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First published online March 7, 2008; 10.1104/pp.108.117572 Plant Physiology 147:331-339 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Construction of a Functional CMP-Sialic Acid Biosynthesis Pathway in Arabidopsis1,[OA]Institute of Applied Genetics and Cell Biology (A.C., C.V., L.M., J.G., R.S., H.S.) and Department of Chemistry (M.P., R.L., F.A.), University of Natural Resources and Applied Life Sciences, BOKU-Vienna, A–1190 Vienna, Austria
Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g–1 fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g–1 fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.
In recent years, plants have become an attractive alternative for the production of therapeutically relevant proteins (Saint-Jore-Dupas et al., 2007  ). Several studies have demonstrated that plants can efficiently produce complex mammalian proteins that need extensive posttranslational modifications (for a recent review, see Fischer et al., 2004). N-Glycosylation is one of the most common and important posttranslational modifications of proteins, and an advantage of using plants as an expression system is their ability to perform N-glycosylation similar to mammalian cells. However, besides a common core structure, plant N-glycans differ from their mammalian counterparts to some extent as they (1) carry plant-specific immunogenic β1,2-Xyl and  1,3-Fuc residues and (2) lack terminal β1,4-Gal and N-acetylneuraminic acid (Neu5Ac) residues. Hence, the production of recombinant therapeutic glycoproteins in plants requires modifications of the plant N-glycosylation pathway not just to overcome the possible immunogenicity of nonmammalian residues but also to enable the addition of terminal Neu5Ac. Engineering of the N-glycosylation pathway in plants by knockout and RNA interference approaches resulted in lines that synthesize complex N-glycan structures lacking the plant-specific glycan epitopes (Koprivova et al., 2004; Strasser et al., 2004, 2008; Cox et al., 2006; Schähs et al., 2007). The predominant N-glycan structure in these plants is GlcNAc2Man3GlcNAc2 (GnGn), which is the required acceptor substrate for its sequential elongation with β1,4-Gal and Neu5Ac residues. Functional expression of human β1,4-galactosyltransferase in plants already has been achieved (Palacpac et al., 1999; Bakker et al., 2001).
The majority of N-glycans on mammalian glycoproteins are terminated by Neu5Ac and other sialic acids linked to terminal β1,4- or β1,3-Gal residues. These negatively charged sugars affect the biological activities and half-lives of many therapeutic glycoproteins (Schauer, 2000
Initial attempts to introduce Neu5Ac residues into plant N-glycans have concentrated mainly on the heterologous expression of enzymes and proteins that act in a late stage of the biosynthetic pathway. Rat 2,6-sialyltransferase expressed in transgenic Arabidopsis was found to be enzymatically active (Wee et al., 1998). In addition, human β1,4-galactosyltransferase was functionally expressed in plants (Palacpac et al., 1999; Bakker et al., 2001), and successful expression of a mammalian CMP-sialic acid transporter and a CMP-sialic acid synthetase in cultured plant cells has been reported (Misaki et al., 2006). However, with the exception of β1,4-galactosyltransferase, in planta activity has not been demonstrated for any of these proteins.
In this study, we report the in planta expression of three enzymes required for the synthesis of CMP-Neu5Ac in mammals: mouse UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE; Horstkorte et al., 1999
Heterologous Expression of Mouse GNE
In mammals, one of the initial steps in the Neu5Ac biosynthesis pathway is the conversion of the nucleotide sugar UDP-GlcNAc, which is naturally present in plant cells (Jiang et al., 2005
For activity assays, total soluble proteins from seedling cultures were extracted and incubated with UDP-GlcNAc in the presence of ATP. The reaction products were derivatized with 2-aminobenzoic acid (AA) and analyzed by reverse-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (RP-LC-ESI-MS/MS). A peak eluting at a similar position as the standard (GlcNAc-6-P-AA) was found in 35S:GNE plants but not in wild-type plants. The fragmentation pattern of this peak was characteristic of HexNAc-P-AA (Fig. 4A ), showing that the enzyme efficiently converts UDP-GlcNAc to ManNAc-6-P in vitro (generating 528 pmol ManNAc-6-P h–1 mg–1 total soluble protein). We also investigated the in vivo functionality of GNE by determining the amounts of ManNAc-6-P produced in Arabidopsis seedling cultures by ESI-MS/MS analysis. To corroborate the in vitro findings with a different methodology, underivatized samples were analyzed in this case. The fragmentation pattern of the HexNAc-P peak obtained for 35S:GNE-expressing plants was indicative of ManNAc-6-P (Fig. 4B). Significant amounts of ManNAc-6-P were found (10 nmol g–1 fresh weight seedlings). These results demonstrate that mouse GNE can synthesize ManNAc-6-P in planta entirely from endogenous precursors, presumably UDP-GlcNAc.
