An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers

Kaori, Furuta

Megumi, Maeda

Tanabe, Chinatsu

Yoshinobu, Kimura

Bioscience Biotechnology and Biochemistry

Okayama University Scientific Achievement Repository

 1 Running title: N-Glycans of bamboo shoot glycoproteins 1  2 Structural feature of N-glycans of bamboo shoot glycoproteins: Useful source of plant antigenic 3 N-glycans  4  5 Chinatsu TANABE, Kaori FURUTA, Megumi MAEDA, and Yoshinobu KIMURA† 6  7 Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, 8 Okayama University, 1-1-1 Tsushima-Naka, Okayama 700-8530, Japan 9  10 † To whom correspondence should be addressed. E-mail: yosh8mar@okayama-u.ac.jp 11  12 Abbreviations: RP-HPLC, reversed-phase HPLC; SF-HPLC, size-fractionation HPLC; PA-, 13 pyridylamino; PCT, plant complex type; Hex, hexose; HexNAc, N-acetylhexosamine; Pen, 14 pentose; Deoxyhex, deoxyhexose; Man, D-mannose; GlcNAc, N-acetyl-D-glucosamine; Xyl, s-15 xylose; Fuc, L-fucose; Lea, Lewis a (Galb1-3(Fuca1-4)GlcNAc); PCT, plant complex type; 16 M3FX, Mana1-6(Mana1-3)(Xylb1-2)Manb1-4GlcNAcb1-4(Fuca1-3)GlcNAc-PA; 17 GN2M3FX, GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb1-4GlcNAcb1-18 4(Fuca1-3)GlcNAc-PA; (Lea)1GN1M3FX, Galb1-3(Fuca1-4)GlcNAc1-2 Mana1-19 6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb1-4GlcNAcb1-4(Fuca1-3)GlcNAc-PA or GlcNAc1-20 2Mana1-6(Galb1-3(Fuca1-4)GlcNAc1-2Mana1-3)(Xylb1-2)Manb1-4GlcNAcb1-4(Fuca1-21 3)GlcNAc-PA; (Lea)2M3FX, Galb1-3(Fuca1-4)GlcNAc1-2 Mana1-6(Galb1-3(Fuca1-22 4)GlcNAc1-2Mana1-3)(Xylb1-2)Manb1-4GlcNAcb1-4(Fuca1-3)GlcNAc-PA. 23   24  2   An effective method to prepare plant complex type N-glycans in large amounts has been 1 required to evaluate their immunological activity. In this study, we found that glycoproteins in 2 bamboo shoots predominantly carry plant complex type N-glycans including the Lewis a 3 epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant 4 antigenic glycans to synthesize immuno-active neoglycopolymers. 5  6 Keywords: Antigenic N-glycans, plant N-glycans, plant glycoproteins, Bamboo shoot, 7 Phyllostachys edulis.  8  9    Plant complex type (PCT) N-glycans are well known to have antigenicity toward 10 mammalians and are commonly found on Cupressaceae pollen allergens, such as Cup a1, Cry 11 j1, Cha o1, and Jun a1.1-5) We have already found that these PCT N-glycans suppressed the 12 production of IL-4 from Th2 cells in patients with Japanese cedar pollinosis,6) although the 13 clinical significance of these PCT N-glycans involved in pollinosis has not been clarified. Our 14 findings suggested a possibility that free PCT N-glycans (FNGs) have potential anti-pollinosis 15 activity for treatment of cedar pollinosis. For evaluation of the putative anti-pollinosis activity 16 or other immunological activities of PCT N-glycans, the effective method to prepare PCT-FNGs 17 in large amount has been required but only a few good sources have been reported.7,8) For 18 preparation of Lewis a epitope-containing PCT N-glycans with high efficiency, we have 19 analyzed the structures of N-glycans linked to plant glycoproteins expressed in various plant 20 materials including edible seeds,7,9) vegetables,10) fresh-water plants,8) and seaweeds.11) In the 21 course of the glycan-screening, we found that bamboo shoot, in addition to fresh-water plants, is 22 a useful source for preparation of immuno-active plant N-glycans or N-glycopeptides containing 23 Lewis a epitope. Hence, in this report, we describe the structural analysis of N-glycans of 24 bamboo glycoproteins. 