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Exploiting Bacterial Glycosylation Machineries for the Synthesis of a Lewis Antigen-containing Glycoprotein*

Open AccessPublished:August 30, 2011DOI:https://doi.org/10.1074/jbc.M111.287755
      Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.

      Introduction

      The discovery of general protein glycosylation systems in Gram-negative bacteria inaugurated a new era in the production of recombinant glycoproteins (
      • Wacker M.
      • Feldman M.F.
      • Callewaert N.
      • Kowarik M.
      • Clarke B.R.
      • Pohl N.L.
      • Hernandez M.
      • Vines E.D.
      • Valvano M.A.
      • Whitfield C.
      • Aebi M.
      ,
      • Wacker M.
      • Linton D.
      • Hitchen P.G.
      • Nita-Lazar M.
      • Haslam S.M.
      • North S.J.
      • Panico M.
      • Morris H.R.
      • Dell A.
      • Wren B.W.
      • Aebi M.
      ). Exploitation of these systems via glycoengineering has the potential to overcome many hurdles of the techniques currently available for the synthesis of glycoconjugates (
      • Langdon R.H.
      • Cuccui J.
      • Wren B.W.
      ,
      • Feldman M.
      ). In contrast to protein glycosylation in eukaryotes, bacterial cells tolerate the manipulation of glycan moieties. In higher eukaryotes even minimal alterations in the glycan structure can be lethal, primarily because eukaryotic N-glycosylation is required for protein folding control (
      • Helenius A.
      • Aebi M.
      ). Although yeasts appear to be a promising system for production of humanized N-glycoproteins, it seems unlikely that these cells can be efficiently used for the production of glycoproteins containing glycans other than the high-mannose type (
      • Hamilton S.R.
      • Davidson R.C.
      • Sethuraman N.
      • Nett J.H.
      • Jiang Y.
      • Rios S.
      • Bobrowicz P.
      • Stadheim T.A.
      • Li H.
      • Choi B.K.
      • Hopkins D.
      • Wischnewski H.
      • Roser J.
      • Mitchell T.
      • Strawbridge R.R.
      • Hoopes J.
      • Wildt S.
      • Gerngross T.U.
      ). On the other hand, chemical synthesis of conjugates often requires harsh conditions for the attachment of the sugars, which usually affects the properties of the carriers (
      • Jones C.
      ). Furthermore, chemical cross-linking is difficult to control and therefore glycoconjugates produced via this method often present reproducibility and homogeneity issues (
      • Jones C.
      ). Utilization of living bacteria for the production of glycoproteins combines the specificities of enzymatic reactions for both, the assembly of the glycan and its conjugation to the polypeptide.
      Oligosaccharyltransferases (OTases)
      The abbreviations used are: OTase
      oligosaccharyltransferases
      UndPP
      undecaprenyl pyrophosphate
      Ni-NTA
      nickel-nitrilotriacetic acid
      FTICR
      Fourier-transform ion cyclotron resonance.
      are the key enzymes involved in such bacterial synthesis pathways. They are responsible for the transfer of glycans from the carrier lipid undecaprenyl pyrophosphate (UndPP) onto acceptor proteins (
      • Nothaft H.
      • Szymanski C.M.
      ). Importantly, some of the best characterized OTases display relaxed substrate specificity. Glycans attached to the carrier lipid can be transferred onto acceptor proteins with very limited or no requirements toward the structure of the oligo- or polysaccharide (
      • Faridmoayer A.
      • Fentabil M.A.
      • Haurat M.F.
      • Yi W.
      • Woodward R.
      • Wang P.G.
      • Feldman M.F.
      ,
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      ). For instance, the presence of an acetamido group at C-2 of the reducing sugar residue and probably a linkage other than 1–4 between the two proximal sugars are the only known substrate requirements of the Campylobacter jejuni OTase PglB (
      • Wacker M.
      • Feldman M.F.
      • Callewaert N.
      • Kowarik M.
      • Clarke B.R.
      • Pohl N.L.
      • Hernandez M.
      • Vines E.D.
      • Valvano M.A.
      • Whitfield C.
      • Aebi M.
      ,
      • Chen M.M.
      • Glover K.J.
      • Imperiali B.
      ). Therefore, if the desired glycan structure fulfills these requirements and can be assembled onto UndPP, it will be transferred onto acceptor proteins in the presence of PglB. No structural requirement for the glycan has been found for the Neisseria OTase PglL (
      • Faridmoayer A.
      • Fentabil M.A.
      • Haurat M.F.
      • Yi W.
      • Woodward R.
      • Wang P.G.
      • Feldman M.F.
