Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11

doi:10.13523/j.cb.20180604

China Biotechnology

2018, Vol. 38

Issue (6): 26-33 DOI: 10.13523/j.cb.20180604

Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11

Qing-meng LI,Sheng-tao LI,Ning WANG,Xiao-dong GAO(

)

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology,Jiangnan University, Wuxi 214122, China

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Abstract

Glycosyltransferase Alg11, which is an important protein in N-glycosylation pathway, transfers the mannose moiety from GDP-Man to DPGn₂M₃ (Dolichyl-pyrophosphate-GlcNAc₂Mannose₃), forming DPGn₂M₄ and DPGn₂M₅ LLO (lipid-linked oligosaccharide) precursors. The structural analysis of Saccharomyces cerevisiae Alg11 showed the prediction of a hydrophobic N-terminal transmembrane domain. Thus, truncated Alg11 lacking the first 44 amino acid was designed and successfully overexpressed in Escherichia coli. The induction time and inducer concentration were optimized and the recombinant protein Alg11_45-548 was purified. After the transferase activity assay, reaction mixture was applied to the liquid chromatography tandem mass spectrometry (LC-MS), which showed Alg11_45-548 was capable to generate PPGn₂M₅ (Phytanyl-pyrophosphate-GlcNAc₂Mannose₅) from substrate PPGn₂M₃. Structural analysis of Gn₂M₅ showed the newly formed two glycosidic bonds could be cleaved by α-1,2 mannosidase, meaning the two mannose moieties were attached to Gn₂M₃ by α-1,2 linkages. Substrate specificity assay indicated the recombinant Alg11 specifically recognized PPGn₂M₃ rather than other LLOs, such as PPGn₂ and PPGn₂M₁. Additionally, oligosaccharide Gn₂M₃ was not elongated by Alg11_45-548, suggesting the lipid chain in the substrate PPGn₂M₃ was critical for the recognition. The achievement of active Alg11 provides an effective tool for producing Gn₂M₅, as well as for the further investigation of kinetic and mechanistic features of related mannosyltransferases.

Key words： N-glycosylation Glycosyltransferase Alg11 Liquid chromatography tandem mass spectrometry (LC-MS) Induction condition

Received: 23 January 2018 Published: 06 July 2018

ZTFLH:

Q786

Corresponding Authors: Xiao-dong GAO E-mail: xdgao@jiangnan.edu.cn

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	Qing-meng LI
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Cite this article:

Qing-meng LI,Sheng-tao LI,Ning WANG,Xiao-dong GAO. Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11. China Biotechnology, 2018, 38(6): 26-33.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180604 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I6/26

Fig.1 Analysis of the transmembrane domain structure of Alg11 protein

Fig.2 Construction of pET28a-Alg11_45-548 (a) Plasimid map of pET28a-Alg11_45-548 (b) PCR amplification of Alg11_45-548 from genome M: 1kb plus DNA marker; 1: PCR product of Alg11_45-548 (c) Identification of recombinant expression vector M: 1kb plus DNA marker; 2: pET28a-Alg11_45-548 digested with BamH I和Hind III

Fig.3 Effect of induction time (a) and IPTG concentration (b) on protein expression Expression of recombinant protein in different time and IPTG concentration (a) 0: 0 h; 1: 1 h; 2: 2h; 3: 3 h; 4: 4 h; 5: 5 h; 6: 6h; 7: 7 h; 8: 20 h, 100μmol/L IPTG (b) 0: 0μmol/L IPTG; 1: 10μmol/L IPTG; 2: 25μmol/L IPTG; 3: 50μmol/L IPTG; 4: 75μmol/L IPTG; 5: 100μmol/L IPTG; 6: 500μmol/L; 7: 1 000μmol/L IPTG; 8: 2 000μmol/L IPTG, 20h

Fig.4 SDS-PAGE (a) and Western blot (b) of recombinant cell and purified Alg11_45-548 (a) Samples were applied to gel electrophoresis followed by staining with Coomassie brilliant blue R-250 (b) Western blot verification of the purified protein M: Marker; 1: Whole cell before inducing; 2: Whole cell after inducing with 100μmol/L IPTG; 3: Purified Alg11_45-548

Fig.5 The oligosaccharide was detected by LC-MS (a) A schematic diagram of glycosylation of Alg1ΔTM, Trx-Alg2 and Alg11_45-548 (b) Elution time of various oligosaccharide in ultra performance liquid chromatography (c) Ion peaks of different oligosaccharides in mass spectrometry

