Bacterial glycosidases for the production of universal red blood cells

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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Figure 1: Structural basis of the ABO blood group antigens.
Figure 2: Overall structure of the E. meningosepticum α-N-acetylgalactosaminidase.
Figure 3: FACS of native and ECO RBCs.
Figure 4: TLC analysis of native and ECO RBC glycolipids.

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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.

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Authors

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.

Corresponding authors

Correspondence to Gerlind Sulzenbacher or Henrik Clausen.

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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.

Supplementary information

Supplementary Fig. 1

Maximum Likelihood phylogenetic tree of the GH109 α-N-acetylgalactosaminidase (a) and the GH110 α-galactosidase (b) families. (PDF 131 kb)

Supplementary Fig. 2

Influence of different additives on the recombinant E. meningosepticum α-N-acetylgalactosaminidase. (PDF 94 kb)

Supplementary Fig. 3

Analysis of pH optimum of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase using AMC-labeled tetrasaccharides by TLC. (PDF 147 kb)

Supplementary Fig. 4

Substrate specificities of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase. (PDF 142 kb)

Supplementary Fig. 5

Analysis of hydrolysis of GalNAcβ-pNP by the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 175 kb)

Supplementary Fig. 6

The E. meningosepticum α-N-acetylgalactosaminidase is dependent on NAD+ as cofactor. (PDF 130 kb)

Supplementary Fig. 7

NAD+ dependence of E. meningosepticum α-N-acetylgalactosaminidase activity with monosaccharide pNP substrates. (PDF 193 kb)

Supplementary Fig. 8

Cartoon representation of the active site of the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 35 kb)

Supplementary Fig. 9

Multiple sequence alignment analysis of novel glycosidase families GH109 and 110. (PDF 638 kb)

Supplementary Fig. 10

Proposed mechanism of the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 137 kb)

Supplementary Table 1

Data collection and refinement statistics (PDF 22 kb)

Supplementary Methods (PDF 151 kb)

Supplementary Results (PDF 94 kb)

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Liu, Q., Sulzenbacher, G., Yuan, H. et al. Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol 25, 454–464 (2007). https://doi.org/10.1038/nbt1298

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