Wet & Dry labCommunication
Relevance and ethicsRhesus protein systemProtein structure and complex predictionFluidic dynamics and system optimizationRelevance and ethicsRhesus protein systemProtein structure and complex predictionFluidic dynamics and system optimizationThe team embarked on this project with a plethora of questions. To effectively navigate our project's intricacies, we diligently delved into each and every one of these inquiries. Our initial focus was on gaining a deep understanding of the ethical considerations and established norms pertaining to blood donations. We proactively sought insights from industry leaders and experts to ensure that our project's relevance and alignment with ethical standards were well-established. Additionally, we encountered technical challenges that required demanded the guidance of seasoned experts in the field. By rigorously addressing these ethical, pertinence, and technical questions, we fortified the foundation upon which our Blood Cell Barber Shop project stands.
At the beginning of the year, with our project freshly decided, we set out to find out more about this field from Héma-Québec, the local nonprofit organization in charge of blood donations and the distribution of blood products here in the province of Québec.
First, we wanted to know more about the need for such a project. Héma-Québec informed us that enzymatic removal of antigens was tried several years ago with poor results. The reasons were the low efficiency of the enzymes used, the need for a matrix, and the requirement of cofactors in the hydrolysis reaction. They did however confirm that there is indeed a need for a supplementary supply of O- type blood in high-demand periods such as summer and winter. Furthermore, this accentuates the need for personalised blood transfusion, especially, for people with recurrent need of transfusion.
Knowing the extensive variety of different antigen groups, they informed us that it would be wiser to tackle specifically ABO type and Rh type antigens, as they are by far the most immunogenic. These groups are followed by Kell type antigens, which are only detected in roughly 2% of the population. Additionally, they warned us that some glycohydrolases could create unusual epitopes after enzymatic degradation that could lead to some immune response. Moreover, they advised us to keep the working condition of our treatment close to physiological conditions (pH 7,40 ± 0,05) and if possible at a temperature near 4 °C.
In consideration of the past trials on enzymatic removal of antigens, we set out to identify and use more efficient enzymes. Previous degradation of A antigen (review; Taipei 2021) relying on the α-N-acetylgalactosaminidase from Elizabethkingia meningoseptica suffered from the low efficiency of the enzyme, the need for a dextran matrix to bring closer the enzyme to the blood cells, and NADH for the reaction turnover. In that regard, we found that a bipartite degradation of A antigen using enzymes from Flavonifractor plautii showed higher efficiency of antigen removal and, most particularly, without the need of a matrix and cofactors. As for degradation of B antigen, we decided to use enzymes from Bacteroides fragilis
Adding the enzymes directly in blood procured from donations was also one of their main concerns. They recommended that we find a way to ensure that the final blood product would be free of our enzymes. Instead of adding our enzymes to blood donation bags, we thought of making the blood pass through a tubular system containing the fixated enzymes on the inner layer. The resulting blood product would be free of any enzymes and ABO-type antigens. We thus redirected our efforts into instead developing a fluidic system with our enzymes coating the inner layer of the system.
Additionally, Héma-Québec also informed us that the targeting of the Rh antigen was a main focus for this type of research on blood donations, convincing us to work on both the ABO and the Rh systems. We were reluctant at first to work with Rh, as little information in regard to its structure was available and its immunogenicity does not arise from post-translational glycosylation like for the ABO system. We considered directed mutagenesis in somatic cells or even capping to remove or hide the recognised residues.
This meeting with our local experts, Héma-Québec, caused us to change the primary focus of the project in favor of the development of an innovative fluidic system geared toward the enzymatic removal of antigens.
With our new design for the project based on the comments we got from Héma-Québec, we reached out to a professor that acted as a Principal Investigator for the team up to 2022: Steve Charette. We discussed with him our new strategy of degrading the specific glycosylation corresponding respectively to A and B antigens and our second strategy of hiding the Rh antigens via mutagenesis or capping.
On the subject of Rh antigens, he informed us that he worked in the past on Rh-like proteins in amoeba. Those proteins were involved with the contractile vacuole. Out of curiosity, he looked at the similarity of sequence and the phylogeny between the amoeba Rh-like proteins and the human RhD protein isoform 1, which incited us to integrate the human Rh homologues. We then discussed with him the results, which briefly showed RhAG is more closely related to RhBG and RhCG which show a similarity to amoeba Rh-like proteins and that RhD and RhCE are highly identical. These results are consistent with previous reports suggesting they arise from genome duplication. Consequently, he recommended that we expand the phylogeny analyses to other Rh-like proteins, as it would be interesting to follow its evolution.