      ). Interestingly, UndPP serves as a carrier lipid for glycan assembly in many bacterial glycoconjugate pathways (
      • Hug I.
      • Feldman M.F.
      ). One example is the lipopolysaccharide biosynthesis pathway, using UndPP for the assembly of its distal polysaccharide, named O antigen. The O antigen is then conjugated en bloc onto the lipid A-core by the O antigen ligase (
      • Raetz C.R.
      • Whitfield C.
      ). Indeed, it was demonstrated that UndPP-linked, fully polymerized O antigens can also be transferred onto acceptor proteins by bacterial OTases (
      • Faridmoayer A.
      • Fentabil M.A.
      • Haurat M.F.
      • Yi W.
      • Woodward R.
      • Wang P.G.
      • Feldman M.F.
      ,
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      ).
      OTases recognize specific acceptor sites on acceptor proteins, making enzymatic glycan transfer superior to chemical conjugation in terms of precision and reproducibility. C. jejuni PglB attaches glycans in an N-linkage, recognizing the extended eukaryotic consensus sequence (D/E)XNX(S/T) (
      • Kowarik M.
      • Young N.M.
      • Numao S.
      • Schulz B.L.
      • Hug I.
      • Callewaert N.
      • Mills D.C.
      • Watson D.C.
      • Hernandez M.
      • Kelly J.F.
      • Wacker M.
      • Aebi M.
      ). Any polypeptide engineered to contain this glyco-tag may serve as a C. jejuni PglB glycosylation target in the bacterial periplasm (
      • Kowarik M.
      • Young N.M.
      • Numao S.
      • Schulz B.L.
      • Hug I.
      • Callewaert N.
      • Mills D.C.
      • Watson D.C.
      • Hernandez M.
      • Kelly J.F.
      • Wacker M.
      • Aebi M.
      ). The recognition determinants for other OTases are less clear. Desulfovibrio desulfuricans PglB does not recognize the same sequon as its Campylobacter counterpart (
      • Ielmini M.V.
      • Feldman M.F.
      ) and it is unknown how Neisseria PglL recognizes its substrates (
      • Vik A.
      • Aas F.E.
      • Anonsen J.H.
      • Bilsborough S.
      • Schneider A.
      • Egge-Jacobsen W.
      • Koomey M.
      ).
      Although the best characterized bacterial glycosylation systems originate from the pathogens C. jejuni, Pseudomonas aeruginosa, and Neisseria spp., these glycoprotein assembly machineries were functionally reconstituted in nonpathogenic and fast growing Escherichia coli laboratory strains (
      • Wacker M.
      • Linton D.
      • Hitchen P.G.
      • Nita-Lazar M.
      • Haslam S.M.
      • North S.J.
      • Panico M.
      • Morris H.R.
      • Dell A.
      • Wren B.W.
      • Aebi M.
      ,
      • Faridmoayer A.
      • Fentabil M.A.
      • Mills D.C.
      • Klassen J.S.
      • Feldman M.F.
      ). This has opened up new avenues for the exploitation of these systems for the synthesis of engineered glycoproteins with potential therapeutic applications.
      The flexibility of the bacterial glycosylation pathways prompted us to develop a strategy to exploit these systems for the synthesis of customized glycoproteins. The Lewis x (Lex) antigen is a glycan structure (Galβ1–4[Fucα1–3]GlcNAc-) that is abundantly expressed on a variety of human cells, as well as on some parasitic helminthes and the gastric pathogen Helicobacter pylori (
      • Chan N.W.
      • Stangier K.
      • Sherburne R.
      • Taylor D.E.
      • Zhang Y.
      • Dovichi N.J.
      • Palcic M.M.
      ,
      • Oriol R.
      • Le Pendu J.
      • Mollicone R.
      ,
      • Srivatsan J.
      • Smith D.F.
      • Cummings R.D.
      ). Glycoconjugates containing Lex have promising applications for the treatment of autoimmune diseases due to their immunosuppressant effect via interaction with the dendritic cell receptor DC-SIGN (
      • van Die I.
      • van Vliet S.J.
      • Nyame A.K.
      • Cummings R.D.
      • Bank C.M.
      • Appelmelk B.
      • Geijtenbeek T.B.
      • van Kooyk Y.
      ). Experiments in animal models have demonstrated that symptoms associated with autoimmune disorders are ameliorated upon treatment with Lex-containing glycoproteins (
      • Atochina O.
      • Harn D.
      ). The “old friend hypothesis” elaborates on this idea, proposing that reduced exposure to immune suppressive signals from microbes due to improved hygiene and generalized use of antibiotics has increased the prevalence of human immune disorders in modern times (
      • Rook G.A.
      ). The use of bacteria for the generation of immunosuppressant and other glycodrugs may be a valuable future technology. In this work, we describe a combined in vivo and in vitro approach for the production of a glycoprotein containing the Lex antigen by exploiting bacterial enzymes.