Heterologous Expression of Human NANS In mammals, ManNAc-6-P is converted by NANS into Neu5Ac-9-P through condensation with phosphoenolpyruvate (PEP). In order to generate plants that express an active form of human NANS, transgenic Arabidopsis plants carrying a 35S:NANS construct (Fig. 2B) were generated. The integration of the transgene was confirmed by genomic PCR (data not shown). Positive lines were analyzed for the expression of the recombinant protein by western blotting using anti-myc antibodies. A line exhibiting strong NANS expression was chosen for further analysis. Immunoblotting exhibited a prominent band with the expected size of approximately 42 kD in this line (Fig. 3). To test the in vitro activity of recombinant NANS, soluble protein extracts from seedling cultures were incubated with ManNAc-6-P and PEP. The reaction products were then derivatized with 1,2-diamino-4,5-methylene dioxybenzene (DMB). RP-HPLC analysis of the samples obtained using extracts from 35S:NANS plants showed a peak eluting at the same retention time as the standard (DMB-Neu5Ac; Fig. 5 ). This peak was not detected in samples incubated without ManNAc-6-P or in samples derived from extracts of nontransformed plants incubated with ManNAc-6-P and PEP (WT+ManNAc-6-P). RP-LC-ESI-MS/MS analysis revealed that only the reaction product obtained with 35:NANS extracts exhibited a fragmentation pattern corresponding to DMB-Neu5Ac, whereas the peak seen in WT+ManNAc-6-P samples was clearly not DMB-Neu5Ac (Fig. 5B). This also demonstrates that Arabidopsis plants are essentially devoid of endogenous NANS activity.
Our results show that crude extracts of Arabidopsis plants expressing human NANS are able to efficiently convert ManNAc-6-P to Neu5Ac. In mammals, Neu5Ac-9-P formed by NANS is dephosphorylated by a Neu5Ac-9-P phosphatase (NANP; Maliekal et al., 2006 ). The accumulation of Neu5Ac rather than Neu5Ac-9-P in our assays suggests that plants contain a NANP-like enzyme.
To test whether ManNAc-6-P produced by GNE can be further converted into Neu5Ac by NANS in planta, the 35S:GNE and 35S:NANS Arabidopsis lines were crossed. Western-blot analysis of the protein extracts from the resulting F1 progeny revealed the presence of the 80- and 42-kD bands (Fig. 3), indicating the expression of both GNE and NANS. After derivatization with DMB, the presence of Neu5Ac in extracts from leaves of 35S:GNE/35S:NANS and wild-type plants was analyzed by RP-HPLC and ESI-time of flight (TOF)-MS/MS (Fig. 6 ). No Neu5Ac (detection limit, 10 pmol g–1 fresh weight) was detected in wild-type plants; however, significant amounts (1,275 nmol g–1 fresh weight) were detected in leaves from the transgenic Arabidopsis line expressing both GNE and NANS (Table I ). Together, these data show that Neu5Ac can be produced in quantitative amounts in plants coexpressing mammalian GNE and NANS.
In Vivo Synthesis of CMP-Neu5Ac in Plants
In mammals, Neu5Ac is finally converted to CMP-Neu5Ac, a reaction catalyzed by CMAS. Since we failed to functionally express a full-length version of human CMAS in Arabidopsis, a construct encoding a truncated form of the enzyme lacking its 40 N-terminal amino acids (35S:tCMAS) was used to transform Arabidopsis. This construct lacks a Pro/Gly-rich domain that has been described to destabilize the protein (Krapp et al., 2003
Total N-Glycan Analysis in Neu5Ac- and CMP-Neu5Ac-Producing Plants
Since our ultimate aim is to generate plants capable of synthesizing N-glycans with terminal Neu5Ac residues, we tested whether the coexpression of GNE, NANS, and CMAS interferes with the N-glycosylation machinery (e.g. by depriving the UDP-GlcNAc-dependent Golgi enzymes GlcNAc transferase I and II of their donor substrate). We first analyzed total protein extracts from leaves of different transgenic lines and wild-type plants by western blot using antibodies recognizing plant complex N-glycans carrying β1,2-Xyl and core
Previous studies in our laboratory have shown that Neu5Ac is essentially undetectable in plants (Zeleny et al., 2006 ). These findings suggested that metabolic engineering of the Neu5Ac biosynthesis pathway is required to enable plants to synthesize significant amounts of Neu5Ac-containing glycoproteins. As an important step toward this goal, we have now successfully expressed three key enzymes in Arabidopsis: mouse GNE for the conversion of endogenous UDP-GlcNAc into ManNAc-6-P, human NANS for the synthesis of Neu5Ac-9-P from ManNAc-6-P, and human CMAS for the subsequent transformation of Neu5Ac into the sugar nucleotide CMP-Neu5Ac.
