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Stable Expression of Human ß1,4-Galactosyltransferase in Plant Cells Modifies N-Linked Glycosylation Patterns

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Stable Expression of Human ß1,4-Galactosyltransferase in Plant Cells Modifies N-Linked Glycosylation Patterns Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4692–4697, April 1999 Plant Biology Stable expression of human b1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns NIRIANNE Q. PALACPAC*, SHOHEI YOSHIDA*†,HIROMI SAKAI*, YOSHINOBU KIMURA‡,KAZUHITO FUJIYAMA*§, TOSHIOMI YOSHIDA*, AND TATSUJI SEKI* *The International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565, Japan; and ‡Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-naka 1-1-1, Okayama 700, Japan Communicated by Charles J. Arntzen, Boyce Thompson Institute for Plant Research, Ithaca, NY, February 23, 1999 (received for review November 25, 1998) b ABSTRACT 1,4-Galactosyltransferase (UDP galactose: N-glycans in plants are mostly of a Man3GlcNAc2 structure b-N-acetylglucosaminide: b1,4-galactosyltransferase; EC with or without b1,2-xylose andyor a1,3-fucose residues (8– 2.4.1.22) catalyzes the transfer of galactose from UDP-Gal to 10). Larger, complex-type plant N-glycans are rare and re- N-acetylglucosamine in the penultimate stages of the terminal cently have been identified as additional a1,4-fucose and glycosylation of N-linked complex oligosaccharides in mam- b1,3-galactose residues, giving rise to mammalian Lewis a malian cells. Tobacco BY2 cells lack this Golgi enzyme. To (Lea) structures (9). The presence of xylose andyor fucose determine to what extent the production of a mammalian residues makes plant recombinant therapeutics less desirable glycosyltransferase can alter the glycosylation pathway of (8–10). Complementing the N-glycan machinery with heter- plant cells, tobacco BY2 suspension-cultured cells were stably ologous glycosyltransferases may help achieve the production transformed with the full-length human galactosyltransferase of glycoproteins with human-compatible-type oligosaccharide gene placed under the control of the cauliflower mosaic virus structures. In plants, transformation of mutant Arabidopsis cgl 35S promoter. The expression was confirmed by assaying cells with the cDNA encoding human N-acetylglucosaminyl enzymatic activity as well as by Southern and Western blot- transferase I (GnT-I) resulted in the conversion of high- ting. The transformant with the highest level of enzymatic mannose glycans into complex glycans that may be fucose-rich activity has glycans with galactose residues at the terminal and xylose-poor, implying that the human enzyme can be nonreducing ends, indicating the successful modification of integrated functionally in the normal pathway for biosynthesis the plant cell N-glycosylation pathway. Analysis of the oligo- of complex glycans in Arabidopsis (8, 12). saccharide structures shows that the galactosylated N-glycans We expressed the human b1,4-galactosyltransferase gene account for 47.3% of the total sugar chains. In addition, the (UDP galactose: b-N-acetylglucosaminide: b1,4 galactosyl- absence of the dominant xylosidated- and fucosylated-type transferase; EC 2.4.1.22), placed under the control of the sugar chains confirms that the transformed cells can be used cauliflower mosaic virus 35S (CaMV35S) promoter, in Nico- to produce glycoproteins without the highly immunogenic tiana tabacum L. cv. Bright Yellow 2 (BY2) cells as a first step glycans typically found in plants. These results demonstrate to evaluate the possibility of enlarging the spectrum of glyco- the synthesis in plants of N-linked glycans with modified and syltransferases in plant suspension-cultured cells, which po- defined sugar chain structures similar to mammalian glyco- tentially could lead to transgenic plants. We chose the mam- proteins. malian glycosyltransferase for two reasons: b1,4-galactosyl- transferase is the first glycosyltransferase in mammalian cells Transgenic plants are one of the promising systems for the that initiates the further branching of complex N-linked gly- production of human therapeutic proteins because of ease of cans after the action of GnT-I and -II (9, 11, 13); and though genetic manipulation, lack of potential contamination with the glycosyltransferase is ubiquitous in the vertebrate kingdom human pathogens, low-cost biomass production, and the in- (13), its presence has not yet been conclusively proven in plants (9, 10). Moreover, we recently have shown by HPLC and herent capacity to carry out most posttranslational modifica- y tions similar to mammalian cells (1–3). Among the posttrans- ion-spray tandem (IS)-MS MS analyses that tobacco BY2 lational modifications, glycosylation has been shown to play suspension-cultured cells do not contain any galactosylated N-glycan (14), suggesting that the glycosyltransferase either critical roles for various physiological activities of mammalian may be absent or too low to be effective in these cells. The glycoproteins (2, 4–6). Modifying the glycosylation activities is transformed cells expressed b1,4-galactosyltransferase activity important not only for medical and biotechnological purposes and possess glycans that bind to Ricinus communis agglutinin (7) but also as a tool to investigate the role of plant glycan (RCA ; specific for b1,4-linked galactose). Galactosylated structures and the N-link glycosylation pathway in plants (8). 