Bacterial glycosidases for the production of universal red blood cells


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes



Protein Data Bank


  1. 1

    Landsteiner, K. Agglutination phenomena of normal human blood. Wien. Klin. Wochenschr. 113, 768–769 (2001).

    CAS  PubMed  Google Scholar 

  2. 2

    Watkins, W.M. Biochemistry and Genetics of the ABO, Lewis, and P blood group systems. Adv. Hum. Genet. 10, 1–136, 379–185 (1980).

    CAS  PubMed  Google Scholar 

  3. 3

    Clausen, H. & Hakomori, S. ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sang. 56, 1–20 (1989).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Yamamoto, F., Clausen, H., White, T., Marken, J. & Hakomori, S. Molecular genetic basis of the histo-blood group ABO system. Nature 345, 229–233 (1990).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Sazama, K. Transfusion errors: scope of the problem, consequences, and solutions. Curr. Hematol. Rep. 2, 518–521 (2003).

    CAS  PubMed  Google Scholar 

  6. 6

    Stainsby, D. et al. Serious Hazards of Transfusion Annual Report 2004 (Serious Hazards of Transfusion Office, Manchester Blood Centre, Manchester, UK; 2005).

    Google Scholar 

  7. 7

    Olsson, M.L. et al. Universal red blood cells–enzymatic conversion of blood group A and B antigens. Transfus. Clin. Biol. 11, 33–39 (2004).

    Article  PubMed  Google Scholar 

  8. 8

    Goldstein, J., Siviglia, G., Hurst, R., Lenny, L. & Reich, L. Group B erythrocytes enzymatically converted to group O survive normally in A, B, and O individuals. Science 215, 168–170 (1982).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Kruskall, M.S. et al. Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. Transfusion 40, 1290–1298 (2000).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Vosnidou, N.C. et al. Seroconversion of type B to O erythrocytes using recombinant Glycine max α-D-galactosidase. Biochem. Mol. Biol. Int. 46, 175–186 (1998).

    CAS  PubMed  Google Scholar 

  11. 11

    Bakunina, I.Y. et al. Alpha-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM 701. Biochemistry (Mosc.) 63, 1209–1215 (1998).

    CAS  Google Scholar 

  12. 12

    Clausen, H., Levery, S.B., Kannagi, R. & Hakomori, S. Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). I. Isolation and chemical characterization. J. Biol. Chem. 261, 1380–1387 (1986).

    CAS  PubMed  Google Scholar 

  13. 13

    Clausen, H., Levery, S.B., Nudelman, E., Tsuchiya, S. & Hakomori, S. Repetitive A epitope (type 3 chain A) defined by blood group A1-specific monoclonal antibody TH-1: chemical basis of qualitative A1 and A2 distinction. Proc. Natl. Acad. Sci. USA 82, 1199–1203 (1985).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Zhu, A., Monahan, C., Wang, Z.K. & Goldstein, J. Expression, purification, and characterization of recombinant α-N-acetylgalactosaminidase produced in the yeast Pichia pastoris. Protein Expr. Purif. 8, 456–462 (1996).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Hsin-Yeh, H., Chapman, L.F., Calcutt, M.J. & Smith, D.S. Recombinant Clostridium perfringens α-N-acetylgalactosaminidase blood group A2 degrading activity. Artif. Cells Blood Substit. Immobil. Biotechnol. 33, 187–199 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. 16

    Bakunina, I.Y. et al. α-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes. Biochemistry (Mosc.) 67, 689–695 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Landry, D. Isolation and composition of a novel glycosidase from chryseobacterium. US patent 6,458,525. (2002).