While Neu5Ac is generated in bacteria by sialic acid synthase through the condensation of ManNAc with PEP (Vann et al., 1997
Previous attempts to generate sialic acid in plants by expressing microbial sialic acid synthase showed a relatively low expression level of the heterologous protein, and although the bacterial enzyme was able to synthesize Neu5Ac in vitro, no in vivo accumulation of sialic acid was detected (Paccalet et al., 2007
We show that the amounts of Neu5Ac produced in transgenic GNE-, NANS-, and CMAS-expressing Arabidopsis are quantitatively significant (41 nmol g–1 fresh weight, equivalent to approximately 3 nmol mg–1 total soluble protein) and comparable to the levels found in mammalian cells (1 nmol mg–1 protein; Tietze et al., 1989
The amount of detected CMP-Neu5Ac (2.4 nmol g–1 fresh weight, equivalent to approximately 200 pmol mg–1 total soluble protein) is 13 times lower than the amount found in rat liver (32 nmol g–1 wet weight; Pels Rijcken et al., 1990
It has been reported for rice roots that a reduction of their UDP-GlcNAc content interferes with protein N-glycosylation and causes cell shrinkage (Jiang et al., 2005
The generation of Arabidopsis plants able to synthesize significant amounts of CMP-Neu5Ac, in combination with the already achieved in planta expression of the CMP-Neu5Ac transporter (Misaki et al., 2006
Construction of myc and DsRed Plant Expression Vectors
The binary plant expression vector p19 was generated by insertion of a myc tag linker fragment containing a stop codon into the BamHI site of pPT2 (Strasser et al., 2005 Bacterial strains containing the cDNAs of mouse GNE (clone IRAKp961N12108Q), human NANS (clone IRALp962F017Q), and human CMAS (clone DKFZp666I142Q) were purchased from the German Resource Centre for Genome Research (http://www.rzpd.de). All clones were completely sequenced to confirm the presence of the full-length open reading frame. The coding regions lacking the stop codons of the GNE and NANS genes were amplified by PCR with primers GNE3F (XbaI, 5'-TATATCTAGAATGGAGAAGAACGGGAACAAC-3')/GNE4R (BglII, 5'-TATAAGATCTGTGGATCCTGCGCGTTGTGTA-3') and NANS3F (XbaI, 5'-TATATCTAGAATGCCGCTGGAGCTGGAGCTG-3')/NANS4R (BamHI, 5'-TATAGGATCCAGACTTGATTTTTTTGCCATG-3'). A truncated form of human CMAS lacking the first 40 amino acids was amplified by PCR with primers CMAS12F (XbaI, 5'-TATATCTAGAATGAAGCCCCCGCACCTGGCAGCCCTA-3') and CMAS5R (BamHI, 5'-TATAGGATCCCTATTTTTGGCATGAATTATTAAC-3'). The PCR products were purified from the gel using the Invisorb Spin PCRapid kit (Invitek), digested with XbaI/BglII (GNE) or with XbaI/BamHI (NANS and CMAS), and cloned into the XbaI/BamHI-digested p19 (GNE and NANS) and pMS (CMAS) vectors. The resulting plant expression vectors were named p19GNE (35S:GNE), p19NANS (35S:NANS), and pMS-tCMAS (35S:tCMAS). Individual clones were sequenced using the PRISM BigDye terminator cycle sequence kit and ABI 3100 genetic analyzer (Applied Biosystems).
Transgenic Arabidopsis (Arabidopsis thaliana) plants were generated by floral dipping as described by Strasser et al. (2005) Individual transgenic plants carrying the 35S:GNE and 35S:NANS constructs were crossed for the coexpression of both proteins in a single plant. The presence of the cDNA sequences in F1 hybrid plants was confirmed by PCR from genomic DNA. The F2 generation of transgenic plants was propagated on soil and as seedling cultures in liquid Murashige and Skoog basal medium (Sigma-Aldrich), pH 5.8, containing 3% (w/v) Suc. The flasks were incubated for 2 d at 4°C in the dark and then transferred to constant light under agitation (130 rpm) at 20°C for 12 d. For coexpression of the three mammalian genes, F2 hybrid plants growing on soil were transformed with pMS-tCMAS by floral dipping, and potential CMAS transgenic Arabidopsis seeds were selected for DsRed expression with a Leica MZ FLIII stereomicroscope equipped with a DsRed filter set. The presence of CMAS was confirmed by PCR with the primers CMAS8F/CMAS9R.
