Abstract
A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.
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All data generated or analyzed during this study are included in this Article (and its Supplementary Information) or are available from the corresponding authors on reasonable request. All unique materials used in this work are available from the authors. Source Data are provided with this paper.
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Acknowledgements
We thank R. Lee and S. Murphy for their contributions working with GT enzymes, L. Yates for helpful discussions with glyco-recoding, D. Mills for helpful discussions regarding O-OSTs, M. Paszek, J. Hershewe, K. Warfel, J. Stark, and M. Jewett for helpful discussions and provision of reagents, M. Li for technical advice and J. Wilson, J. Brooks and J. Merritt for help with vector design and yeast-based recombineering. We are also grateful to R. Bhawal and S. Zhang of the Proteomics and Metabolomics Core Facility in the Cornell Institute of Biotechnology for assistance with LC-MS. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D.), National Science Foundation (grant no. CBET-1605242 to M.P.D.) and National Institutes of Health (grant no. 1R01GM127578-01 to M.P.D.). Glycomics analysis was supported in part by the National Institutes of Health (grant no. 1S10OD018530 to P.A.). The work was also supported by seed project funding (to M.P.D.) through the National Institutes of Health-funded Cornell Center on the Physics of Cancer Metabolism (supporting grant no. 1U54CA210184-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. T.J. was supported by a Royal Thai Government Fellowship and also a Cornell Fleming Graduate Scholarship. E.C.C. was supported by a National Institutes of Health Chemical-Biology Interface (CBI) training fellowship (supporting grant no. T32GM008500).
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A.N. designed and performed all research, analyzed all data and wrote the paper. T.J. designed and performed research related to cell-free glycosylation and analyzed data. M.C.-S. and O.Y. performed research related to constructing and testing glycan biosynthetic pathways. J.C.M. performed research related to testing different proteins for glycosylation. E.C.C. performed research related to antibody-based detection of different MUC1 glycoforms. A.S., M.V., S.V., J.D.V. and P.A. performed mass spectrometry analysis and aided in data interpretation. M.P.D. directed research, analyzed data and wrote the manuscript.
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M.P.D. has a financial interest in Glycobia, Inc. and Versatope, Inc. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict of interest policies. All authors declare no other competing interests.
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Extended data
Extended Data Fig. 1 MS/MS fragmentation analysis of Tn-modified glycoprotein.
EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(HexNAc)APQPGKPPR418 derived from MBPMOOR by trypsin digestion. The spectrum identifies the neutral loss pattern of the single HexNAc monosaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.
Extended Data Fig. 2 Flow cytometric screening of Gal transferases for biosynthesis of T antigen.
(a) Schematic of flow cytometric screen to evaluate candidate Gal transferases (GalTs) for their ability to generate lipid-linked T antigen. Once formed, the T antigen is subsequently flipped to periplasm by the native E. coli flippase, Wzx, transferred to lipid A core by the promiscuous O-antigen ligase WaaL native to E. coli, and ultimately displayed on the cell surface. Cells are labeled with FITC-conjugated PNA that specifically binds the T antigen. (b) Flow cytometric analysis of PNA-labeled E. coli MC4100 ΔwecA (MCΔw) (yellow) or MC4100 ΔwecA ΔwaaL (MCΔww) (gray) carrying no plasmid, plasmid pOG-Tn, or plasmid pOG-Tn modified with one of the candidate GalT enzymes as indicated. (c) Flow cytometric analysis of PNA-labeled MCΔw (yellow) or MCΔww (gray) carrying no plasmid, plasmid pOG-T (producing T antigen glycan with EcWbwC), or plasmid pOG-TΔgne (encoding T antigen pathway but lacking CjGne epimerase). In (b) and (c), unlabeled MCΔw cells (white) were included as negative controls. Inset histograms show representative flow cytometric data used to generate mean fluorescence intensity data. See Supplementary Fig. 1 for flow cytometry gating strategy.
Extended Data Fig. 3 MS/MS fragmentation analysis of T-modified glycoprotein.
EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(HexHexNAc)APQPGKPPR418 derived from MBPMOOR by trypsin digestion. The spectrum identifies the neutral loss pattern of the HexHexNAc disaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.
Extended Data Fig. 4 Orthogonal biosynthesis of sialylated O-glycoforms in E. coli.
(a) Nano-LC-MS/MS analysis of purified acceptor protein generated by nanA-deficient E. coli cells carrying plasmid pConNeuDBAC for CMP-NeuNAc biosynthesis along with pOG-T-NgPglO and pEXT-spDsbA-MBPMOOR-EcWbwA. Sequence coverage of 88% was obtained for the MBPMOOR protein in the analysis. Spectrum reveals a predominant species (80% abundance) corresponding to the indicated peptide fragment bearing a single HexHexNAc modification as well as three less abundant species bearing a single NeuNAcHexHexNAc, a single HexNAc, and no modification (16%, 2%, and 2%, respectively). (b) Same as in (a) but with purified acceptor protein generated by nanA-deficient glyco-recoded cells carrying pOG-Tn-NgPglO and pEXT-spDsbA-MBPMOOR-PspST6. Sequence coverage of 92% was obtained for MBPMOOR in the analysis. Spectrum reveals a predominant species (90% abundance) corresponding to the indicated peptide fragment bearing a single HexNAc modification as well as two less abundant species bearing a single NeuNAcHexNAc and no modification (2% and 9%, respectively). Arrow denotes modified serine (bold underlined font) as determined by EThcD fragmentation analysis.