25    Bamboo shoots were collected at Yakage Town (Oda-Gun, Okayama Prefecture, Japan).  26 Chopped bamboo shoots (732 g) were homogenized in 5% formic acid (2 L) with a waring 27 blender and digested with 1.2 g of pepsin at 37℃ for 72 h. After neutralization with ammonium 28  3 solution, the peptic digests were filtered with two-layered gauze and the filtrates were 1 centrifuged at 12,450 g for 20 min. The supernatants were concentrated to 415 mL with a rotary 2 evaporator and were dialyzed (using Visking tubing UC24/32, Viskase Co. IL, USA) against 3 deionized water (2.5 L twice) at 4oC for three days. Outer solution was concentrated to about 4 200 mL with a rotary evaporator and applied to Dowex 50 × 2 resin (200 ml) to remove the 5 neutral oligosaccharides. After washing the resin with 1% formic acid, adsorbed positive-6 charged compounds containing glycopeptides were eluted with 3L of 0.2N NH4OH and 7 concentrated to a small amount of solution with a rotary evaporator. The glycopeptide-fractions 8 were applied to a Sephadex G-25 fine column (4.0 × 80 cm) equilibrated with 0.1N NH4OH. 9 The glycopeptide-fractions (elution volume, 390-700 ml) were detected by the Phenol-H2SO4 10 method,12) and were concentrated with a rotary evaporator and lyophilized. The glycopeptide-11 fraction (483 mg) was dissolved in deionized water (25 mL) and applied to a Wako Gel 100C18 12 column (3.2 x 42.5 cm). Glycopeptides were eluted by a linear gradient of acetonitrile from 0 to 13 25% in deionized water, and the glycopeptide-fraction eluted between 7 to 20 % of acetonitrile 14 was concentrated with a rotary evaporator and lyophilized. The partially purified peptic 15 glycopeptides (233 mg) were further digested with actinase (20 mg) in 25 mL of 0.1 M Tris-HCl 16 (pH 8.0) containing 5 mM CaCl2 at 37℃ for 72 h. After incubation, the reaction mixture was 17 concentrated to small amount and desalted by a gel-filtration with a Sephadex G-25 fine column 18 (4.0 × 80 cm) equilibrated with 0.1N NH4OH. The glycopeptide-fraction (elution volume, 400-19 700 ml) was concentrated with a rotary evaporator and lyophilized. The lyophilized 20 glycopeptides (95.1 mg) were dissolved in 50% acetonitrile/water, and Asn-glycopeptides were 21 further purified by the hydrophilic partitioning with Shodex Asahipak NH2P-50 resin as 22 described in our previous paper,7) Asn-glycopeptides were predominantly eluted with 0.1% 23 TFA/water and the final amount of Asn-glycopeptides purified in this study was 15. 7 mg.  24      To identify structures of N-glycans linked to the bamboo shoot glycopeptides, N-25 glycans were liberated by hydrazinolysis (100oC, 10 hr) from the peptides (about 1 mg).  The 26  4 liberated N-glycans were N-acetylated and pyridylaminated.13) After gel filtration to remove of 1 excess amount of 2-aminopyridine, the PA-sugar chains were partially purified by RP-HPLC 2 using a Cosmosil 5C18 AR column (6.0 x 250 mm, Nacalai Tesque, Kyoto), 7) and PA-sugar 3 chains were pooled as indicated by a horizontal bar in Fig. 1-I. Structural features of the PA-4 sugar chains obtained by RP-HPLC were analyzed by SF-HPLC using a Asahipak NH2P-50 5 column (0.46 x 25 cm, Showa Denko, Tokyo). 