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Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage

Nature Chemical Biology volume 17, pages 806–816 (2021)Cite this article

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Abstract

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA ‘barcoded’ M13 virions that display 30–1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

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Fig. 1: Synthesis and characterization of LiGA components.
Fig. 2: A colormetric PFU assay for rapid validation of the LiGA.
Fig. 3: Summary of lectin binding to LiGA composed of 65 different glycophage constructs.
Fig. 4: LiGA measures affinity- and avidity-based responses in purified GBPs.
Fig. 5: LiGAs measure affinity- and avidity-based responses in cell-displayed GBPs.
Fig. 6: LiGA measures glycan binding response of GBP in vivo.

Data availability

All raw deep-sequencing data are publicly available at http://ligacloud.ca/ with data-specific URLs listed in Supplementary Table 3. DNA sequences of the three LiGA phage constructs were deposited at GenBank (MN865131, MN865132, MN872303). All public CFG data were downloaded from the CFG website using an automated Python script as described in the section Access to CFG data. Each dataset can be accessed manually on the CFG website at http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp. Source data are provided with this paper.

Code availability

MATLAB, Python and R scripts were deposited at https://github.com/derdalab/liga.

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Acknowledgements

We thank the staff at the University of Alberta mass spectrometry facility (Chemistry Department) for help with MALDI analysis and S. Dang at the molecular biology service unit for assistance with Illumina sequencing. Cell sorting was performed at the University of Alberta, Faculty of Medicine and Dentistry Flow Cytometry Facility with financial support from the Faculty of Medicine and Dentistry and Canada Foundation for Innovation awards to contributing investigators. We thank K. Drickamer (Imperial College, London), B. Turnbull (University of Leeds), D. Bundle, C. Cairo and L. West (University of Alberta) for provision of critical reagents. We acknowledge funding from the NSERC (RGPIN-2018-04365 to T.L.L., RGPIN-2018-03815 to M.S.M. and RGPIN-2016-402511 to R.D.) and the NSERC Accelerator Supplement (to R.D.), GlycoNet (SD-1 to T.L.L., TP-22 to R.D.), the Alberta Innovates Strategic Research Project to R.D. and NIH projects (AI118842 to M.S.M. and GM062116 and AI050143 to J.C.P.). Infrastructure support was provided by the Canada Foundation for Innovation New Leader Opportunity (to R.D. and M.S.M.). J.M. acknowledges a summer research fellowship from GlycoNet and Alberta Innovates Health Solutions. Many compounds were prepared by the CFG, supported by NIH GM061126.

Author information

Author notes

  1. Amira Khalil

    Present address: Pharmaceutical Chemistry Department, Faculty of Pharmacy, The British University in Egypt, El Sherouk, Egypt

Authors and Affiliations

  1. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Mirat Sojitra, Susmita Sarkar, Jasmine Maghera, Emily Rodrigues, Eric J. Carpenter, Shaurya Seth, Daniel Ferrer Vinals, Nicholas J. Bennett, Revathi Reddy, Amira Khalil, Xiaochao Xue, Michael R. Bell, Ruixiang Blake Zheng, Justin J. Bailey, Todd L. Lowary, Matthew S. Macauley & Ratmir Derda

  2. Department of Chemistry, University of Calgary, Calgary, Alberta, Canada

    Ping Zhang & Chang-Chun Ling

  3. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA

    Corwin Nycholat & James C. Paulson

  4. Institute of Biological Chemistry, Academia Sinica, Nangang, Taipei, Taiwan

    Todd L. Lowary

  5. Institute of Biochemical Sciences, National Taiwan University, Da’an, Taipei, Taiwan

    Todd L. Lowary

  6. Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

    Matthew S. Macauley

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Contributions

N.J.B. constructed and characterized all LiGA plasmids and libraries; M.S., S. Sarkar, J.M., D.F.V. and R.R. isolated phage clones and performed modifications of phage by glycans, MALDI analysis, protein-binding experiments and in vitro assays. M.S., E.R., J.M. and R.D. performed cell-based assays; M.S.M. and S. Sarkar performed animal experiments; E.J.C., M.S. and R.D. performed statistical analysis; S. Seth performed custom analysis of public CFG data; T.L.L., P.Z., C.-C.L., C.N., J.J.B. and J.C.P. contributed synthetic glycan reagents; A.K. and M.R.B. constructed synthetic antigens to raise and characterize anti-Galf4 antibodies, which were produced by S. Sarkar. X.X. and R.B.Z. constructed antigens for incorporation into the LiGA. R.D. and M.S. wrote the manuscript; R.D., M.S.M., T.L.L. and J.C.P. edited the final manuscript and contributed intellectual and strategic input. All authors approved the final manuscript.

