N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a lipid-linked oligosaccharide (LLO), and (ii) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided an insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.
Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.
more » « less- NSF-PAR ID:
- 10381484
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Glycobiology
- ISSN:
- 1460-2423
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐
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null (Ed.)Abstract Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins, and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate Xyloglucan Xylosyltransferase 1 (XXT1), a GT involved in synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from UDP-Xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-Xylose and UDP-Glucose, while reactions with UDP-Arabinose and UDP-Galactose are over 10-fold slower. Using kcat/Km as a measure of efficiency, UDP-Xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-Glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific, and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.more » « less
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