Surprisingly, the discussions we had with Steve Charette with the purpose of getting a second opinion on our project led to the addition of significant content to it. It brought us to investigate the relation of known Rh-like proteins across different species to better understand the evolution of such proteins. The preliminary phylogeny we made based on those discussions led to identification of important similarities of different Rh-like proteins that we would later use for structural modelling. Furthermore, we followed his recommendation of expanding our phylogeny to other and more distanced Rh-like proteins. In that regard, it is currently ongoing.
This overall added a new dimension in our project to expand the current knowledge on Rh-like proteins.
Our meetings with the experts from Héma-Québec established the need for a lot of work to be done on the Rh protein complex and potential methods to treat it for blood transfusions. After reviewing available literature on the subject, we determined that the RhD subunit was the most immunogenic, and therefore the best candidate for eventual mutations or protein capping. We started to look for the published 3D structures of RhD, with and without its surrounding protein complex. To our surprise, there was no published tri-dimensional structure of RhD, alone or in a complex.We decided to determine that structure using bioinformatics methods.
The first expert we consulted on this subject was Stéphane Gagné, a local researcher with great expertise in protein structure prediction. He helped us determine what had already been done in terms of modeling and assembly of the Rh protein complex. We asked him what would be the best strategy to model the different Rh proteins and its complex, as well as advice on specific bioinformatics tools to use. He suggested a de novo approach with elements of protein homology. Additionally, he informed us on common quality control measures and working parameters, which we integrated into our structure prediction methods. We also discussed with him our possible strategy to hide our substitute immunogen segments via directed mutagenesis
The second expert we consulted was Patrick Lagüe, a local researcher with extensive expertise in molecular dynamics, with a specialization in proteins and membranes. We presented him the results of our protein structure predictions so he could advise us on the subsequent steps to take. He confirmed that our predictions were high quality. We consulted him on ways to obtain information about the stability of the predicted protein complexes. He told us to contact François Rouleau to estimate their folding energy using FoldX. He suggested we perform molecular dynamics for the complexes with the best folding energy and most interesting biological implications.
François Rouleau is a PhD candidate and former iGEM member who was recommended to us for advice on the use of FoldX and ways to compute subunit affinities within the Rh complex. He helped us determine which functions to use within the FoldX suite, how to perform predictions that reflect biological reality, and how to evaluate the results’ quality. He also provided additional confirmation of the high quality of the protein models produced until that point.
Our discussions with Stéphane Gagné helped develop our de novo approach. We used a combination of Blast, varied secondary structure prediction tools and AlphaFold2 Multimer. Those recommendations led us to the 3-dimensional simulation of each monomer, dimer, trimer and specific tetramer combinations of the human Rh complex. Such simulations represent a significant contribution to our understanding of the Rh complex for further analysis, but also they confer significant knowledge advance in the field.
The FoldX calculations, guided by François Rouleau and Patrick Lagüe, determined which protein complex predictions should be used for subsequent steps and any implementation. It also confirmed our previous assumptions from the complex structure prediction step, that certain combinations were much more unstable and biologically improbable. This allowed us to then identify the most probable biological conformation of the Rh complex, 3RhAG, 2RhAG-1RhCE and 2RhAG-1RhD, for further investigation of immunogenicity of exposed segments.
The molecular dynamics were then performed using Charmm-gui, using a protocol suggested to us and modified by Patrick Lagüe. His advice was most helpful in designing the asymmetrical lipid membrane within the constraints of Charmm-gui and the OPM-PPM3 servers. This enabled us to visualise the aforementioned exposed segments of the Rh complex when integrated in a human plasma membrane. Although the current version of this modelling is still in its preliminary form, we are currently improving the modelling based on their recommendations.
In order to better implement our project, we thought that having a modular system with fixed enzymes would enable a higher enzymatic efficiency. For that purpose, we reached out to Hossein Hassanzadeh, whom we had collaborated with in past iGEM projects.
We discussed with him our implementation, as he is an expert in fluidic systems. Although such systems would be quite a challenge to simulate, it would be intriguing to investigate and could lead to more concrete therapeutic avenues. So we set out to identify the different parameters to work with, in order to obtain a preliminary model. