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Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris

  • Biotechnologically relevant enzymes and proteins
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Abstract

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.

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References

  • Bhan N, Xu P, Koffas MAG (2013) Pathway and protein engineering approaches to produce novel and commodity small molecules. Curr Opin Biotechnol 24:1137–1143. doi:10.1016/j.copbio.2013.02.019

    Article  CAS  PubMed  Google Scholar 

  • Chapman E, Best MD, Hanson SR, Wong C-H (2004) Sulfotransferases: structure, mechanism, biological activity, inhibition, and synthetic utility. Angew Chem (Int ed) 43:3526–3548. doi:10.1002/anie.200300631

    Article  CAS  Google Scholar 

  • Cheng J, Randall AZ, Sweredoski MJ, Baldi P (2005) SCRATCH: a protein structure and structural feature prediction server. Nucleic Acids Res 33:W72–W76. doi:10.1093/nar/gki396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng J, Sweredoski MJ, Baldi P (2006) DOMpro: protein domain prediction using profiles, secondary structure, relative solvent accessibility, and recursive neural networks. Data Min Knowl Disc 13:1–10. doi:10.1007/s10618-005-0023-5

    Article  Google Scholar 

  • Cimini D, De Rosa M, Carlino E, Ruggiero A, Schiraldi C (2013) Homologous overexpression of rfaH in E.coli K4 improves the production of chondroitin-like capsular polysaccharide. Microb Cell Factories 12:46. doi:10.1186/1475-2859-12-46

    Article  CAS  Google Scholar 

  • Cimini D, Fantaccione S, Volpe F, De Rosa M, Restaino OF, Aquino G, Schiraldi C (2014) IS2-mediated overexpression of kfoC in E. coli K4 increases chondroitin-like capsular polysaccharide production. Appl Microbiol Biotechnol 98:3955–3964. doi:10.1007/s00253-014-5506-0

    Article  CAS  PubMed  Google Scholar 

  • Cimini D, Restaino OF, Catapano A, De Rosa M, Schiraldi C (2010) Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications. Appl Microbiol Biotechnol 85:1779–1787. doi:10.1007/s00253-009-2261-8

    Article  CAS  PubMed  Google Scholar 

  • Englaender JA, Zhu Y, Shirke AN, Lin L, Liu X, Zhang F, Gross RA, Koffas MAG, Linhardt RJ (2017) Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris. Appl Microbiol Biotechnol 101:2843–2851. doi:10.1007/s00253-016-8047-x

    Article  CAS  PubMed  Google Scholar 

  • He W, Fu L, Li G, Andrew Jones J, Linhardt RJ, Koffas M (2015) Production of chondroitin in metabolically engineered E. coli. Metab Eng 27:92–100. doi:10.1016/j.ymben.2014.11.003

    Article  CAS  PubMed  Google Scholar 

  • Hiraoka N, Nakagawa H, Ong E, Akama TO, Fukuda MN, Fukuda M (2000) Molecular cloning and expression of two distinct human chondroitin 4-O-sulfotransferases that belong to the HNK-1 sulfotransferase gene family. J Biol Chem 275:20188–20196. doi:10.1074/jbc.M002443200

    Article  CAS  PubMed  Google Scholar 

  • Jin P, Zhang L, Yuan P, Kang Z, Du G, Chen J (2016) Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis. Carbohydr Polym 140:424–432. doi:10.1016/j.carbpol.2015.12.065

    Article  CAS  PubMed  Google Scholar 

  • Kang H, Gyoo H-G, Evers MR, Xia G, Baenziger JU, Schachner M (2002) Molecular cloning and characterization of chondroitin-4-O-sulfotransferase-3: a novel member of the HNK-1 family of sulfotransferases. J Biol Chem 277:34766–34772. doi:10.1074/jbc.M204907200

    Article  CAS  PubMed  Google Scholar 

  • Kluppel M (2005) Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis. Development 132:3989–4003. doi:10.1242/dev.01948

