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N-Linked glycoengineering for human therapeutic proteins in bacteria

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Abstract

Approx. 70% of human therapeutic proteins are N-linked glycoproteins, and therefore host cells for production must contain the relevant protein modification machinery. The discovery and characterisation of the N-linked glycosylation pathway in the pathogenic bacterium Campylobacter jejuni, and subsequently its functional transfer to Escherichia coli, presents the opportunity of using prokaryotes as cell factories for therapeutic protein production. Not only could bacteria reduce costs and increase yields, but the improved feasibility to genetically control microorganisms means new and improved pharmacokinetics of therapeutics is an exciting possibility. This is a relatively new concept, and progress in bacterial N-glycosylation characterisation is reviewed and metabolic engineering targets revealed.

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References

  • Abu-Qarn M, Eichler J, Sharon N (2008) Not just for Eukarya anymore: protein glycosylation in bacteria and Archaea. Curr Opin Struct Biol 18:544–550

    Article  PubMed  CAS  Google Scholar 

  • Apweiler R, Hermjakob H, Sharon N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1473:4–8

    PubMed  CAS  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  PubMed  CAS  Google Scholar 

  • Bickel T, Lehle L, Schwarz M et al (2005) Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol. J Biol Chem 280:34500–34506

    Article  PubMed  CAS  Google Scholar 

  • Burda P, Aebi M (1999) The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta 1426:239–257

    PubMed  CAS  Google Scholar 

  • Chantret I, Dancourt J, Barbat A et al (2005) Two proteins homologous to the N- and C-terminal domains of the bacterial glycosyltransferase Murg are required for the second step of dolichyl-linked oligosaccharide synthesis in Saccharomyces cerevisiae. J Biol Chem 280:9236–9242

    Article  PubMed  CAS  Google Scholar 

  • Dempski RE Jr, Imperiali B (2002) Oligosaccharyl transferase: gatekeeper to the secretory pathway. Curr Opin Chem Biol 6:844–850

    Article  PubMed  CAS  Google Scholar 

  • Egrie JC, Browne JK (2001) Development and characterization of novel erythropoiesis stimulating protein (NESP). Br J Cancer 84(Suppl1):3–10

    Article  PubMed  CAS  Google Scholar 

  • Elliott S, Lorenzini T, Asher S et al (2003) Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 21:414–421

    Article  PubMed  CAS  Google Scholar 

  • Fanger MW, Smyth DG (1972) Oligosaccharide units of rabbit IgG multiple CHO attachment sites. Biochem J 127:757–765

    PubMed  CAS  Google Scholar 

  • Feldman MF, Wacker M, Hernandez M et al (2005) Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci USA 102:3016–3021

    Article  PubMed  CAS  Google Scholar 

  • Fussenegger M, Schlatter S, Datwyler D et al (1998) Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol 16:468–472

    Article  PubMed  CAS  Google Scholar 

  • Gao XD, Tachikawa H, Sato T et al (2005) 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. J Biol Chem 280:36254–36262

    Article  PubMed  CAS  Google Scholar 

  • Glover KJ, Weerapana E, Chen MM et al (2006) Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway. Biochemistry 45:5343–5350

    Article  PubMed  CAS  Google Scholar 

  • Gomord V, Faye L (2004) Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol 7:171–181

    Article  PubMed  CAS  Google Scholar 

  • Guerry P, Ewing C, Schirm M et al (2006) Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence. Mol Microbiol 60:299–311

    Article  PubMed  CAS  Google Scholar 

  • Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18:387–392

    Article  PubMed  CAS  Google Scholar 

  • Hamilton SR, Bobrowicz P, Bobrowicz B et al (2003) Production of complex human glycoproteins in yeast. Science 301:1244–1246

    Article  PubMed  CAS  Google Scholar 

  • Hitchen PG, Dell A (2006) Bacterial glycoproteomics. Microbiology 152:1575–1580

    Article  PubMed  CAS  Google Scholar 

  • Houdebine LM (2002) Antibody manufacture in transgenic animals and comparisons with other systems. Curr Opin Biotechnol 13:625–629

