support the idea that the posttranslational modifications are necessary to result in an active adiponectin protein. Full-length adiponectin produced by mammalian cells can acutely decrease hyperglycemia in several diabetic animal models, whereas E. coli-derived adiponectin has no such activities (Berg et al., 2001). The fact that the adiponectin protein produced in sweet potato occurs

ARTICLE IN PRESS Adiponectin production in sweet potato also as a larger form of 31 kDa, suggests that it is a post-translationally modified form of the 29 kDapolypeptide. At this moment, we could not evaluate the physiological activity of the heterologously produced adiponectin protein because of its insufficient amounts. From the morphological appearance, we could not find significant differences between wild type and transgenic plants expressing adiponectin protein. Thus, it can be assumed that in case of large-scale adiponectin production in sweet potato there will be at least the same quantity of biomass production as is usually harvested from wild-type plants. In summary, we showed that the pharmaceutically interesting protein adiponectin could be successfully produced in the edible plant sweet potato, even though the expression level is quite low, and that the protein is probably post-translationally modified. Further analysis is necessary to determine whether adiponectin derived from sweet potato is post-translationally modified in the correct way and whether this protein represents the physiologically active form. A plant-friendly modified adiponectin gene is currently designed and generated. Transgenic plants expressing this modified gene sequence will then also be used for expression analysis of adiponectin in tuber tissue. In the study presented here we solely examined the expression in leaf tissue which has been available long before tuberization of the transgenic plants started. Using the codon-modified adiponectin gene, organ-specific and intracellular-specific targeting in sweet potato plants will be worth to try for future works.

Acknowledgment We thank Drs. K. Ohashi and K. Mizuno for kindly providing mouse cDNAs. We also thank Drs. K. Nakamura and E. Hood for providing the binary vector, pIGHm121 and A. tumefaciens EHA101 strain, respectively. This work was supported in parts by a grant for the project ‘Establishment of Transgenic Agro-Biofarm System’ from the Ministry of Agriculture, Forestry and Fisheries and a grant from The Saito Gratitude Foundation Museum of Natural History.

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