Fig.6 Product verification by Mannosidases Ultra Performance Liquid Chromatography profiles of: (a)Gn₂M₅ (b)~(e): Oligosaccharide after digesting Gn₂M₅ by different mannosidase b: α-1,2 mannosidase; c: α 1-2,3 mannosidase; d: α 1-2,3 and α-1,6 mannosidase; e: α-1,6 mannosidase

Fig.7 The substrate specificity of Alg11_45-548 Reactions with different substrates (a) PPGn₂M₃ (b) PPGn₂ (c) PPGn₂M₁ (d) Gn₂M₃


[1]	Wiedwerschain G Y . Essentials of glycobiology. Biochemistry, 2009,74(9):1056-1056.

[2]	Larkin A, Imperiali B . The expanding horizons of asparagine-linked glycosylation. Biochemistry, 2011,50(21):4411-4426. doi: 10.1021/bi200346n pmid: 21506607

[3]	Bickel T, Lehle L, Schwarz M , et al. Biosynthesis of lipid-linked oligosaccharides in saccharomyces cerevisiae Alg13p and Alg14p from a complex required for the formation of GlcNAc2-PP-dolichol. Journal of Biological Chemistry, 2005,280(41):34500-34506. doi: 10.1074/jbc.M506358200

[4]	Gao X D, Tachikawa H, Sato T , et al. Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation. Journal of Biological Chemistry, 2005,280(43):36254-36262. doi: 10.1074/jbc.M507569200

[5]	Gao X D, Moriyama S, Miura N , et al. Interaction between the C termini of Alg13 and Alg14 mediates formation of the active UDP-N-acetylglucosamine transferase complex. Journal of Biological Chemistry, 2008,283(47):32534-32541. doi: 10.1074/jbc.M804060200

[6]	O’Reilly M K, Zhang G F, Imperiali B . In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-Linked glycoprotein biosynthesis. Biochemistry, 2006,45(31):9593-9603. doi: 10.1021/bi060878o pmid: 16878994

[7]	Ramírez A S, Boilevin J, Lin C W , et al. Chemo-enzymatic synthesis of lipid-linked GlcNAc2Man5 oligosaccharides using recombinant Alg1, Alg2 and Alg11 proteins. Glycobiology, 2017: 1-8. doi: 10.1093/glycob/cwx045 pmid: 28575298

[8]	Burda P, Aebi M . The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta, 1999,1426(2):239-257. doi: 10.1016/S0304-4165(98)00127-5

[9]	Schwarz F, Aebi M . Mechanisms and principles of N-linked protein glycosylation. Current Opinion in Structural Biology, 2011,21(5):576-582. doi: 10.1016/j.sbi.2011.08.005 pmid: 21978957

[10]	Jr D R, Imperiali B . Oligosaccharyl transferase: gatekeeper to the secretory pathway. Current Opinion in Chemical Biology, 2002,6(6):844-850. doi: 10.1016/S1367-5931(02)00390-3 pmid: 12470740

[11]	Cipollo J F, Trimble R B, Chi J H , et al. The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum. Journal of Biological Chemistry, 2001,276(24):21828-21840. doi: 10.1074/jbc.M010896200

[12]	Absmanner B, Schmeiser V, Kämpf M , et al. Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an alpha1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide. Biochemical Journal, 2010,426(2):205-217. doi: 10.1042/BJ20091121

[13]	Flitsch S L, Pinches H L, Taylor J P , et al. Chemo-enzymatic synthesis of a lipid-linked core trisaccharide of N-linked glycoproteins. Cheminform, 1992,23(48):2087-2093.

[14]	Wilson I B, Taylor J P, Webberley M C , et al. A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase. Biochemical Journal, 1993, 295 (Pt 1)( 1):195-201. doi: 10.1042/bj2950195 pmid: 8216216

[15]	Li S T, Wang N, Xu S , et al. Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation. Biochimica et biophysica acta, 2016,1861(1):2934-2941. doi: 10.1016/j.bbagen.2016.09.023 pmid: 27670784

[16]	Li S T, Wang N, Xu X X , et al. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase. Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology, 2017: fj.201701267R. doi: 10.1096/fj.201701267R pmid: 29273674

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