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Using enzymes to convert all donated blood into universal type O
The 4 main blood types and their importance
An individual’s blood type is most commonly determined by the presence or absence of different types of antigens on their red blood cells (RBCs). The 3 antigens that determine blood group are antigens A, B and H, which are found attached to the oligosaccharides (short carbohydrate chains) projecting from glycoproteins and glycolipids in the plasma membrane of RBCs. If a RBC has antigen A attached to it, the blood is type A, if there is antigen B attached, the blood type is B and if both antigens A and B are present the blood type is AB. When neither A or B are attached to the RBC, only antigen H, the blood type is referred to as O. The plasma of blood will contain anti-A and/or anti-B antibodies if the corresponding antigen is absent, for example type A blood contains anti-B, but not anti-A antibodies in the plasma 1. There is no anti-H as all RBCs have some H antigens on them (as well as antigens A and B in types A, B and AB).

left80645Figure 1 4 showing which blood types can safely donate to and receive blood from one another.

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00Figure 1 4 showing which blood types can safely donate to and receive blood from one another.

When an individual receives a blood transfusion, they must not receive a blood type containing antigens matching the antibodies in their blood plasma. If the blood transfused is incompatible, the recipient’s antibodies will attack the foreign antigens and destroy the donor RBCs. This not only defeats the purpose of a transfusion, but also causes symptoms such as nausea, headaches, muscle and joint pain and breathing difficulties 2 and in 10 to 50% of cases results in death 3. Figure 1 shows the ABO compatibility of donors and recipients, highlighting that type O blood can be donated to everyone and is therefore a ‘universal donor type’. As a result, type O blood is constantly needed in hospitals, especially within emergency medicine because the blood type of a patient is often not immediately known. This high demand for universal blood has resulted in several studies attempting to convert types A, B and AB blood into type O.

Composition of antigens A, B and H
Antigens are composed of monosaccharides (carbohydrate units). As seen in figure 2, antigen H is a disaccharide composed of galactose (Gal) and fucose (Fuc) connected by an ?-1,2-link. Antigens A and B also have the same structure, but with an additional saccharide unit attached at the end. Antigen A has the terminal N-acetylgalactosamine (GalNAc) joined to Gal by an ?-1,3-link, whereas antigen B has the terminal Gal also joined by an ?-1,3-link 5. Antigen H is the precursor of A and B, all antigens on RBCs start off as H. Then, depending on the individuals genotype, a terminal monosaccharide (GalNAc or Gal) may be added to the chain. People with type A blood have an allele coding for the enzyme GalNAc-transferase which catalyses the addition of GalNAc to H antigens, while those with type B blood have an allele that codes for Gal-transferase, which catalyses the addition of Gal. The type O allele codes for an inactive enzyme meaning H antigens are not converted, and individuals that are type AB produce both GalNAc-transferase and Gal-transferase 11.

2136140145415Figure 2 5 showing the molecular composition of A, B and H antigens.

00Figure 2 5 showing the molecular composition of A, B and H antigens.

The use of enzymes to convert blood type
Numerous studies have attempted altering the structure of antigens A and B, to generate type O RBCs. A method of doing so that has been explored for over 3 decades, is the removal of the terminal saccharide of antigens A and B, leaving the underlying H. This method has been carried out with glycosidases (enzymes that catalyse the hydrolysis (breaking) of glycosidic bonds) 6 which are essentially doing the opposite of GalNAc and Gal-transferase.

In the early 1980’s Jack Goldstein lead a study using ?-galactosidase, an exoglycosidase (exoglycosidases are enzymes that break the bond linking the terminal monosaccharide and the rest of the saccharide chain). ?-galactosidase, which is found in green coffee beans, converted B antigens into H antigens without impairing the function of RBC. These ‘enzymes converted O’ (ECO) RBCs were successfully transfused into humans, functioning normally in type A and O recipients 7. Although the study established that enzymes could be used to generate the highly desirable type O blood, unsustainably large quantities of ?-galactosidase was required with 1-2 grams of enzyme needed to convert each unit of RBCs 1. Furthermore, the attempts to convert type A blood were largely unsuccessful, suggesting that type A and type AB blood (which 37% of individuals worldwide have 8) blood could not be converted to universal blood.

N-acetylgalactosamine105651118639There are 2 types of A antigen, A1 and A2, which is why converting type A RBCs has been more challenging. A1 antigens have 2 repeats of Fuc-?-1,2-Gal-?-1,3-GalNAc in their structure, whereas A2 have just one. Additionally, A1 RBCs have around 5 times more antigens covering their surface. In previous research, the use of the enzyme ?-N-acetylgalactosaminidase to convert A antigens was ineffectual, only partially converting the RBC leaving it with several A antigens.

-122062565463Figure 3 10 comparing the structure of A1, A2, B ; H antigens.

00Figure 3 10 comparing the structure of A1, A2, B ; H antigens.