7) As shown in Fig. 1-II, seven PA-sugar chains 6 (peak-a, peak-b, peak-c, peak-d, peak-e, peak-f, and peak-g) were observed, and each elution 7 position was compared with those of authentic PCT N-glycans purified the water-plant 8 glycoproteins.7) The elution position of peak-a on SF-HPLC coincided with that of 9 Man2Xyl1Fuc1GlcNAc2-PA (M2FX), peak-b with that of M3FX, peak-c with that of 10 GlcNAc1Man3Xyl1Fuc1GlcNAc2-PA (GN1M3FX), peak-d with that of 11 GlcNAc2Man3Xyl1Fuc1GlcNAc2-PA (GN2M3FX), peak-e with that of 12 Gal1GlcNAc2Man3Xyl1Fuc1GlcNAc2-PA (G1GN2M3FX), peak-f with that of 13 Gal1Fuc1GlcNAc2Man3Xyl1Fuc1GlcNAc2-PA ((Lea)1GN1M3FX), and peak-g with that of 14 Gal2Fuc2GlcNAc2Man3Xyl1Fuc1GlcNAc2-PA ((Lea)2M3FX). These deduced structures were 15 confirmed by ESI-MS analyses as follows: peak-a, m/z 1105.4 [(M+H) +]; peak-b, m/z 1267.5 16 [(M+H) +]; peak-c, m/z 735.8 [(M+2H) 2+]; peak-d, m/z 837.3 [(M+2H) 2+]; peak-e, m/z 918.4 17 [(M+2H) 2+]; peak-f, m/z 991.4 [(M+2H) 2+]; peak-g, m/z1145.4 [(M+2H) 2+]. These structures 18 were further analyzed by exoglycosidase digestions (Fig. 2). Peaks-f and -g were converted to 19 peak-d (GN2M3FX) by the sequential digestion with a-1,3/4-fucosidase and b-1,3/6-20 galactosidase, suggesting that PA-sugar chains in peak-f and peak-g must be modified with 21 Lewis a epitope(s) at their non-reducing terminals. Finally, PA-sugar chains, peaks-c and -d, 22 and the enzyme digests obtained from peaks-f and -g, were converted to peak-b by b-N-23 acetylglucosaminidase, suggesting modification with N-acetylglucosamine (GlcNAc) 24 residue(s). Since peak-h emerged after digestion of the b-1,3/6-galactosidase digest with b-N-25 acetylglucosaminidase, xylosyl and afucosyl N-glycans (GlcNAc1Man3Xyl1GlcNAc2-PA or 26 Fig. 1 Fig. 2  5 GlcNAc2Man3Xyl1GlcNAc2-PA), which are eluted at almost the same elution positions to 1 M3FX and GN1M3FX respectively, must be contaminated in peak-b or peak-c. These results 2 indicate that almost all glycoproteins expressed in bamboo shoot carry PCT N-glycans but not 3 high-mannose type ones, and some of them are modified with Lewis a epitope(s). This 4 glycoform of bamboo N-glycans is basically similar to that of N-glycans linked to 5 glycoproteins in a fresh-water plant, Elodea nuttallii,8) although the relative amount of M3FX 6 in the bamboo PCT N-glycans (32.6%) was higher than that in the water plant PCT N-glycans 7 (12.1%). Since we have already confirmed that Lewis a epitope-containing N-glycans and the 8 truncated type N-glycans (M3FX and M2FX) were separated by a gel-filtration with Sephadex 9 G-25 column used in our previous study,7) the Lewis a epitope-containing N-glycans can be 10 easily prepared from the mixture of bamboo N-glycans or N-glycopeptides. The yield of each 11 PCT N-glycans is summarized in Table. The yield of the Lea epitope-containing PCT N-12 glycans as pyridylaminated derivatives was more than 300 nmol from 700 g of bamboo shoot, 13 indicating that bamboo shoot is one of good sources for preparation of Lea epitope-containing 14 PCT N-glycans to evaluate their putative immunological activities. In previous study,8) we 15 found that the fresh-water plants, Elodea nuttallii, Egeria densa, Ceratophyllum demersum, 16 are good sources for preparation of immunogenic PCT