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Abstract

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the α-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

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Acknowledgements

This paper is dedicated to the memory of Margot Kruskall. We thank Phil Robbins for his invaluable help throughout this work. We are grateful to Annika Hult at the Blood Center in Lund for technical assistance with FACS analysis and to Etienne Danchin, AFMB, for preparing Supplementary Fig. 1 online. This work was supported by ZymeQuest Inc. and the Centre National de la Recherche Scientifique. Work performed in M.L.O.'s laboratory was supported by the Swedish Research Council (project no. K2005-71X-14251), governmental ALF research grants to Lund University Hospital, the Inga and John Hain Foundation for Medical Research and Region Skåne, Sweden. The European Synchrotron Radiation Facility (ESRF) is acknowledged for beam time allocation.

Author information

Author notes

    • Qiyong P Liu
    •  & Gerlind Sulzenbacher

    These authors contributed equally to this work.

Affiliations

  1. ZymeQuest Inc., 100 Cummings Center, Suite 436H, Beverly, Massachusetts 01915, USA.

    • Qiyong P Liu
    • , Huaiping Yuan
    • , Greg Pietz
    • , Kristen Saunders
    • , Jean Spence
    • , Edward Nudelman
    •  & Thayer White
  2. Architecture et Fonction des Macromolécules Biologiques, UMR6098, CNRS, Universités Aix-Marseille I & II, Case 932, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France.

    • Gerlind Sulzenbacher
    • , Yves Bourne
    •  & Bernard Henrissat
  3. Departments of Cellular and Molecular Medicine and Oral Diagnostics, University of Copenhagen, Blegdamsvej, DK-2200 Copenhagen N, Denmark.

    • Eric P Bennett
    • , Greg Pietz
    •  & Henrik Clausen
  4. Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.

    • Steven B Levery
  5. Harvard Microchemistry and Proteomics Analysis Facility, Harvard University, Cambridge, Massachusetts 02138, USA.

    • John M Neveu
    •  & William S Lane
  6. Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University and University Hospital Blood Center, SE-22185, Lund, Sweden.

    • Martin L Olsson
  7. Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

    • Martin L Olsson
  8. Hematology Division Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Henrik Clausen

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Contributions

Q.P.L. contributed to screening and purification of enzymes, planning and design of the project, and manuscript writing; G.S. contributed to the X-ray crystallography and manuscript writing; H.Y. contributed to purification of enzymes; E.P.B. and K.S. contributed to cloning of genes; G.P. contributed to cloning of genes; J.S. and K.S. contributed to development of enzyme conversion protocol; E.N. contributed to glycolipid analysis; S.B.L. contributed with NMR analysis; T.W. contributed to planning and design of the project; J.M.N. and W.S.L. contributed to sequencing of purified enzyme; Y.B. contributed to manuscript writing; M.L.O. contributed to FACS analysis, planning and design of the project and manuscript writing; B.H. contributed to planning and manuscript writing; H.C. contributed to planning and design of the project, and manuscript writing.

Competing interests

Authors (except for G.S., J.M.N., W.S.L. and Y.V.) are employees, consultants and/or shareholders in Zymequest Inc., which holds patents covering the described technologies.

Corresponding authors

Correspondence to Gerlind Sulzenbacher or Henrik Clausen.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Maximum Likelihood phylogenetic tree of the GH109 α-N-acetylgalactosaminidase (a) and the GH110 α-galactosidase (b) families.

  2. 2.

    Supplementary Fig. 2

    Influence of different additives on the recombinant E. meningosepticum α-N-acetylgalactosaminidase.

  3. 3.

    Supplementary Fig. 3

    Analysis of pH optimum of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase using AMC-labeled tetrasaccharides by TLC.

  4. 4.

    Supplementary Fig. 4

    Substrate specificities of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase.

  5. 5.

    Supplementary Fig. 5

    Analysis of hydrolysis of GalNAcβ-pNP by the E. meningosepticum α-N-acetylgalactosaminidase.

  6. 6.

    Supplementary Fig. 6

    The E. meningosepticum α-N-acetylgalactosaminidase is dependent on NAD+ as cofactor.

  7. 7.

    Supplementary Fig. 7

    NAD+ dependence of E. meningosepticum α-N-acetylgalactosaminidase activity with monosaccharide pNP substrates.

  8. 8.

    Supplementary Fig. 8

    Cartoon representation of the active site of the E. meningosepticum α-N-acetylgalactosaminidase.

  9. 9.

    Supplementary Fig. 9

    Multiple sequence alignment analysis of novel glycosidase families GH109 and 110.

  10. 10.

    Supplementary Fig. 10

    Proposed mechanism of the E. meningosepticum α-N-acetylgalactosaminidase.

  11. 11.

    Supplementary Table 1

    Data collection and refinement statistics

  12. 12.

    Supplementary Methods

  13. 13.

    Supplementary Results

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DOI

https://doi.org/10.1038/nbt1298