120 glycans did not react with an antibody specific for complex The asparagine-linked (N-linked) glycosylation mechanism glycans containing b1,2-xylose residues, indicating that the in mammalian and plant systems is conserved evolutionarily; sugar chains do not contain b1,2-xylose residues. Glycans with however, the fine details in the oligosaccharide trimming and a1,3-fucose were not observed based on HPLC and IS-MSyMS further modification of glycans in the Golgi differ (5, 8–10). determinations. Structural analysis of the oligosaccharide moi- Thus, high-mannose-type N-glycans in plants have structures eties from glycoproteins of transformed cells provides proof of identical to those found in other eukaryotic cells; however, plant complex N-linked glycans differ substantially (8). In the Abbreviations: hGT, human b1,4-galactosyltransferase; BY2, Nicoti- mammalian system, the Man3GlcNAc2 (M3) core structure is ana tabacum L. cv. Bright Yellow 2; RCA120, Ricinus communis120 extended further to contain penultimate galactose and termi- agglutinin; PA, pyridylamino; RP- and SF-HPLC, reversed-phase and nal sialic acid residues (5, 11). In contrast, typically processed size fractionation HPLC; IS-MSyMS, ion-spray tandem MSyMS; GnT-I and -II, N-acetylglucosaminyl transferase I and II; CaMV35S, The publication costs of this article were defrayed in part by page charge cauliflower mosaic virus 35S. †Present address: EZAKI Glico Co., Ltd., 4-6-5 Utajima, Nishiyo- payment. This article must therefore be hereby marked ‘‘advertisement’’ in dogawa, Osaka 565, Japan. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. e-mail: fujiyama@ PNAS is available online at www.pnas.org. icb.osaka-u.ac.jp. 4692 Downloaded by guest on September 29, 2021 Plant Biology: Palacpac et al. Proc. Natl. Acad. Sci. USA 96 (1999) 4693 the changes in plant N-glycan structure and confirms that tories) and washed, and the horseradish peroxidase color galactosylation influenced and improved the N-linked pathway reaction was carried out by using the POD immunostain kit in tobacco BY2 cells. (Wako Chemicals, Osaka). Immunoblot analysis for plant-specific complex glycans was MATERIALS AND METHODS carried out by using a polyclonal antiserum raised against carrot cell-wall b-fructosidase (27). Construction of the Plant Expression Vector. The gene for b1,4-Galactosyltransferase activity was assayed by using human b1,4-galactosyltransferase (hGT) was amplified by UDP-Gal and pyridylamino-labeled GlcNAc2Man3GlcNAc2 PCR using two sets of primers based on the cDNA sequence 9 (GlcNAc2Man3GlcNAc2-PA) as substrate (28). The enzyme reported by Masri et al. (15). Primers 1 (5 -AAGACTAGT- reaction contained 1–120 mg protein, 25 mM sodium cacody- GGGCCCCATGCTGATTGA-39, SpeI site in italics) and 2 late (pH 7.4), 10 mM MnCl , 200 mM UDP-Gal, and 100 nM (59-GTAGTCGACGTGTACCAAAACGCTAGCT-39, SalI 2 GlcNAc Man GlcNAc -PA. Reaction products were analyzed site in italics) were used to amplify an 813-bp fragment from 2 3 2 by HPLC using a PALPAK Type R and a Type N column human placenta cDNA (CLONTECH) by using Taq polymer- ase (Takara Shuzo, Kyoto). For the N-terminal portion of the (Takara Shuzo) according to the manufacturer’s recommen- gene, primers 3 (59-AAAGAATTCGCGATGCCAG- dation. PA-labeled standards GlcNAc2Man3GlcNAc2-PA and GCGCGTCCCT-39, EcoRI site in italics) and 4 (59- Gal2GlcNAc2Man3GlcNAc2-PA and two isomers of AATACTAGTAGCGGGGACTCCTCAGGGCA-39, SpeI GalGlcNAc2Man3GlcNAc2-PA were from Takara Shuzo and site in italics) were used to obtain a 376-bp fragment from Honen Co. (Tokyo), respectively. human genomic DNA (CLONTECH). The PCR products Affinity Chromatography on RCA120. Crude cell extracts were cloned in M13mp18, sequenced with an AutoRead and microsome fractions of transformed cells with highest Sequencing Kit (Pharmacia), and analyzed by using an ALF enzymatic activity were loaded onto an RCA120-agarose col- DNA sequencer (Pharmacia). The truncated coding sequences umn (Wako). The column was washed with 15 volumes of 10 were juxtaposed to obtain the complete b1,4-galactosyltrans- mM ammonium acetate, pH 6.0. Bound proteins were eluted ferase gene (1.2 kbp). Sequence alignment of the entire gene with 0.2 M lactose and fractionated on SDSyPAGE
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