    Article  CAS  PubMed  Google Scholar 

  • Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD, Lin-Cereghino GP (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38:44–46 48

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mikami T, Kitagawa H (2013) Biosynthesis and function of chondroitin sulfate. Biochim Biophys Acta - Gen Subj 1830:4719–4733. doi:10.1016/j.bbagen.2013.06.006

    Article  CAS  Google Scholar 

  • Mikami T, Mizumoto S, Kago N, Kitagawa H, Sugahara K (2003) Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis. J Biol Chem 278:36115–36127. doi:10.1074/jbc.M306044200

    Article  CAS  PubMed  Google Scholar 

  • Okuda T, Mita S, Yamauchi S, Matsubara T, Yagi F, Yamamori D, Fukuta M, Kuroiwa A, Matsuda Y, Habuchi O (2000) Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase. J Biochem 128:763–770

    Article  CAS  PubMed  Google Scholar 

  • Sterner E, Li L, Paul P, Beaudet JM, Liu J, Linhardt RJ, Dordick JS (2014) Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity. Anal Bioanal Chem 406:525–536. doi:10.1007/s00216-013-7470-4

    Article  CAS  PubMed  Google Scholar 

  • Sugiura N, Shioiri T, Chiba M, Sato T, Narimatsu H, Kimata K, Watanabe H (2012) Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions. J Biol Chem 287:43390–43400. doi:10.1074/jbc.M112.412676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suflita M, Fu L, He W, Koffas MAG, Linhardt RJ (2015) Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering. Appl Microbiol Biotechnol 99:7465–7479. doi:10.1007/s00253-015-6821-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang J, Guleria S, Koffas MAG, Yan Y (2016) Microbial production of value-added nutraceuticals. Curr Opin Biotechnol 37:97–104. doi:10.1016/j.copbio.2015.11.003

    Article  PubMed  Google Scholar 

  • Xu P, Bhan N, Koffas MAG (2012) Engineering plant metabolism into microbes: from systems biology to synthetic biology. Curr Opin Biotechnol 24:291–299. doi:10.1016/j.copbio.2012.08.010

    Article  PubMed  Google Scholar 

  • Yusa A, Kitajima K, Habuchi O (2005) N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1. Biochem J 388:115–121. doi:10.1042/BJ20041573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou X, Chandarajoti K, Pham TQ, Liu R, Liu J (2011) Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3′-phosphoadenosine-5′-phosphosulfate. Glycobiology 21:771–780. doi:10.1093/glycob/cwr001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors gratefully acknowledge funding from the National Institutes of Health (HL096972), the National Science Foundation (MCB-1448657, CBET-1604547), and the China Scholarship Council.

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Author notes

  1. Wenqin He and Yuanyuan Zhu equally contributed in this study.

Authors and Affiliations

  1. Departments of Chemistry and Chemical Biology, Biology, Chemical and Biological Engineering, and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Wenqin He, Abhijeet Shirke, Richard A. Gross, Mattheos A. G. Koffas & Robert J. Linhardt

  2. Department of Chemical Processing Engineering of Forest Products, Nanjing Forestry University, Nanjing, China

    Yuanyuan Zhu

  3. National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, 2500100, China

    Xiaojun Sun

  4. Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA

    Jian Liu

  5. Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, China

    Ming Li

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  1. Wenqin He
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  2. Yuanyuan Zhu
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  4. Xiaojun Sun
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  6. Richard A. Gross
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  7. Mattheos A. G. Koffas
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  9. Ming Li
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Corresponding authors

Correspondence to Mattheos A. G. Koffas, Robert J. Linhardt or Ming Li.

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He, W., Zhu, Y., Shirke, A. et al. Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris . Appl Microbiol Biotechnol 101, 6919–6928 (2017). https://doi.org/10.1007/s00253-017-8411-5

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  • DOI: https://doi.org/10.1007/s00253-017-8411-5

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