Corresponding author

Correspondence to Ratmir Derda.

Ethics declarations

Competing interests

R.D. and N.J.B. are shareholders of the start-up company 48Hour Discovery Inc. that licensed the patent application (WO2018141058A1) describing LiGA technology. R.D., N.J.B. and S. Sarkar are co-inventors on the aforementioned application.

Additional information

Peer review information Nature Chemical Biology thanks Matthew DeLisa, Ten Feizi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Cloning of silent double barcode (SDB) regions.

a, Location of SB1 and SVEK Peptide region (SB2) on initial template. b, Primer 1 and 4 were used to convert M13KE QFT*LHQ to M13KE SDBlib QFT*LHQ. c, Frequency plot analysis of the top leader region of 100 unique sequences observed in deep sequencing of M13KE SDBlib vector; notably nucleotide C was completely suppressed in position 8 even though it was possible by design (CTN codon in Primer 1); d, Distribution and cumulative distribution of the unique sequences in deep sequencing of M13KE SDBlib vector. From 6144 possible sequences the most abundant ~200 unique sequences dominated 90% of the available diversity. e, Primer 5 and 6 were used to convert M13KE SDB QFT*LHQ to M13KE SDB SVEK library. f, Summary of degenerate sites in M13KE SB1 and SVEK Peptide (SB2) regions. g, Location of Illumina Sequencing Primers. h, Sequences of the Illumina sequencing primers.

Extended Data Fig. 2 MALDI spectra of phage at different concentrations.

Spectra were acquired as described in section ‘Analysis of glycosylation of phage samples by MALDI-TOF MS’ using indicated concentrations of unmodified phage (left) or (right) phage that was partially acylated by DBCO-NHS and then partially modified (Fig. 1d).

Extended Data Fig. 3 Regioselectivity of the modification of pVIII protein.

a, Scheme of acid induced cleavage of Asp-5-Pro-6 (D5-P6)1 bond in pVIII to yield pVIII[1–5] and pVIII[6–55] fragments. b, Scheme of the modification of the phage clone with the azido-glycan Galf4 (Galfβ1-5Galfβ1-5Galfβ1-5Galfβ-(CH2)-N3). Modified phage was purified by Zeba column to remove unreacted glycan. c, MALDI-TOF analysis of modified and unmodified phage clones after incubation in 70% (v/v) TFA for 1 h (‘High TFA’) or without any pre-treatment with TFA (‘Low TFA’). Analysis detects unmodified pVIII[6–55] fragment but does not detect presence of glycosylated pVIII[6–55] fragment. Glycosylation, thus, occurs preferentially at the N-terminus residue. d, Scheme of the cleavage of the pVIII modified with the azido-glycan Galf4 on the N-terminus and ε-amine of Lys-8 residue. 1. Crimmins, D.L., Mische, S.M. & Denslow, N.D. Chemical cleavage of proteins in solution. Curr. Protoc. Protein Sci. 11(2005).

Extended Data Fig. 4 ELISA measuring the binding of glycosylated phage to lectins.

a, MALDI characterization of the tested phage constructs. b, For ConA, phage displaying branched trimannoside (Manα1-6[Manα1-3]Manα-s6) exhibited the highest binding, the clone displaying a similar copy number of Manα glycan exhibited lower binding, while phage conjugated with LNT and unconjugated blank phage showed no detectable binding. c, In Galectin 3 (Gal3)-coated wells, the phage modified with LNT exhibited the highest binding. Minor non-specific binding of unmodified phage was observed at 1010 PFU/ml concentration. Interestingly, Manα showed a significantly lower binding to Gal3 compared to unmodified phage. The top x-axis in b-c indicates the total phage particles (PFU) and the bottom x-axis indicates the concentration (PFU/ml). Data from (b,c) are represented as mean ± s.d. (n = 3) and p-values at the indicated concentrations were measured using two-tailed student t-test.