    Article  PubMed  CAS  Google Scholar 

  • Inga B, Schmidt MA (2002) Never say never again: protein glycosylation in pathogenic bacteria. Mol Microbiol 45:267–276

    Article  Google Scholar 

  • Jacobs PP, Callewaert N (2009) N-glycosylation engineering of biopharmaceutical expression systems. Curr Mol Med 9:774–800

    Article  PubMed  CAS  Google Scholar 

  • Johnson IS (1983) Human insulin from recombinant DNA technology. Science 219:632–637

    Article  PubMed  CAS  Google Scholar 

  • Kelleher DJ, Gilmore R (2006) An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology 16:47R–62R

    Article  PubMed  CAS  Google Scholar 

  • Kelly J, Jarrell H, Millar L et al (2006) Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer. J Bacteriol 188:2427–2434

    Article  PubMed  CAS  Google Scholar 

  • Kowarik M, Numao S, Feldman MF et al (2006) N-linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase. Science 314:1148–1150

    Article  PubMed  CAS  Google Scholar 

  • Legardinier S, Klett D, Poirier J-C et al (2005) Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic™ insect cells. Glycobiology 15:776–790

    Article  PubMed  CAS  Google Scholar 

  • Lehle L, Tanner W (1978) Glycosyl transfer from dolichyl phosphate sugars to endogenous and exogenous glycoprotein acceptors in yeast. Eur J Biochem 83:563–570

    Article  PubMed  CAS  Google Scholar 

  • Li H, Sethuraman N, Stadheim TA et al (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215

    Article  PubMed  CAS  Google Scholar 

  • Linton D, Allan E, Karlyshev AV et al (2002) Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni. Mol Microbiol 43:497–508

    Article  PubMed  CAS  Google Scholar 

  • Linton D, Dorrell N, Hitchen PG et al (2005) Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway. Mol Microbiol 55:1695–1703

    Article  PubMed  CAS  Google Scholar 

  • Mescher MF, Strominger JL, Watson SW (1974) Protein and carbohydrate composition of the cell envelope of Halobacterium salinarium. J Bacteriol 120:945–954

    PubMed  CAS  Google Scholar 

  • Messner P (2004) Prokaryotic glycoproteins: unexplored but important. J Bacteriol 186:2517–2519

    Article  PubMed  CAS  Google Scholar 

  • Mobili P, Serradell Mde L, Trejo S et al (2009) Heterogeneity of S-layer proteins from aggregating and non-aggregating Lactobacillus kefir strains. Antonie Van Leeuwenhoek 95:363–372

    Article  PubMed  CAS  Google Scholar 

  • Müller D, Bayer K, Mattanovich D (2006) Potentials and limitations of prokaryotic and eukaryotic expression systems for recombinant protein production—a comparative view. In: The 4th recombinant protein production meeting. Microbial Cell Factories, Barcelona, Spain

  • Parkhill J, Wren BW, Mungall K et al (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665–668

    Article  PubMed  CAS  Google Scholar 

  • Peng Z, Wu H, Ruiz T et al (2008) Role of gap3 in Fap1 glycosylation, stability, in vitro adhesion, and fimbrial and biofilm formation of Streptococcus parasanguinis. Oral Microbiol Immunol 23:70–78

    PubMed  CAS  Google Scholar 

  • Potgieter TI, Cukan M, Drummond JE et al (2009) Production of monoclonal antibodies by glycoengineered Pichia pastoris. J Biotechnol 139:318–325

    Article  PubMed  CAS  Google Scholar 

  • Price NP, Momany FA (2005) Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases. Glycobiology 15:29R–42R

    Article  PubMed  CAS  Google Scholar 

  • Rangarajan ES, Bhatia S, Watson DC et al (2007) Structural context for protein N-glycosylation in bacteria: the structure of PEB3, an adhesin from Campylobacter jejuni. Protein Sci 16:990–995