      DISCUSSION

      The synthesis of defined glycoconjugates is an important but challenging task for the pharmaceutical industry. In the future, the exploitation of glycoconjugate biosynthesis pathways functionally expressed in nonpathogenic bacteria may become a valuable technology for the production of such compounds. In this work, we have produced a glycoprotein containing an epitope commonly found in human tissues, using a combined approach consisting of in vivo and in vitro glycosylation steps. The strategy used is presented in Fig. 1.
      Fucosylation of free oligosaccharides with LacNAc-epitopes for the assembly of the Lex antigen was previously accomplished in live E. coli cells using H. pylori fucosyltransferases (
      • Dumon C.
      • Bosso C.
      • Utille J.P.
      • Heyraud A.
      • Samain E.
      ). The design of the glycoengineering approach followed here, however, imposes that the glycans must be built onto the UndPP carrier to allow en bloc transfer to acceptor proteins. This requires the participation of a distinct class of initiating glycosyltransferases, capable of adding a sugar residue onto the lipid carrier (
      • Hug I.
      • Feldman M.F.
      ). The substrate for the next glycosyltransferases is therefore fixed to the inner membrane. It is possible that due to sterical hindrance the fixed glycans might not be able to reach the active sites of some glycosyltransferases, which prevents the use of some enzymes for this second step. Based on previous work (
      • Phillips N.J.
      • Miller T.J.
      • Engstrom J.J.
      • Melaugh W.
      • McLaughlin R.
      • Apicella M.A.
      • Gibson B.W.
      ), it was correctly predicted that the set of H. influenzae glycosyltransferases selected for the in vivo glycosylation step was able to generate the required glycolipid anchored in the inner membrane. In the presence of the C. jejuni OTase PglB, the tetrasaccharide was transferred onto the AcrA acceptor protein. Some side products were generated, having slightly elongated glycans with an additional HexNAc residue or a Hex-HexNAc disaccharide. This was surprising and indicated a partial reduction in acceptor specificity of the responsible glycosyltransferases in the heterologous expression system.
      Because of its terminal LacNAc structure, the tetrasaccharide assembled with the H. influenzae glycosyltransferases may be modified in various ways to generate humanized antigens. Addition of sialic acid, galactose, or GalNAc residues could result in epitopes mimicking gangliosides or blood group antigens. The goal of this work was the synthesis of a Lex-containing glycoprotein by fucosylation of the LacNAc acceptor. The fucose incorporation was successfully accomplished by in vitro fucosylation of the in vivo assembled glycoprotein. Although MS/MS peaks at 900 and 1062 Da misleadingly indicated an unexpected site of fucosylation (LacNAc-Lea-), the analysis of the permethylated product confirmed that the expected Lex-epitope was indeed produced. Curiously, fucose rearrangement in the gas phase was prominent using Q-TOF MS (supplemental Fig. S2), but only minor using MALDI-MS (Fig. 5A). This can be explained considering that the compounds spend several milliseconds in the collision cell of a Q-TOF but only microseconds of interaction time in a MALDI TOF/TOF.
      The presented work demonstrates a step toward a novel technology for the production of glycoproteins. Our work was preceded by an analogous, although conceptually different approach for the synthesis of homogenous N-glycoproteins. The strategy, also consisting of an initial in vivo glycosylation step, and several modification steps in vitro, was recently reported by Schwarz et al. (
      • Schwarz F.
      • Huang W.
      • Li C.
      • Schulz B.L.
      • Lizak C.
      • Palumbo A.
      • Numao S.
      • Neri D.
      • Aebi M.
      • Wang L.X.
      ). In that work, AcrA was glycosylated in vivo, the glycan was trimmed by glycanases in vitro, and subsequently subjected to an enzymatic transglycosylation reaction to obtain N-glycoproteins carrying oligosaccharides of the high mannose type. Unlike the strategy presented by Schwarz et al. (
      • Schwarz F.
      • Huang W.
      • Li C.
      • Schulz B.L.
      • Lizak C.
      • Palumbo A.
      • Numao S.
      • Neri D.
      • Aebi M.
      • Wang L.X.
      ), our approach can be adapted to generate glycoproteins carrying human glycan epitopes of diverse nature. This includes glycans unlikely to be synthesized in engineered yeast or mammalian cells such as those present on glycolipids. The ultimate goal of the glycoengineering approach is to achieve complete synthesis in vivo, followed by simple purification of the customized glycoconjugates. Using an optimized set of glycosyltransferases, the all-enzymatic in vivo synthesis is expected to assure the highest product quality and low production costs. With this approach an almost limitless combination of designed glycan and protein acceptor structures appears possible. One limitation is that bacterial strains will have to be engineered to produce the activated sugar donors not available in E. coli under standard growth conditions, for example, GDP-fucose and CMP-sialic acid. However, strains producing these activated sugars have been constructed (
      • Antoine T.
      • Priem B.
      • Heyraud A.
      • Greffe L.
      • Gilbert M.
      • Wakarchuk W.W.
      • Lam J.S.
      • Samain E.
      ). A main focus of future work should be the thorough analysis and optimization of metabolic pathways and the expression and control of activity of glycosyltransferases originating from various species, in E. coli. These efforts are currently ongoing in our laboratory.

      Acknowledgments

      We thank M. Veronica Ielmini and M. Florencia Haurat for critical reading of the manuscript.

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