Enzymatic Conversion of Red Blood Cell Antigens to the Universal Blood Group O

Every year, millions of blood transfusion are performed worldwide. Miss-matching of donor blood type to the recipient can result in hemolytic transfusion reactions, which can be deadly. These reactions occur when recipient plasma antibodies recognize non-self ABO antigens on the surfaces of donor Red Blood Cells (RBCs). Therefore, correct matching of donor and recipient ABO blood group is essential for safe transfusions. Increasing blood demand for the ageing populations, and limited donor numbers presents a blood supply challenge. An attractive solution encompasses the use of exo-glycosidases to convert the A and B antigens into the universal type, O, in order to maintain stocks of only universal blood. This would alleviate issues related to keeping stocks of different blood groups and minimize spill of unused units. Efficient enzymes targeting the A and B antigens have been reported, but unexplained incompatibility issues between recipient plasma and the Enzyme Converted O (ECO) RBCs, raise questions about the safe implementation of the method. Strikingly, the clinical significance of four extensions of type A and B antigens on transfusion compatibility has remained unexplored.

In this thesis, we discover and employ two cocktails of 5 enzymes, collectively targeting both the canonical and the extended A and B antigens. We show for the first time that targeting the extended A and B antigens significantly decreases the level and severity of positive crossmatches, as a proxy for transfusion compatibility, between ECO RBCs and recipient plasma, in comparison to only targeting the canonical A and B antigens. We highlight the complex nature of the glycan phenotype that decorate surface glycoconjugates of human RBCs and the matching complexity and diversity in plasma antibody repertoires. This work contributes to changing the current paradigm in regards to what constitutes universal donor blood. Additionally, we provide evidence for the evolutionary adaptation of enzymes from the mucin-associated human gut microbiota symbiont, Akkermansia muciniphila, to efficiently target human glycoconjugates. We report the crystal structures of three novel enzymes that exhibit high efficacy in the conversion of ABO antigens on RBCs, allowing us to dissect enzymatic signatures that correlate with activity on RBCs. Herein, we report the first structure of a blood-active GH110 with an ancillary CBM-51 domain. We also report a structure of a GH20 β-N-acetylhexosaminidase appended to a novel putative CBM with GalNAc binding activity. Altogether, we provide novel insights into structural features that correlate to high efficacy on RBC surface glycoconjugates and unravel the hitherto unknown importance of targeting the extended A and B antigens. Here, we set the foundation for a new generation of enzymes for truly ABO-universal enzymatically generated blood.