A study published in 2007 describes the discovery of bacterial enzymes that could convert A antigens (and B antigens more efficiently). The research hypothesised that ?-N-acetylgalactosaminidases that had been used in the past were not specific to the A antigen, due to being discovered through testing on simple substrates. So, they screened around 2500 bacteria and fungi to find exoglycosidases with complementary active sites to the complex A antigen, and better suited ?-galactosidase. Enzymes produced by Bacteroides fragilis which remove the terminal of B antigens, and Elizabethkingia meningosepticum which can convert both A1 and A2 antigens were identified. The genes coding for the ?-galactosidase and ?-N-acetylgalactosaminidase were cloned, and expressed in in E. coli with a yield of 1 g/l of recombinant ?-N-acetylgalactosaminidase and 0.5 g/l of recombinant ?-galactosidase. These enzymes were then used to create A-ECO and B-ECO RBCs, which were converted efficiently in all ;250 donor units. Around 60 mg of ?-N-acetylgalactosaminidase was required to hydrolyse 1 unit (approximately 200 ml) of A1 RBCs, and just 15 mg to convert a unit of A2 RBCs. The hydrolysis of type B RBCs only needed 2 mg of ?-galactosidase per unit, which is 1000 times less than was used by Goldstein. These enzymes were also able to convert AB type RBCs, hydrolysing antigens A and B simultaneously, as they were selected during the screening so that they would function efficiently in the same environment 1.

51092106459745158105155357722447255651500A recent study offered a different approach, using an endoglycosidase to remove the whole antigen from RBCs, to leave them antigen-less which cannot trigger an immune reaction. Streptococcus pneumoniae, a bacterium, secretes endo-?-galactosidase (an EABase) which can efficiently hydrolyse the Gal?-1,4-GlcNAc linkage connecting the majority of antigens to the RBC’s oligosaccharides, this is referred to as a type 2 core chain. There are also type 1 chains which have a Gal?-1,3-GlcNAc linkage, and type 3 and 4 chains which have Gal?-1,3-GalNAc linkages, and are attached only to glycolipids. The bonds between antigens and type 1, 3 and 4 core chains are hydrolysed extremely slowly by the EABase making the process of converting RBCs ineffective.

225044043457Figure 4 10 showing the different oligosaccharides projecting from RBCs.

0Figure 4 10 showing the different oligosaccharides projecting from RBCs.

The researchers have been developing an EABase with advantageous mutations through directed evolution. They then isolated and replicated a mutant enzyme with enhanced activity towards type 1 chains. The mutant is able to hydrolyse the bond between the antigens and type 1 chains 170 times faster while having the same catalytic effect on the breaking of type 2 chains. Although there is currently no increase in activity towards type 3 and 4 chains, the study has shown it is possible to engineer an EABase that is more efficient and intents to develop an enzyme that can remove antigens from type 1, 2, 3 and 4 chains 10.

Concluding remarks
Although ECO RBCs are currently not available for use, the ongoing research into glycosidases is promising. The obstacle to overcome is not finding a way to convert A, B and AB RBCs, but instead a method that is cost effective. Progress has been made since the 1980’s when trials first began, with 1000 times less enzyme being required to convert type B blood, and research is ongoing.

However, there is the issue of the Rh factor which is not being accounted for at the present. If an individual has D antigens on their RBCs they are said to be Rh positive (Rh+), and if antigen D is absent they are Rh negative (Rh-). Rh- blood contains anti-D, an antibody, meaning that Rh+ RBCs cannot be transfused to someone that is Rh negative because an immune response would be triggered, doing more harm than good. Despite this, the studies so far have proven that it is possible to alter or remove antigens from RBCs, so it is very likely that methods to convert Rh+ blood to Rh- is possible 11.Bibliography
1 Liu, Q P., et al. Bacterial glycosidases for the production of universal red blood cells. Nature Publishing Group. 2007.
2 Colledge, H., Boskey, E. ABO Incompatibility Reaction. Date published unknown, medically reviewed by D Murrell on 02/05/2017.
3 Harrahill, M., Boshkov, L. Journal of Emergency Nursing, ABO incompatible transfusion: What you need to know. Elsevier. August 2000.
4 Author unknown. Blood Types. American Red Cross. Date published unknown.
5 Creator and publication date unknown.
6 Olsson, M., et al. Universal red blood cells—enzymatic conversion of blood group A and B antigens. Elsevier. February 2004.
7 Goldstein, J. Conversion of ABO blood groups. Elsevier. June 1989.
8 Author Unknown. ABO Blood Types Distribution. Aqua for Balance. Date published unknown.
9 Kruskall, M., et al. Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. November 2000.
10 Kwan, D., et al. Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution. ACS publications. April 2015.
11 Daniels, G., Bromilow, I. Essential Guide to Blood Groups. John Wiley ; Sons. 2014.

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