Source data

Extended Data Fig. 5 Binding of G3C to glycans in LiGA of different complexity.

a, Composition of LiGA mixtures used in this experiment: LiGA YT contains 75 clones and includes glycans ligands with known affinity for G3C; LIGA UX is a subset of LiGA YT composed of 11 ligands with known affinity for G3C. b, Schematic description two reference phage clones LNT-NAc-[green] and Man3–[red]: a clonal phage containing mNeonGreen reporter gene modified with LNT-NAc (Galβ1-3GlcNAcβ1-3 Galβ1-4GlcNAcβ1) and a clonal phage containing mCherry gene modified Man3 (Manα1-6(Manα1-3)Manα1). Both reference clones we added into a LiGA mixture produced from phage clones with LacZ reporter gene. c-e, Plaque forming assay (PFU) measured the titer of LiGA of three compositions and reference clones before binding (input) and after binding (output) to the biotinylated Galectin-3 CRD immobilized on streptavidin beads (‘G3C’), in the presence of 60 mM lactose or to control streptavidin beads (‘beads’). Recovery is calculated as 100%×PFUoutput/ PFUinput. f, Summary of recovery from (c-d) confirming that addition of 60 mM lactose significantly abrogated the interaction between G3C and LiGA YT (c), LiGA UX (d) and LNT-NAc-[green] admixed to LiGA YT or UX (c-d). g, Summary of recovery from (c-e) confirming that binding of reference phage LNT-NAc-[green] to G3C is similar in the presence of LiGA YT, LiGA UX or azidoethanol modified phage. Binding of the reference clone to G3C is not influenced by the presence of other G3C binding clones in the mixture and only a minor decrease in recovery was observed in the presence of LiGA UX composed of G3C-binding clones (a). Another reference phage Man3–[red] does not bind to the G3C in these conditions. h, Summary of recovery from c and e confirming the indistinguishable (low) binding of LiGA YT or azidoethanol-modified phage to streptavidin beads. All figures represent n = 3 independent biological replicate with p-values from two-tailed t-tests. Hollow points indicate no observable plaque in the replicate. Data is represented as mean+s.d, and the s.d for recovery is propagated from variance of input and output PFU.

Source data

Extended Data Fig. 6 Binding of G3C to LiGA and CFG glass array.

a, Binding of LiGA YT to biotinylated G3C immobilized on streptavidin agarose beads (see Extended Fig. 5a and Supplementary Table 3 for composition of LiGA YT). Heat map describes Fold Change (FC) values observed in DE analysis using LiGA binding to G3C-coated beads as ‘test’ and LiGA binding to beads alone as a control. Each row represents an independently conducted screening campaign and DE-analysis (n = 3 for ‘test’ and ‘control’ independent biological replicates in each campaign). Third row in the heat map is mirrored from Fig. 2h for consistency. FC and FDR and were calculated using negative binomial model, TMM-normalization and BH-correction for FDR (* designate FDR < 0.05, FC > 4). b, Binding of different concentrations of fluorescently-labeled G3C to glass-based glycan array (n = 6); public microarray data, CFG request #2564; searchable as ‘primscreen_6003’ to ‘primscreen_6011’ at CFG website. Data represent an average response fluorescent units (RFU, n = 6).

Source data

Extended Data Fig. 7 Significance of the differences in binding of targets to glycans of different density.

Data was adapted from Fig. 4b-d, and Fig. 5c and analyzed using ANOVA with a post-hoc test. MALDI spectra of the phage glycoconjugates of low and high density. a, The analysis confirmed significance of differences (p < 0.05) between binding to medium density and high density for mAb-Galf4 with exception of binding to galectin 3. Galectin-3 exhibited p=0.17. b, The binding of medium density and high density for ConA and DC-SIGN was significance of differences (p < 0.05). LiGA components b1, b2, b3, c1, c2, d1 and d2 described in (a-b). Fold change was determined as the ratio of normalized average copy numbers in LiGA associated with positive and negative samples in respective target protein. Galectin-3, n = 4. mAb-Galf4, n = 3. DC-SIGN, n = 3. ConA, n = 3 test, n = 4 control. FC and FDR were calculated by EdgeR DE analysis using its negative binomial model with TMM-normalization and BH-correction for FDR; asterisks designate FDR < 0.05. Error bars represent s.d. propagated from the variance of the TMM-normalized sequencing data. All respective n are independent biological replicates.

Source data

Extended Data Fig. 8 Steric occlusions in the interactions between protein ConA and multivalent glycans on phage.