N-glycans. However, large amount 17 contamination of water soluble pigments contained in these fresh-water plants interfered the 18 extraction and purification steps, and we had to remove these pigments by repeat gel-filtration 19 or hydrophilic chromatographic steps. On the other hand, the bamboo shoot extract was 20 almost uncolored and the decolorization steps to purify PCT N-glycans or N-glycopeptides 21 were not required. Furthermore, chips of bamboo shoot can be easily homogenized with 5% 22 formic acid using a waring blender compared with the bulky water-plants.  23      Using bamboo shoot glycopeptides obtained by the method established in this study, we 24 have already succeeded in synthesis of a neoglycopolymer consisting of g-poly glutamic acid 25 and plant antigenic N-glycans (Itano, S., et al., Proceedings of the Japanese Society for 26 Table  6 Immunology. 45, 166 (2016)). The immunological activities of this neoglycopolymer will be 1 described elsewhere.  2  3 Author contribution 4  Y.K. shared responsibility for the writing of the manuscript with C.T., K.F., and M.M. All 5 authors were responsible for the study concept and design. C.T., K.F., and M.M. carried it out.  6 All authors contributed to the critical revision of the manuscript. 7  8 Funding. 9 This work was supported in part by grants from the Ministry of Education, Culture, Sports, 10 Science, and Technology of Japan(Basic Research C (no. 15K07841 to M.M.), Fostering Joint 11 International Research (no. 15KK0282) to M.M, Japan Science and Technology Agency 12 (Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-step, 13 no. AS262Z00115Q to YK), and Takano Life Science Research Foundation (Y.K). 14  15 Acknowledgments 16 We are grateful to the Department of Instrumental Analysis, Advanced Science Research a 17 nonsensical phrase Center, Okayama University, for ESI-MS analysis. 18  19 References 20 [1] Di Felice GD, Barletta B, Tinghino R, et al. Cupressaceae pollinosis: identification, 21 purification and cloning of relevant allergens. Int. Arch. Allergy Immunol. 125, 280-289 22 (2001).  23  [2] Alisi C, Afferni C, Iacovacci P, et al. Rapid isolation, charcterization, and glycan analysis 24 of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen. Allergy, 56, 25 978-984 (2001). 26  7 [3] Kimura Y, Kamamoto M, Maeda M, et al. Occurrence of Lewis a epitope in N-glycans of a 1 glycoallergen, Jun a 1, from mountain cedar pollen. Biosci. Biotechnol. Biochem., 69, 137-2 144 (2005). 3 [4] Maeda M, Kamamoto M, Hino K, et al. Glycoform analysis of Japanese cedar pollen allergen, 4 Cry j1. Biosci. Biotechnol. Biochem., 69, 1700-1705 (2005). 5 [5] Kimura Y, Kuroki M, Maeda M, et al. Glycoform analysis of Japanese cypress pollen allergen, 6 Cha o 1: A comparison of the glycoforms of cedar and cypress pollen allergens. Biosci. 7 Biotechnol. Biochem. 72, 485-491 (2008).  8 [6] Okano, M., Kimura, Y., Maeda, M., Matsuda, F., Michigami, Y., Kimura, M., and Nishizaki, 9 K. Roles of major oligosaccharides on Cry j 1 in human immunoglobulin E and T cell 10 responses. Clin. Exp. Allergy, 34. 770-778 (2004). 11 [7] Maeda M, Takeda N, Mano A, et al. Large-scale preparation of Asn-glycopeptide carrying 12 structurally homologous antigenic N-glycan. Biosci. Biotechnol. Biochem. 77, 1269-1274 13 (2013). 