The ABO blood system groups red blood cells (RBCs) based on the presence or absence of the A and/or B glycan antigens (Fig. 1). This system is paramount for matching donor-blood with recipients to avoid potentially lethal immune reactions between donor RBCs and antibodies in recipients’ plasmas, which recognize non-self A or B antigens. Hospitals and blood centres must maintain stocks of all four ABO blood groups to meet the large demand for patients undergoing medical procedures with intensive blood needs. Group O RBCs only express H-antigens, the precursors of both the A and B antigens. Group O blood can be safely transfused to any patients in medical emergencies, paediatric transfusions, and for uncertain patient blood types, resulting in common shortages of this group. The Enzymatic Conversion of A and B antigens on RBCs to the universal group O (ECO blood concept) offers an attractive solution to these challenges. Since this concept was pioneered by Goldstein et al. in 1982¹, several enzymes with increasing efficiencies towards the A and B antigens have been reported. However, unexplained incompatibility issues between ECO RBCs and recipients have hindered clinical implementation of the method^2,3

A coincidental encounter would eventually lead to my PhD project as a part of a cross-disciplinary collaboration between the labs of Prof. Maher Abou Hachem and Prof. J. Preben Morth at the Technical University of Denmark (DTU) and Prof. Martin L. Olsson at Lund University (LU) in Sweden to address the ‘missing link’ for universal ECO blood.

Before my PhD project started, Prof. Abou Hachem, an expert in the utilization of complex glycans by the human gut microbiota, had a project focusing on understanding the molecular apparatus and strategy of mucin degradation by the dedicated human gut mucin degrader, Akkermansia muciniphila. During a Nordic glycobiology meeting in Lund, a previous guest PhD student in our lab at DTU, Bashar Shuoker, met Dr. Jennifer Ricci Hagman, at the time a PhD student in Prof. Olsson’s lab at LU. Jennifer had recently discovered a new carbohydrate extension of the B antigen, named ExtB (Fig. 1), which she gave a talk about at the meeting. Based on this, a few A. muciniphila enzymes were supplied to Jennifer to evaluate the conversion of the new ExtB structure. Prof. Olsson, who is a specialist in transfusion medicine, has previously worked on the ECO blood concept, and, together with Prof. Henrik Clausen (University of Copenhagen) reported the first microbial A and B antigen-converting enzymes⁴. Besides the extended B antigen, three extensions of the A antigen were previously reported by Clausen and colleagues in the mid-1980s^5–7. Yet, before our work, no one had considered the clinical importance of the A extensions, and consequently none of the extended structures had been targeted for enzymatic conversion.

Based on the successful removal of ExtB by A. muciniphila enzymes and the activity of an α-fucosidase from the same bacterium to remove the H type 3 antigen, one of the extended A structures⁸, my PhD project was born. This was the fruit of the collaboration between my direct mentor Prof. Abou Hachem and Prof. Olsson, where we set the quest to discover enzymes against all extended blood structures to evaluate their impact on ECO-blood compatibility. Our hypothesis was that the adaptation of A. muciniphila to target mucin O-glycans, which display the A and B antigens, may extend to efficient conversion of all ABO antigens on RBCs as these are constantly available substrates in the human gut (Fig. 1).

In 2020, my PhD project started, and keeping the focus on A. muciniphila, in total, 24 enzyme candidates from seven different Carbohydrate Active enZYme (CAZy) families (http://www.cazy.org/Home.html) were produced and screened for activity against the A, B and extended structures. Ultimately, seven enzymes were identified, collectively targeting both the A and B antigens, besides all their known extensions. These enzymes proved to be remarkably efficient, allowing conversions at lower enzyme concentrations and shorter reaction time compared to previous studies. Subsequently, my co-supervisor, Prof. J. Preben Morth, taught me the art of X-ray crystallography. Despite the defiance of many A. muciniphila enzymes to crystallisation, some perseverance and luck led to the determination of the first modular GH110 α-galactosidase and a GH20 β-N-acetylhexosaminidase (Fig. 1). Together, these enzymes converted both the B and the extended B structures on RBCs. Analyses of these two structures revealed shallow active sites flanked by rings of positive charges, which may contribute to enzyme accessibility to the heavily negatively charged mucin and RBC surface glycans. Moreover, both enzymes possess putative carbohydrate binding modules, which may contribute to their efficiency on conjugated glycans, due to divalent binding by both catalytic and ancillary modules.

Excited by these results, we finally set out to answer the key question of whether removing extended A and B antigen structures influences compatibility of ECO blood. Dr. Linn Stenfelt, a Postdoc in the team, performed 1522 crossmatch reactions between RBCs and plasma samples to answer this question in the lab of Prof. Olsson. Remarkably, the ECO RBCs treated for the removal of both A and B, as well as their extended structures, showed a significant improvement in compatibility as compared to conventional ECO RBCs treated with only A or B antigen converting enzymes. 

After more than three years of hard work and thanks to the efficiency of A. muciniphila enzymes and the joint expertise of involved groups, we provided the first evidence that the removal of extended A and B antigens improves ECO blood compatibility, thereby reviving hope for truly ABO-universal blood.

Refs.

1. Goldstein, J. et al.  Group B erythrocytes enzymatically converted to group O survive normally in A , B , and O individuals.  Science 215, 168–170 (1982).
2. 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).
3. Gao, H. W. et al. Evaluation of group A1B erythrocytes converted to type as group O: Studies of markers of function and compatibility. Blood Transfus. 14, 168–174 (2016).
4. Liu, Q. P. et al. Bacterial glycosidases for the production of universal red blood cells. Nat. Biotechnol. 25, 454–464 (2007).
5. Clausen, H. et al.  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).
6. Clausen, H. et al.  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).
7. Clausen, H. et al. 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. U. S. A. 82, 1199–1203 (1985).
8. Shuoker, B. et al. Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria. Nat. Commun. 14, 1833 (2023).