  18. 18

    Pikis, A., Immel, S., Robrish, S.A. & Thompson, J. Metabolism of sucrose and its five isomers by Fusobacterium mortiferum. Microbiology 148, 843–852 (2002).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Yip, V.L. et al. An unusual mechanism of glycoside hydrolysis involving redox and elimination steps by a family 4 beta-glycosidase from Thermotoga maritima. J. Am. Chem. Soc. 126, 8354–8355 (2004).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Rajan, S.S. et al. Novel catalytic mechanism of glycoside hydrolysis based on the structure of an NAD+/Mn2+-dependent phospho-alpha-glucosidase from Bacillus subtilis. Structure 12, 1619–1629 (2004).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Yip, V.L. & Withers, S.G. Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima bglt: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4. Biochemistry 45, 571–580 (2006).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Lesk, A.M. NAD-binding domains of dehydrogenases. Curr. Opin. Struct. Biol. 5, 775–783 (1995).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Kingston, R.L., Scopes, R.K. & Baker, E.N. The structure of glucose-fructose oxidoreductase from Zymomonas mobilis: an osmoprotective periplasmic enzyme containing non-dissociable NADP. Structure 4, 1413–1428 (1996).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Varrot, A. et al. NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides. J. Mol. Biol. 346, 423–435 (2005).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Yip, V.L. & Withers, S.G. Family 4 glycosidases carry out efficient hydrolysis of thioglycosides by an alpha,beta-elimination mechanism. Angew. Chem. Int. Edn. Engl. 45, 6179–6182 (2006).

    CAS  Article  Google Scholar 

  27. 27

    Ishikura, H., Arakawa, S., Nakajima, T., Tsuchida, N. & Ishikawa, I. Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme. J. Med. Microbiol. 52, 1101–1107 (2003).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Goldstein, J., Lenny, L., Davies, D. & Voak, D. Further evidence for the presence of A antigen on group B erythrocytes through the use of specific exoglycosidases. Vox Sang. 57, 142–146 (1989).

    CAS  Article  PubMed  Google Scholar 

  29. 29

    Clausen, H., Holmes, E. & Hakomori, S. Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). II. Differential conversion of different H substrates by A1 and A2 enzymes, and type 3 chain H expression in relation to secretor status. J. Biol. Chem. 261, 1388–1392 (1986).

    CAS  PubMed  Google Scholar 

  30. 30

    Davies, G.J., Gloster, T.M. & Henrissat, B. Recent structural insights into the expanding world of carbohydrate-active enzymes. Curr. Opin. Struct. Biol. 15, 637–645 (2005).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Henrissat, B. & Davies, G.J. Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol. 124, 1515–1519 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Zhu, A. et al. Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. Arch. Biochem. Biophys. 327, 324–329 (1996).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Davis, M.O., Hata, D.J., Johnson, S.A., Walker, J.C. & Smith, D.S. Cloning, expression and characterization of a blood group B active recombinant α-D-galactosidase from soybean (Glycine max). Biochem. Mol. Biol. Int. 39, 471–485 (1996).

    CAS  PubMed  Google Scholar 

  34. 34

    Calcutt, M.J., Hsieh, H.Y., Chapman, L.F. & Smith, D.S. Identification, molecular cloning and expression of an α-N-acetylgalactosaminidase gene from Clostridium perfringens. FEMS Microbiol. Lett. 214, 77–80 (2002).

    CAS  PubMed  Google Scholar 

  35. 35

    Henrissat, B. Glycosidase families. Biochem. Soc. Trans. 26, 153–156 (1998).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Rye, C.S. & Withers, S.G. Glycosidase mechanisms. Curr. Opin. Chem. Biol. 4, 573–580 (2000).

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 26 (1992).

  38. 38

    CCP4. The CCP4 Suite. Programs for protein crystallography. Acta Crystallogr D Biol. Crystallogr. 50, 760–763 (1994).

  39. 39

    Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. 40

    Sheldrick, G.M. Macromolecular phasing with SHELXE. Z. Kristallogr. 217, 644–650 (2002).

    CAS  Google Scholar 

  41. 41

    Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Roussel, A. & Cambillau, C. TURBO-FRODO. in Silicon Graphics Geometry Partners Directory (eds. Silicon Graphics Committee) 77–78 (Silicon Graphics, Mountain View, California, 1989).

    Google Scholar 

  43. 43

    Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240–255 (1997).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Hooft, R.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    CAS  Article  PubMed  Google Scholar 

  45. 45

    Technical Manual (AABB, Bethesda, Maryland, USA; 2005).

  46. 46

    Clausen, H., Levery, S.B., McKibbin, J.M. & Hakomori, S. Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes. Biochemistry 24, 3578–3586 (1985).

    CAS  Article  PubMed  Google Scholar 

Download references


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




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.

Ethics declarations

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading