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Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood

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Data availability

The atomic coordinates of AmGH110A and AmGH20A have been deposited in the Protein Data Bank (https://www.rcsb.org) under the PDB accessions 8PVS for AmGH110A and 8PXT, 8PXU and 8PXV for AmGH20A-GalNAc, unliganded AmGH20A and AmGH20A-lactose, respectively (Supplementary Tables 7 and 11). The GenPept accessions of the characterized enzymes in the study are in Supplementary Table 1. All other data are available in the main text or Supplementary Information. Unmanipulated TLC plates are provided in Supplementary Figs. 28-31. Source data and Supplementary Data (source data for supplementary figures) are provided with this paper.

Code availability

No codes were used to analyse data presented in this paper.

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Acknowledgements

The study has been funded by a Research Fund Denmark, Technology and Production Sciences (FTP Project 2 grant no. 0136-00086B) to M.A.H. with M.L.O. and J.P.M. as co-applicants. Additional funding was from Research Fund Denmark, Natural Sciences (FNU Project 2 grant no. 1026-00386B) to M.A.H. and Swedish Research Council (grant no. 2019-01683), the Knut and Alice Wallenberg Foundation (grant no. 2020.234) and the Swedish government and county councils (grant no. ALFSKANE-446521 and 2022-Projekt0287) to M.L.O., and the Novo Nordic Foundation Interdisciplinary Synergy Programme (grant no. NNF22OC0077684) to M.A.H. and M.L.O. J.R.H. was supported by a research and development grant for PhD students from Region Skåne. We would like to acknowledge B. Shouker, who had cloned most of the A. muciniphila enzymes, produced AmGH20 enzymes and delivered them to J.R.H. for initial testing, during his PhD project, which was supervised by E. Nordberg Karlsson and funded by a scholarship from the Iraqi Ministry of Higher Education and Scientific Research. We acknowledge MAX IV Laboratory for time on the BioMax beamline under proposals 20190334 and 20200120. We thank A. Gonzales and U. Muller for assistance in using the beamline and data collection. We acknowledge DESY for time on the P13 beamline at Petra III under the proposal MX846 and would like to thank I. Bento for assistance in using the beamline and data collection.

Author information

Author notes

  1. These authors contributed equally: Mathias Jensen, Linn Stenfelt.

  2. These authors jointly supervised this work: Martin L. Olsson, Maher Abou Hachem.

Authors and Affiliations

  1. Department of Biotechnology & Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

    Mathias Jensen, Linn Stenfelt, Michael Jakob Pichler, Julia Weikum, Tine Sofie Nielsen, Jens Preben Morth & Maher Abou Hachem

  2. Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden

    Linn Stenfelt, Jennifer Ricci Hagman & Martin L. Olsson

  3. Department of Clinical Immunology and Transfusion Medicine, Office for Medical Services, Region Skåne, Sweden

    Jennifer Ricci Hagman, Annika Hult & Martin L. Olsson

Contributions

M.A.H. and M.L.O. conceptualized this work. M.A.H., M.L.O., A.H. and J.P.M. developed the methodology. M.J., L.S., J.R.H., T.S.N., J.W. and A.H. conducted the investigations. M.J., L.S., J.R.H. and M.J.P. carried out the analysis. M.J. and L.S. created the visualizations. M.A.H., J.P.M. and M.L.O. acquired funding. M.A.H. and M.L.O. carried out the project administration. M.J., L.S., M.L.O. and M.A.H. wrote the original draft. M.J., L.S., J.R.H., M.J.P., T.S.N., A.H., J.P.M., M.L.O. and M.A.H. were involved in reviewing and editing the final paper.

Corresponding authors

Correspondence to Martin L. Olsson or Maher Abou Hachem.

Ethics declarations

Competing interests

A patent application has been filed on the basis of data from this study with the following authors as inventors: M.J., L.S., J.R.H., A.H., M.L.O. and M.A.H. The other authors declare no competing interests.

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Nature Microbiology thanks Ashley Toye and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Enzyme selection strategy and evaluation against group A and B RBCs.

Overview of the discovery and selection of exoglycosidases for conversion of: a B antigen, b ExtB antigen, c A and A type 3 antigens, d H type 3 antigen and e Gal-A antigen. Initial screening against chromogenic substrates guided initial selection of enzymes, which were further assayed against unconjugated saccharides using thin layer chromatography. Normalized rates (Vo/E) of best performing enzymes were determined using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), then these enzymes were evaluated against target antigens on RBCs using flow cytometry. The blood drop symbol denotes top-performing enzymes, which were selected for crossmatch reactivity (compatibility) analyses. f Crossmatch reactivity analyses were performed by enzymatic conversion of RBCs from A1, A2 and B donors. Single-enzyme treatments targeting A or B antigens as well as sequential and one-pot treatments with enzyme cocktails targeting A or B antigens and their respective extended forms were performed. Blood group A or B RBCs of secretor/non-secretor backgrounds were converted with either a single enzyme or enzyme cocktails and converted RBCs from each treatment were crossmatched against 100 group O plasmas, resulting in 1522 crossmatch tests, besides lower numbers of crossmatch assays with plasmas from other ABO groups. Created with BioRender.com.

Extended Data Fig. 2 Activity of enzyme candidates towards B antigens on blood group B RBCs.

Representative flow cytometry histograms of anti-B (clone 9621A8)/anti-RAMκ-Phycoerythrin (PE)-stained blood group B RBCs (filled blue) and native group O RBCs (dotted black) as staining negative controls. a Native B RBCs and b, c, d and e RBCs post-treatment with 1 μM of AmGH110A, AmGH36A, AmGH36C or AmGH27, respectively for 30 min at room temperature (RT) in conversion buffer (200 mM glycine, 3 mM NaCl, pH 6.8). f Overview of the data in a-e. The bars are means of the median fluorescence intensity (MFI) of the PE-fluorescence signal of RBC aliquots from three donors (n = 3). The open circles are the MFI values from each donor. g Native group B RBCs. h and i, RBCs from the same donor as in g, after treatment with 0.12 µM AmGH110A for 60 min at RT in conversion buffer and PBS (phosphate buffered saline), respectively. j Removal of B antigens monitored by changes of MFI at different AmGH110A concentrations over time and with RBCs from a single group B donor. k Removal of B antigens monitored by changes of MFI at different concentrations of AmGH110A in 30 min reactions with RBCs from three donors (n = 3). The MFI on y-axes in j and k are shown in logarithmic scales and the dotted line in each graph mark MFI levels of negative controls (group O RBCs). All reactions were performed with 38% haematocrit (volumetric ratio between RBCs and the source blood suspension).

Source data

Extended Data Fig. 3 Activity of enzyme candidates towards the ExtB antigen on blood group B RBCs.

a Eleven GH20 candidates were evaluated in treatments of ExtB carrying group B RBCs from two donors (n = 2) (1 µM of each enzyme, 30 min, 38% haematocrit in conversion buffer, room temperature). The conversion was monitored by changes in mean median fluorescence intensity (MFI) of enzyme-treated and anti-ExtB-stained RBCs relative to native RBCs. b Representative flow cytometry histograms of anti-ExtB-stained blood group B RBCs (blue filled) and group O RBCs (dotted black line) as negative staining controls. c-e The RBCs from the same donors in b after treatment with AmGH20A, AmGH20C and AmGH20I, respectively. f Overview data from three group B donors (n = 3), including the example shown in panels c-e. Dotted lines in panels a and f graphs are MFI levels of group O RBC, the negative staining control. The data are the mean MFI (blue bars) of three donors (n = 3) shown as open circles.

Source data

Extended Data Fig. 4 Effect of enzyme concentration and reaction buffer on removal of the ExtB antigen on blood group B RBCs.

a-c Conversion of the ExtB antigen as a function of concentration of AmGH20A, AmGH20C and AmGH20I, respectively in one pot 30 min reactions with the B antigen-removing AmGH110A. d,e Effect of the concentration of AmGH20A and AmGH20C, respectively, on conversion of the ExtB antigen. f,g Removal of the ExtB antigen on group B RBCs in conversion buffer and PBS, respectively, using either AmGH20A (0.2 µM) or AmGH20C (0.1 µM). The bars f and g, are mean MFI values and dotted lines mark MFI levels of the negative staining control, O RBCs. Each scatter plot represents data from a single anonymous donor, denoted with donor number.

Source data

Extended Data Fig. 5 Activity of A. muciniphila enzymes towards A antigens on blood group A RBCs.

a,b Representative histograms (blue) of immuno-A-stained RBCs from A1 and A2 donors, respectively, or the same after 30 min incubation at room temperature with 1 μM of c,d AmGH109A; e,f AmGH109B, or g,h AmGH36A, all compared to negative staining control O RBCs (dotted line histograms). i,j Overview of data from three donors (n = 3) of A1 and A2 phenotype, respectively, based on the median fluorescence intensity (MFI). Bars are MFI means from three donors, shown as open circles. k Histograms of A-stained RBCs (blue) and negative staining control (dotted line histogram), l-o RBCs from the same donors in k, post 60 min treatment with AmGH109B (8 µM) or AmGH36A (1.1 µM) in PBS or conversion buffer, respectively. p Levels of A antigens of A1 RBCs after different incubation times and concentrations of AmGH36A. q Levels of A antigens on A1 RBCs from three more donors after incubation with different AmGH36A concentrations for 30 min. r Levels of the extended A type 3 antigen on the same RBCs as in q. In graphs p-q Logarithmic scales starting at 100 are used for the Y-axis. Dotted horizontal line in each graph are MFI levels of O RBCs as negative control. A-antigen levels were assayed by flow cytometry, after staining with anti-A monoclonal antibodies (ES-15) and for anti-A type 3 the monoclonal antibody TH1. For both, a secondary antibody rat anti-mouse-κ conjugated to Phycoerythrin (PE) was used.