Total proteins were extracted from leaves with 1x PBS supplemented with 1% (v/v) Triton X-100. Proteins from young leaves or from four to five Arabidopsis seedlings were extracted in the same way. The protein extracts were centrifuged twice at 8,000 rpm for 10 min at 4°C and subjected to 12.5% SDS-PAGE under reducing conditions. The fractionated proteins were blotted to Hybond-ECL nitrocellulose membranes (GE Healthcare) and blocked for 1 h in 1x PBS supplemented with 3% (w/v) bovine serum albumin and 1% (v/v) Tween 20, and detection was performed using anti-myc antibodies (9E10; Santa Cruz Biotechnology) and SuperSignal West Pico Chemiluminescent substrate (Pierce). Immunoblots for the detection of complex N-glycans carrying β1,2-Xyl and core
Seedlings (2 g) were homogenized in 1 mL of 100 mM Tris-HCl, pH 7.5, and 20 mM MgCl2 in the presence of protease inhibitors (Complete Mini, EDTA free; Roche). After centrifugation at 13,000 rpm for 10 min at 4°C, 93 µL of supernatant was incubated, in a final volume of 100 µL, with 50 nmol of UDP-GlcNAc (Sigma-Aldrich) in the presence of 100 nmol of ATP and 1 mM CaCl2 either for 4 h or overnight. The reaction mixture was centrifuged at 13,000 rpm for 10 min. The liquid was applied to a C18-RP SPE cartridge, whereby the flow through was collected and dried by vacuum. The dried sample was derivatized with AA (Anumula, 1994
Seedlings (0.5 g) were homogenized with an Ultra-Turrax homogenizer in 1 mL of water. After adding 80 µL of concentrated acetic acid, the mixture was centrifuged at 20,000 rpm for 20 min. The supernatant was applied to a C18-RP SPE cartridge. The flow through was directly applied to a gel filtration column (1 x 75 cm) filled with Biogel P2 fine (Bio-Rad) and eluted with 20 mM ammonium acetate, pH 5.5, at a flow rate 1 mL min–1. Fractions were analyzed for the presence of HexNAc-6-P by direct infusion into the ESI source (in 50% [v/v] AcCN and 0.1% [v/v] formic acid). Fragments of the parent mass of 300.0 D were measured with a Q-TOF Ultima Global apparatus operated in the negative ion mode.
Seedlings (2 g) were homogenized with an Ultra-Turrax homogenizer in 1 mL of 50 mM Bicine hydrochloride, pH 8.0, in the presence of protease inhibitors (Complete Mini, EDTA free; Roche) and further centrifuged at 13,000 rpm for 10 min at 4°C. The assay was performed by incubating 83 µL of supernatant with 5 nmol of ManNAc-6-P and 5 nmol of PEP in the presence of 12.5 mM MnCl2 in a final volume of 100 µL. After overnight incubation at 37°C, the reaction mixture was centrifuged and dried as above. The dried sample was derivatized with DMB (Sigma-Aldrich; Hara et al., 1987
The first steps were performed as described for the ManNAc-6-P analysis using Arabidopsis leaves (0.5 g) from 6-week-old plants instead of seedlings. Fractions obtained after separation by gel filtration (see above) were brought to 50% (v/v) AcCN and 0.1% (v/v) formic acid and analyzed for the presence of Neu5Ac by direct infusion into the ESI source. Analytes were detected by MS/MS ([M–H]– = 308.1) in negative ion mode. Additionally, quantification was done by HPLC after DMB derivatization (see above).
Leaves (0.1 g) from 6-week-old Arabidopsis plants were homogenized with an Ultra-Turrax homogenizer (in 100 µL of 1% NH4 solution). The sample was centrifuged at 13,000 rpm for 10 min, and the supernatant was applied to a C18-RP SPE cartridge. The flow through was brought to 40% AcCN (v/v) and applied to a 10-mg HyperSep Hypercarb SPE cartridge (Thermo Scientific). The flow through was collected and freeze-dried, and equivalents of 1% were analyzed on a Hypercarb column (0.32 x 50 mm; Thermo Scientific) using a 65 mM ammonium formiate buffer, pH 3.0, as the aqueous solvent. After sample injection, which was performed with buffer only, a gradient from 4% to 22% AcCN was developed in 30 min with a flow rate of 8 µL/min. Analytes were detected with an ESI-Q-TOF Ultima Global apparatus in MS/MS mode with MS1 set on mass-to-charge ratio = 613.1, the mass of the [M–H]– ion of CMP-Neu5Ac.
N-Glycans were extracted from 500 mg of leaves from 6-week-old transgenic Arabidopsis plants and analyzed by MALDI-TOF-MS as described previously (Strasser et al., 2004
We thank Pia Gattinger and Karin Polacsek (University of Natural Resources and Applied Life Sciences, Vienna) for excellent technical assistance. Received February 7, 2008; accepted March 3, 2008; published March 7, 2008.
1 This work was supported by Grant LS 154 from the Vienna Science and Technology Fund and by a grant from the Austrian Science Fund (grant no. P18314 to H.S.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard Strasser (richard.strasser{at}boku.ac.at).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.117572 * Corresponding author; e-mail richard.strasser{at}boku.ac.at.
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