Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis

Abstract

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.

Figure 1.

Schematic representation of the mammalian pathway for the sialylation of glycoconjugates. The enzymes involved in the process are indicated: GNE, NANS, NANP, CMAS, CMP-Neu5Ac transporter (CST), and sialyltransferase (ST).

Figure 2.

Schematic representation of the different plant expression cassettes used in this study. A, 35S:GNE. B, 35S:NANS. C, 35S:tCMAS. g7T, Agrobacterium tumefaciens gene 7 terminator; Kan^R, neomycin phosphotransferase II; LB, left border; mc, c-myc epitope tag; Pnos, nopaline synthase gene promoter; P35S, promoter of the 35S transcript of the Cauliflower mosaic virus; P35SS, promoter of the 35S transcript of the Cauliflower mosaic virus with double enhancer; RB, right border; TL, translational enhancer (5′ untranslated region of Tobacco etch virus); Tnos, nopaline synthase gene terminator; T35S, terminator of the 35S transcript of the Cauliflower mosaic virus; TP, transit peptide from barley (Hordeum vulgare) granule-bound starch synthase I.

Figure 3.

Immunoblot of Arabidopsis leaf extracts using anti-myc antibodies. Proteins were subjected to SDS-PAGE under reducing conditions. Lane 1, extract from the wild type; lane 2, extract from the 35S:GNE transgenic line; lane 3, extract from the 35S:GNE/35S:NANS transgenic line; lane 4, extract from the 35S:NANS transgenic line. Protein sizes are indicated.

Figure 4.

ManNAc-6-P production in vitro and in planta by GNE expression in Arabidopsis seedling cultures. A, Determination of ManNAc-6-P production in vitro. MS/MS spectra of the HexNAc-6-P peak region acquired from the GlcNAc-6-P standard (S), from extracts of 35S:GNE transgenic plants (GNE), and from wild-type plants (wt) incubated in the presence of UDP-GlcNAc and ATP. Masses of fragments arising from AA-labeled HexNAc-6-P are underlined. B, Demonstration of ManNAc-6-P production in planta. The presence of HexNAc-6-P in Arabidopsis extracts was analyzed by direct infusion ESI-MS/MS analysis of the [M−H]⁻ parent ion of mass 300.0. Characteristic fragmentation products are labeled F1, F2, and F3. Spectra were obtained with GlcNAc-6-P standard (S) and extracts from 35S:GNE transgenic plants (GNE) and wild-type plants (wt).

Figure 5.

A, In vitro NANS activity assay analyzed by RP-HPLC of DMB-Neu5Ac in extracts from Arabidopsis seedlings: DMB-Neu5Ac standard (S), DMB-labeled extract from wild-type plants after incubation in the presence (wt + ManNAc-6-P) or absence (wt) of ManNAc-6-P, and 35S:NANS transgenic plants after incubation in the presence (NANS + ManNAc-6-P) or absence (NANS) of ManNAc-6-P. The elution position of DMB-Neu5Ac is indicated. B, The eluate in the region of DMB-Neu5Ac was collected, concentrated, and subjected to RP-LC-ESI-MS/MS to acquire a fragment spectrum of the presumed DMB-Neu5Ac peaks. All traces are shown with the same y axis setting.

Figure 6.

In planta synthesis of Neu5Ac. A, RP-HPLC of DMB-Neu5Ac in wild-type extract spiked with DMB-Neu5Ac (wt + S), extract from GNE- and NANS-expressing transgenic Arabidopsis (GNE NANS), and wild-type control (wt). The DMB-Neu5Ac peak is indicated. The background peaks are higher in the spiked wild-type extract because 10 times more sample was injected there. B, Mass spectrometric detection (ESI-MS/MS on mass 308.1) of underivatized Neu5Ac standard (S), direct infusion of partially purified extract from GNE- and NANS-expressing transgenic Arabidopsis (GNE NANS), and wild-type control (wt). Characteristic peaks are marked by arrows.

Figure 7.

In planta synthesis of CMP-Neu5Ac. A, LC-ESI-MS/MS analysis. CMP-Neu5Ac standard (S), extract from wild-type control (wt), and extract from GNE-, NANS-, and CMAS-expressing transgenic Arabidopsis (GNE NANS CMAS) were analyzed with parent ion mass set to 613.1 D. B, Mass spectra at the elution position of CMP-Neu5Ac: standard CMP-Neu5Ac (S), wild-type control (wt), and GNE-, NANS-, and CMAS-expressing transgenic Arabidopsis (GNE NANS CMAS). All runs were set to identical absolute intensity.

Figure 8.

Analysis of changes in overall N-glycosylation in Neu5Ac- and CMP-Neu5Ac-producing Arabidopsis plants. A, Proteins were extracted from leaves and subjected to SDS-PAGE under reducing conditions. Immunoblotting was performed using anti-horseradish peroxidase antibodies. Lane 1, Arabidopsis wild type; lane 2, xylt fuct mutant plant (negative control; Strasser et al., 2004); lane 3, GNE NANS plant; lane 4, GNE NANS CMAS plant. The bottom panel shows Ponceau S staining of the membrane. B, MALDI-TOF-MS analysis of total N-glycans present in leaves from wild-type (wt), GNE NANS, and GNE NANS CMAS Arabidopsis plants. For abbreviations of N-glycan structures, see http://www.proglycan.com/.