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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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.
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.
Maximum Likelihood phylogenetic tree of the GH109 α-N-acetylgalactosaminidase (a) and the GH110 α-galactosidase (b) families. (PDF 131 kb)
Influence of different additives on the recombinant E. meningosepticum α-N-acetylgalactosaminidase. (PDF 94 kb)
Analysis of pH optimum of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase using AMC-labeled tetrasaccharides by TLC. (PDF 147 kb)
Substrate specificities of E. meningosepticum α-N-acetylgalactosaminidase and FragA α-galactosidase. (PDF 142 kb)
Analysis of hydrolysis of GalNAcβ-pNP by the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 175 kb)
The E. meningosepticum α-N-acetylgalactosaminidase is dependent on NAD+ as cofactor. (PDF 130 kb)
NAD+ dependence of E. meningosepticum α-N-acetylgalactosaminidase activity with monosaccharide pNP substrates. (PDF 193 kb)
Cartoon representation of the active site of the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 35 kb)
Multiple sequence alignment analysis of novel glycosidase families GH109 and 110. (PDF 638 kb)
Proposed mechanism of the E. meningosepticum α-N-acetylgalactosaminidase. (PDF 137 kb)
Data collection and refinement statistics (PDF 22 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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