Source data

Extended Data Fig. 6 Dependence of α-1,2-fucosidase activity towards H antigens on RBCs on buffer and enzyme concentration.

a Representative histograms of native group O RBCs stained with anti-H (BRIC231) and RAMκ-Phycoerytrhin (PE). b-d are RBCs from the same donor after treatment with of RiGH95, AmGH95A and AmGH95B (each at 1.2 µM) in conversion buffer during 60 min incubations at RT, respectively. e Overview of the data in a-d showing the median fluorescence intensity (MFI). f, g and h Same reactions as b, c and d but performed in PBS. i Overview graph of the MFI in a, f-h. j Activity of AmGH95B after 60 min incubations towards H antigens at different concentration in the presence of AmGH36A.

Source data

Extended Data Fig. 7 Evaluation of GH35 β-galactosidases towards the extended Gal-A antigen.

a Representative histogram (blue) of the Gal-A antigen of A1 RBCs from a single donor, compared with negative control O RBCs (dotted black line histogram), stained with anti-T/Gal-A (clone 3C9) followed by RAMκ-Phycoerythrin (PE). b and c RBCs from the same donor after treatment with AmGH35A (5.8 µM) and AmGH35B (7.2 µM), respectively, during 60 min incubations in conversion buffer at RT. d Overview of the data b and c showing the median fluorescence intensity (MFI). The dashed black line is the MFI level in the negative group O control RBCs. e Activity of AmGH35A at different concentrations in the presence of AmGH36A and AmGH95B after incubation for 30 min. For comparison, RBCs only with AmGH36A and the one-pot treatment with AmGH36A and AmGH95B are included. Logarithmic scales starting at 100 are used on the Y-axis.

Source data

Extended Data Fig. 8 Crossmatch reactivity of group O plasmas towards enzymatically converted B RBCs.

Crossmatches before and after treatment with AmGH110A (0.5 μM) or a one-pot treatment with AmGH110A (0.5 μM) and either AmGH20A or AmGH20C (both at 0.4 μM). The colour-scale (red-pink-white) corresponds to the strength of crossmatch reactivity towards group O plasma samples (n = 100, a-j, 1-10), where red is the strongest reaction (4 + ) and the three shades of pink represent gradually weaker reactions (3 + , 2 + , 1 + ). Minus represents a negative crossmatch. A minus in brackets shows a reaction between negative and very weakly positive but not fulfilling the criteria for a positive (1 + ) reaction. Untreated RBCs of secretor/non-secretor background were crossmatched with only a subset of O plasmas (n = 10, 1a-1j) to get a baseline level of reactivity for comparison with enzyme-treated cells. The figure allows tracking changes in crossmatch reactivity of individual plasmas with the single and one-pot enzyme treatments.

Source data

Extended Data Fig. 9 Crossmatch reactivity of group O plasmas towards enzymatically converted A1 RBCs.

The crossmatches were performed before and after treatment with AmGH36A (1 μM) or a one-pot treatment with AmGH36A (1 μM), AmGH95B (0.02 μM) and AmGH35A (0.2 μM). The colour-scale (red-pink-white) corresponds to strength of crossmatch reactivity towards group O plasma samples (n = 100, a-j, 1-10), where red is the strongest reaction (4 + ) and the shades of pink represent gradually weaker reactions (3 + , 2 + , 1 + ). Minus represents a negative crossmatch. A minus in brackets shows a reaction between negative and very weakly positive but not fulfilling the criteria for a positive (1 + ) reaction. Untreated RBCs of secretor/non-secretor background were crossmatched with only a subset of O plasmas (n = 10, 1a-1j) to get a baseline level of reactivity for comparison with enzyme-treated cells. The figure allows tracking changes in crossmatch reactivity of individual plasmas with the single and one-pot enzyme treatments.

Source data

Extended Data Fig. 10 Phylogenetic analysis of the AmGH20A CBM-like domain.

a Phylogenetic tree generated from a search with the C-terminal CBM-like domain of AmGH20A (aa 555-665) as a query against the non-redundant database using Delta-Blast (Domain enhanced lookup time accelerated blast) algorithm against sequences with 20-95 % sequence identity and 90-100% sequence coverage. The search retrieved 549 sequences, mostly derived from other putative HexNAcases, but also putative M14 peptidases and Gfo/Idh/MocA-like oxidoreductases. Sequences were initially aligned using MAFFT and then trimmed to only include the segment corresponding the CBM-like domain of AmGH20A. The redundancy of the sequences was then reduced using CD-HIT with a 95% identity cutoff and MaxAlign at default settings. A phylogenetic tree was generated from the remaining 253 sequences and annotated based on phylum affiliation. The cluster shaded with blue harbours the AmGH20A CBM. b The N-acetylgalactosamine binding residues that are presented by three loops are shown and polar interactions are denoted with yellow dotted lines. Sequence logos of the loops forming the GalNAc-binding site, show conservation in the AmGH20A CBM cluster, as compared to the conservation in all sequences in the tree, respectively. The positions of residues shown in the cartoon are marked with black arrows.

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Jensen, M., Stenfelt, L., Ricci Hagman, J. et al. Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood. Nat Microbiol 9, 1176-1188 (2024). https://doi.org/10.1038/s41564-024-01663-4

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