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Making glycoproteins: Bacteria go animal

Schematic representation of the protein expression and glycosylation engineering system. For full legend see Schwarz et al. Nat. Chem. Bio. (2010) doi:10.1038/nchembio.314

High diversity among the structures of N-glycans, even at the same protein glycosylation site, complicates functional studies. Although subtle structural changes are known to alter the binding specificities and functions of glycoproteins, the roles of individual glycans are difficult to pinpoint. Pure, defined glycoforms are needed for functional studies and biomedical applications, but natural samples are mixed and recombinant mammalian proteins produced in microorganisms carry non-native glycosylations.

Building on recent advances, Markus Aebi, Lai-Xi Wang and colleagues now describe a new system for production of eukaryotic N-glycoproteins. Their method, published in Nature Chemical Biology, uses engineered bacteria to produce proteins with the correct initial sugar attachment, and in vitro enzymatic methods to complete the mammalian glycosylation.

Although many bacteria do not routinely glycosylate their proteins, some pathogenic species that colonize mammals do have N-glycosylation systems. A well-studied example is found in Campylobacter jejuni and can be transferred into the workhorse of recombinant protein production, Escherichia coli. However, the glycans attached by this machinery are very different to mammalian glycans, and begin with bacillosamine (Bac), a sugar that is not found in eukaryotes. In mammalian proteins, glycosylated asparagine residues are linked to N-acetylglucosamine (GlcNAc).

To introduce the key GlcNAc–Asn linkage to recombinant glycoproteins, the authors altered the glycosylation locus from C. jejuni. Genes required for the synthesis and transfer of bacillosamine were deleted, and the resulting operon was transferred into E. coli. Without the ability to use Bac, glycan assembly – which occurs on a lipid anchor before transfer to an acceptor protein – was expected to begin with GlcNAc. This was confirmed by expressing the C. jejuni glycoprotein AcrA in the glycoengineered E. coli, and analyzing the glycoforms produced. All three AcrA forms identified contained glycans with GlcNAc at the reducing end, elongated with N-acetylgalactosamine (GalNAc) residues. These latter were trimmed enzymatically to yield AcrA carrying just the GlcNAc–Asn residues.

Many carbohydrate processing enzymes have highly specific activity that can be exploited to modify glycans in vitro. Endo-glycoside hydrolases transfer specific glycan structures to acceptor proteins, but also hydrolyze their product, resulting in low yield. The authors recently developed a method to overcome this, by mutating the endo-β-N-acetylglucosaminidase enzymes from Arthrobacter protophormiae (EndoA) and Mucor hiemalis (EndoM) such that they lack product hydrolyzing activity. Using these enzymes, complex-type glycans were transferred to the AcrA GlcNAc–Asn.

Successful attachment of mammalian glycans to a C. jejuni protein only completes part of the challenge, because the bacterium uses an extended consensus sequence for efficient N-glycosylation. To test the system for mammalian proteins, a human antibody fragment and a single-chain antibody were both engineered to contain this extended sequence in place of the shorter NXS/T of human glycoproteins, and were expressed in the glycoengineered E. coli. Although the yield of glycosylated antibody fragment was disappointing, the single chain antibody was about 40% glycosylated by the system. This is an important proof-of-concept that can be adapted and optimized for other proteins.

This study addresses a pressing need for efficient methods of producing homogenous glycoforms of human and other mammalian glycoproteins, and will expedite understanding and clinical applicability of glycan structures.

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Author: Emma Leah