Enzymatic conversion of A and B to O universal type blood

Blood transfusion is one of the most common hospital procedures, with red blood cells (RBCs) being the most requested transfusion product with around 85 million RBC units transfused worldwide per year. This requires correct matching of A, B, AB and O blood group antigens, which are based on distinct sequences of sugars on the surface of the red blood cells. The plasma from individuals of blood group A develop antibodies against B antigen and vice versa. Thus, transfusion with B blood type to A blood type patients potentially results in a massive activation of the immune response by the antibodies against B antigen, RBC destruction and subsequent death. Importantly, the cell surface of O type RBCs -lacking the outermost sugar- displays no antigen and thus the O type can be universally transfused to any patient. A surplus of universal O type blood in blood banks is therefore essential as it can be used for emergency transfusions where the patients blood type is unknown or to overcome potential limited supplies of A and B type blood. Consequently, the option to convert both A and B type red blood cells to universal O type blood using suitable enzymes cleaving the terminal sugar has gained considerable scientific interest over the years. This process involves an enzymatic removal of the A and B antigens on the RBC cell-surfaces, effectively turning the blood into type O. Despite early achievements in converting B to O type, advances towards the more challenging conversion of A to O type blood remained limited due to the very low activity of the enzymes available. Recently, the Withers group at the University of British Columbia in Vancouver discovered an efficient two-enzyme system derived from bacteria in the human gut that effected the conversion of A to O RBCs. The method was based on isolated bacterial genomes from human feces and testing thousands of enzymes in a reaction system with synthetic fluorogenic models resembling the A and B antigens. These fluorogenic substrates consisted of only the terminal sugar of A or B antigens and produced a fluorescent signal when cleaved, allowing for the identification of enzymes with potent activity. Even though this approach was successful in finding new enzymes, it is likely that additional enzymes might exist that operate on the entire sequence of sugars constituting the A and B antigens. As the synthetic access to such substrates is limited due to the complexity of the sugar sequences, much smaller reaction volumes for testing would be needed. The project thus aims to miniaturize the reaction system by using droplet-based microfluidic technologies, defined by pico- to femtoliter sized of water- in-oil droplets allowing to test 108 enzymes per day. Also, broader sources of bacteria will be screened, increasing the chances of discovering new efficient enzymes that convert both A and B type red blood cells to O type blood and providing universal flexibility in blood supply for transfusion in the future.

Correct matching of A, B, AB and O blood group antigens, which are based on distinct sequences of sugars on the surface of the red blood cells is essential as the plasma from individuals of blood group A develop antibodies against B antigen and vice versa. Thus, transfusion with B blood type to A blood type patients potentially results in a massive activation of the immune response by the antibodies against B antigen, RBC destruction and subsequent death. Importantly, the cell surface of O type RBCs -lacking the outermost sugar- displays no antigen and thus the O type can be universally transfused to any patient. A surplus of universal O type blood in blood banks is therefore essential as it can be used for emergency transfusions where the patient's blood type is unknown or to overcome potential limited supplies of A and B type blood. Consequently, the option to convert both A and B type red blood cells to universal O type blood using suitable enzymes cleaving the terminal sugar has gained considerable scientific interest over the years. This process involves an enzymatic removal of the A and B antigens on the RBC cell-surfaces, effectively turning the blood into type O. Recently, the Withers group at the University of British Columbia in Vancouver discovered an efficient two-enzyme system derived from bacteria in the human gut that effected the conversion of A to O RBCs. The method was based on isolated bacterial genomes from human feces and testing thousands of enzymes in a reaction system with synthetic fluorogenic models resembling the A and B antigens. These fluorogenic substrates consisted of only the terminal sugar of A or B antigens and produced a fluorescent signal when cleaved, allowing for the identification of enzymes with potent activity. Even though this approach was successful in finding new enzymes, it is likely that additional enzymes might exist that operate on the entire sequence of sugars constituting the A and B antigens. As the synthetic access to such substrates is limited due to the complexity of the sugar sequences, much smaller reaction volumes for testing would be needed. Thus, I developed a robust system for ultrahigh-throughput droplet-based microfluidic screening to miniaturize the reaction system, defined by pico- to femtoliter sized of water-in-oil droplets allowing to test 9.106 enzymes per hour for B antigen cleaving enzymes. Through the functional metagenomic screening of the human gut microbiome library, after validation and characterization of the hits, a new candidate enzyme for the conversion of B to O RBCs was discovered. Preliminary results on engineering the new B cleaving enzyme showed promise for generating O type blood for patient transfusions.