    Article  PubMed  CAS  Google Scholar 

  • Sarkar A, Fritz T, Taylor W et al (1995) Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1→4GlcNAc beta-O-naphthalenemethanol. Proc Natl Acad Sci USA 92:3323–3327

    Article  PubMed  CAS  Google Scholar 

  • Schaffer C, Messner P (2004) Surface-layer glycoproteins: an example for the diversity of bacterial glycosylation with promising impacts on nanobiotechnology. Glycobiology 14:31R–42R

    Article  PubMed  CAS  Google Scholar 

  • Schwarz F, Huang W, Li C et al (2010) A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat Chem Biol 6(4):264–266

    Article  PubMed  CAS  Google Scholar 

  • Sethuraman N, Stadheim TA (2006) Challenges in therapeutic glycoprotein production. Curr Opin Biotechnol 17:341–346

    Article  PubMed  CAS  Google Scholar 

  • Shao J, Zhang J, Kowal P et al (2002) Donor substrate regeneration for efficient synthesis of globotetraose and isoglobotetraose. Appl Environ Microbiol 68:5634–5640

    Article  PubMed  CAS  Google Scholar 

  • Sheeley DM, Merrill BM, Taylor LC (1997) Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose. Anal Biochem 247:102–110

    Article  PubMed  CAS  Google Scholar 

  • Skretas G, Carroll S, DeFrees S et al (2009) Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli. Microb Cell Fact 8:50

    Article  PubMed  CAS  Google Scholar 

  • Strasser R, Altmann F, Mach L et al (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett 561:132–136

    Article  PubMed  CAS  Google Scholar 

  • Szymanski CM, Burr DH, Guerry P (2002) Campylobacter protein glycosylation affects host cell interactions. Infect Immun 70:2242–2244

    Article  PubMed  CAS  Google Scholar 

  • Tatar LD, Marolda CL, Polischuk AN et al (2007) An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling. Microbiology 153:2518–2529

    Article  PubMed  CAS  Google Scholar 

  • van Berkel PH, Welling MM, Geerts M et al (2002) Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol 20:484–487

    Article  PubMed  CAS  Google Scholar 

  • Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130

    Article  PubMed  CAS  Google Scholar 

  • Varki A, Cummings R, Esko J et al (1999) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Wacker M, Linton D, Hitchen PG et al (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790–1793

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Brock A, Herberich B et al (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500

    Article  PubMed  CAS  Google Scholar 

  • Warner TG (1999) Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by metabolic engineering endogenous gene control with antisense DNA and gene targeting. Glycobiology 9:841–850

    Article  PubMed  CAS  Google Scholar 

  • Weerapana E, Imperiali B (2006) Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16:91R–101R

    Article  PubMed  CAS  Google Scholar 

  • Young NM, Brisson JR, Kelly J et al (2002) Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J Biol Chem 277:42530–42539

    Article  PubMed  CAS  Google Scholar 

  • Yuen CT, Storring P, Tiplady R et al (2005) Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Adv Exp Med Biol 564:141–142

    Article  PubMed  CAS  Google Scholar 

  • Yurist-Doutsch S, Chaban B, VanDyke DJ et al (2008) Sweet to the extreme: protein glycosylation in Archaea. Mol Microbiol 68:1079–1084

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Gildersleeve J, Yang YY et al (2004) A new strategy for the synthesis of glycoproteins. Science 303:371–373

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Gildersleeve J, Yang YY et al (2009) Retraction. Science 326:1187

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors acknowledge funding from the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) through the Bioprocess Research Industry Club (BRIC) programme (ref BBF0048421), and also from the Engineering and Physical Sciences Research Council (EPSRC) (ref EP/E036252/1).

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  1. ChELSI Institute, Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK

    Jagroop Pandhal & Phillip C. Wright

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  1. Jagroop Pandhal
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  2. Phillip C. Wright
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Correspondence to Phillip C. Wright.

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Pandhal, J., Wright, P.C. N-Linked glycoengineering for human therapeutic proteins in bacteria. Biotechnol Lett 32, 1189–1198 (2010). https://doi.org/10.1007/s10529-010-0289-6

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