a, A DBCO modified phage clone was split into three aliquots and reacted with different ratio of Man3 and Galf4 azido-glycans to produce three hybrid clones. (i) 120 copies of Man3 and 1380 copies of Galf4 per phage (measured by MALDI). (ii) 380 copies of Man3 and 1125 copies of Galf4 per phage (measured by MALDI). (iii) 500 copies of Man3 and 1000 copies of Galf4 per phage (measured by MALDI). We incubated each hybrid phage with biotinylated ConA immobilized on the streptavidin beads, washed the beads, boiled the beads and measured the released DNA using qPCR Cq values. Binding of each clone was estimated as Cqinput – Cqoutput. We use the same assay to measure the binding of phage clones that display only Man3 ligand at a different density (same clones were used in main text Fig. 4): 140, 300 and 1500 copies of Man3 per phage as well as negative control phage (no glycosylation). Recall that Man3 is a ligand for ConA whereas Galf4 does not bind to ConA (Fig. 4). b, We observed that binding of any hybrid phage to ConA was significantly lower than binding of any Man3-decorated phage. Binding of hybrid phage to ConA was also statistically indistinguishable from binding of non-glycosylated phage to ConA. Access of ConA protein to Man3 ligands is inhibited by neighboring Galf4 glycans present on the same phage (steric occlusion). This experiment also confirmed that binding of phage displaying 1500 copies of Man3 to ConA is significantly lower (p=0.01) than binding of phage with 300 copies of Man3. Steric occlusion may also play a role in this decrease: access of ConA protein to Man3 ligands is inhibited by neighboring Man3 glycans present on the same phage. Mean values with error representing s.d propagated from the variance in Cqinput and Cqoutput. Man3 − Galf4 hybrid are n = 4 independent biological replicates and Man3, Wildtype are n = 3 independent biological replicates.

Source data

Extended Data Fig. 9 Binding of LiGA to anti-A and anti-B antibodies.

Murine monoclonal anti-A IgM and anti-B IgM (generous gift of Lori West, University of Alberta) were coated on polystyrene well and binding of LiGA YZ (Supplementary Table 3) was performed (n = 5 and 6 independent biological replicates). The data was analyzed using EdgeR differential enrichment analysis using anti-A and anti-B as two sets. The volcano plot describes fold change (FC) difference and p-value of glycans enriched on anti-A (FC < 0) and anti-B (FC > 0) antibodies. Red circles denote four DNA barcodes that exhibited enrichment with FDR < 0.05. The structures of the glycans associated with these barcodes are known blood group glycans targeted by anti-A and anti-B antibodies.

Source data

Extended Data Fig. 10 Binding of DC-SIGN to LiGA and glass-based glycan array.

a, Binding of LiGA of three different compositions (YO, YX and YZ) to DC-SIGN on the surface of rat fibroblasts measured as differential enrichment of LiGA pulled down by DC-SIGN+ fibroblasts and DC-SIGN− fibroblasts. LiGA YZ data in heat map is mirrored from bar chart in Fig. 5c for consistency. n = 3,2,4 for YO, YX and YZ, respectively. b, Alignment to data describing binding of fluorescently labeled monomeric and tetrameric DC-SIGN protein to glass-based glycan array (publicly available CFG data under CFG request #2569 and searchable as ‘primscreen_5273’ at: http://www.functionalglycomics.org/static/index.shtml). c, Overview of the full CFG array describing the binding of monomeric DC-SIGN protein and structures of the top binding glycans. *FDR < 0.05. Bars represent an average response fluorescent units (RFU, n = 6). All respective n are independent biological replicates.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Notes 1 and 2, Supplementary Schemes 1–6 and Supplementary Figs. 1–15

Reporting Summary

Supplementary Data

Source data

Source Data Fig. 2

Plaque count used to generate data for Fig. 2c–f. DE analysis for Fig. 2g,h. Each panel’s source data are tab separated in the excel file.

Source Data Fig. 3

DE analysis of sequencing data for Fig. 3a. Additional data for each buffer condition are provided in a separate tab, as requested in prior revisions.

Source Data Fig. 4

DE analysis of sequencing data for Fig. 4b–d. Each panel’s source data are tab separated in the excel file.

Source Data Fig. 5

DE analysis of sequencing data for Fig. 5a–d. Each panel’s source data are tab separated in the excel file.

Source Data Fig. 6

Plaque count source data for Fig. 6a.

Source Data Extended Data Fig. 4

ELISA absorbance source data used to generate Extended Data Fig. 4. Each panel’s source data are tab separated in the excel file.

Source Data Extended Data Fig. 5

Plaque count source data used to generate Extended Data Fig. 5c–h. Each panel’s source data are tab separated in the excel file.

Source Data Extended Data Fig. 6

DE analysis of sequencing data for Extended Data Fig. 6a.

Source Data Extended Data Fig. 7

Analysis of data from Fig. 4 using ANOVA with a post hoc test.

Source Data Extended Data Fig. 8

Statistical source data for the hybrid phage-binding assay using qPCR.

Source Data Extended Data Fig. 9

DE analysis of sequencing data for Extended Data Fig. 9.

Source Data Extended Data Fig. 10

DE analysis of sequencing data for Extended Data Fig. 10a.

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Sojitra, M., Sarkar, S., Maghera, J. et al. Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage. Nat Chem Biol 17, 806–816 (2021). https://doi.org/10.1038/s41589-021-00788-5

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