14 [8] Maeda M, Tani M, Yoshiie T, et al. Structural features of N-glycans linked to glycoproteins 15 expressed in three kinds of water plants: Predominant occurrence of the plant complex 16 type N-glycans bearing Lewis a epitope. Carbohydr. Res. 435, 50-57 (2016). 17 [9] Kimura M, Hara T, and Kimura Y. N-Glycans linked to glycoproteins in edible beans 18 (Zatsu-mame): Natural resources for bioactive oligosaccharides. Biosci. Biotechnol. 19 Biochem., 74, 155-158 (2011). 20 [10] Nakamura K, Inoue M, Hosoi K, et al. Predominant occurrence of truncated complex 21 type N-glycans among glycoproteins in mature-red tomato. Biosci. Biotechnol. Biochem., 22 73 221-223 (2009). 23 [11] Yoshiie T, Maeda M, Kimura M, et al. Structural features of N-glycans of seaweed 24 glycoproteins: predominant occurrence of high-mannose type N-glycans in marine plants. 25 Biosci. Biotechnol. Biochem., 76, 1996-1998 (2012). 26 [12] Dubois M, Gilles KA, Hamilton JK, et al. Colorimetric method for determination of 27  8 sugar and related substances. Anal. Chem. 28, 350-356 (1956). 1 [13] Natsuka S, and Hase S. Analysis of N- and O-glycans by pyridylamination. Methods Mol. 2 Biol. 76 101-113 (1998). 3 [14] Itano S, Maeda M, Kimura Y. Immunodulatory activity of glycopolymers bearing highly 4 clustered N-glycans for Th 1 and Th 2 immune response. Proc Jpn Soc Immunol. 2016; 5 45:166. 6  7 Legends to figures 8 Fig. 1. RP-HPLC and SF-HPLC profiles of PA-sugar chains obtained from bamboo shoot 9 glycopeptides. 10 I. RP-HPLC profile of PA-sugar chains obtained from bamboo shoot glycopeptides. 11 II. SF-HPLC profiles of PA-sugar chains obtained in I.  12  13 Fig. 2. SF-HPLC profiles of exoglycosidase digests of PA-sugar chains obtained from 14 bamboo shoot glycopeptides 15 1, PA-sugar chains from bamboo shoot glycopeptides. 2, Streptomyces a-1,3/4-Fuc’ase digest 16 of 1, 3, b-1,3/6-Gal’ase digest of 2; 4, Jack bean b-GlcNAc’ase digest of 3. 17  18 Table. Comparison of relative amount of N-glycans linked to Elodea nuttallii and bamboo 19 shoot glycoproteins  20 Fluorescence intensity (Ex 310 nm / Em380 nm )20  40Retention time ( min )Fluorescence intensity (Ex 310 nm / Em380 nm )20  40Retention time ( min )bM3FXm/z 1267.5 [M+H]+cGN1M3FXm/z 735.8[M+2H]2+dGN2M3FXm/z 837.3[M+2H]2+g(Lea)2M3FXm/z 1145.4[M+2H]2+f(Lea)1GN1M3FXm/z 991.4[M+2H]2+aM2FXm/z 1105.4 [M+H]+eG1GN2M3FXm/z 918.4[M+2H]2+I II Fig. 1 Tanabe et al.Fluorescence intensity (Ex 310 nm / Em380 nm )20  40Retention time ( min )1234M2FX M3FXGN1M3FXGN2M3FXG1GN2M3FX(Lea)1GN1M3FX(Lea)2M3FXa-1,3/4-Fuc’aseb-1,3-Gal’aseb-GlcNAc'aseORM2FXM3FXGN1M3FXGN2M3FXG1GN2M3FX(Lea)1GN1M3FX(Lea)2M3FXabcde f ghFig. 2 Tanabe et al.Elodea nuttallii a)    (nmol/g)      (%)　　　　 M2FX 0.09         3.70 0.11         4.60  Peak-a　　　　 M3FX                GN1M3X　　　　 GN1M3FX　　　　 GN2M3X　　　　 GN2M3FX 0.94          39.8 0.63          26.1 Peak-d　　　　 G1GN2M3FX ND 0.17          7.10 Peak-e　　　　 (Lea)1GN1M3FX 0.38          16.0 0.22          9.00 Peak-f　　　　 (Lea)2M3FX 0.39          16.7 0.24          10.0 Peak-gStructure bamboo shoot    (nmol/g)     (%)         　 Peaksa) Reference [8]Peak-c0.26         10.60.28         11.7Peak-b0.79         32.60.29         12.1

Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans

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Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans

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