Extended Data Fig. 5 MS/MS fragmentation analysis of ST- and STn-modified glycoproteins.
EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(NeuNAcHexHexNAc)APQPGKPPR418 derived from (a) ST-modified MBPMOOR and (b) STn-modified MBPMOOR that were subjected to trypsin digestion. The spectrum identifies the neutral loss pattern of the single NeuNAc and Hex monosaccharides, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.
Extended Data Fig. 6 Yield determination for MBPMOOR modified with different O-glycans.
(a) Coomassie-stained SDS-PAGE gel showing MBPMOOR proteins purified from different strains. MBPMOOR bearing Tn or T antigens was produced in CLM25 cells co-transformed with pEXT-based plasmid for acceptor protein and appropriate sialyltransferase expression and either pOG-Tn-NgPglO or pOG-T-NgPglO plasmids, respectively. MBPMOOR bearing STn or ST antigens was produced in glyco-recoded cells carrying the CMP-NeuNAc biosynthesis pathway in the genome and co-transformed with pEXT-based plasmid for acceptor protein expression and either pOG-Tn-NgPglO or pOG-T-NgPglO plasmids, respectively. CLM25 cells co-transformed with only the pEXT-based plasmid for expressing MBPMOOR (agly) and appropriate sialyltransferase served as the control. Molecular weight (Mw) marker included on the left. SDS-PAGE gel is representative of three biological replicates. See Source Data for uncropped version of the image. (b) Yield of each glycoprotein calculated by multiplying the total yield times the percentage glycosylated (% gly), the latter of which was determined from nano-LC-MS/MS analysis of each glycoprotein product. Yield values are the average of three biological replicates and the error is the standard deviation of the mean.
Extended Data Fig. 7 O-linked glycosylation of diverse protein targets.
(a) Immunoblot analysis of acceptor proteins purified from CLM25 cells co-transformed with pOG-T-NgPglO (+, top), pOG-T-NmPglL (+, bottom), or pOG-T without an O-OST (-) along with pEXT-based plasmid encoding each of the different protein targets as indicated. MBPMOOR and MBPMOORmut derived from the same cells served as positive and negative control, respectively. Blots were probed with anti-hexa-histidine antibody (6xHis) to detect acceptor proteins and PNA lectin to detect the T antigen. Molecular weight (Mw) markers are indicated on the left of each blot. All immunoblot results are representative of at least three biological replicates. (b, c) Same as in (a) with pOG-T-NmPglL (+) or pOG-T without NmPglL (-) along with pEXT-based plasmid encoding each of the different protein targets as indicated. See Source Data for uncropped versions of the images.
Extended Data Fig. 8 Secretion of O-glycoproteins in the culture supernatant.
Immunoblot analysis of culture supernatants derived from CLM24 ΔyaiW cells co-transformed with pOG-T-NgPglO or pOG-T-NmPglL along with pEXT-based plasmid encoding YebF-MBPMOOR or YebF-MBPMOORmut as indicated. Mutation of acceptor serine to glycine in YebF-MBPMOORmut served as negative control. Blots were probed with anti-hexa-histidine antibody (6xHis) to detect acceptor proteins and PNA lectin to detect the T antigen. Molecular weight (Mw) markers are indicated on the left of each blot. Immunoblot results are representative of at least three biological replicates. See Source Data for uncropped versions of the images.
Extended Data Fig. 9 Orthogonal biosynthesis of different MUC1 O-glycoforms in E. coli.
Nano-LC-MS/MS analysis of purified acceptor protein generated by CLM25 cells carrying plasmid pOG-T-NgPglO along with pEXT-based plasmid for expression of different MUC1 constructs including: (a) MUC1_8; (b) MUC1_20; (c) MUC1_24; and (d) MUC1_41. Sequence coverage of 77% was obtained for MUC1_8, 78% for MUC1_20, 88% for MUC1_24, and 75% for MUC1_41 in the analysis. All spectra reveal a predominant species corresponding to the indicated peptide fragments bearing a single HexHexNAc modification. Additional less abundant species bearing a single HexNAc and no modification were observed in all cases. For MUC1_41, several doubly glycosylated species were also identified as minor species. Arrow denotes modified serine (bold underlined font) as determined by EThcD fragmentation analysis.
Extended Data Fig. 10 MS/MS fragmentation analysis of MUC1 O-glycoforms bearing the T antigen.
EThcD fragmentation analysis of glycosylated peptides derived by trypsin digestion. The spectrum identifies the neutral loss pattern of HexHexNAc disaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the sites of glycosylation (S409 in MUC1_8; S415 in MUC1_20; S417 in MUC1_24 and S417 of MUC1_41) within relevant MUC1 peptides as indicated in the inset sequences.
Supplementary information
Supplementary Information
Supplementary Table 1 and Fig. 1.
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Natarajan, A., Jaroentomeechai, T., Cabrera-Sánchez, M. et al. Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria. Nat Chem Biol 16, 1062–1070 (2020). https://doi.org/10.1038/s41589-020-0595-9
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DOI: https://doi.org/10.1038/s41589-020-0595-9
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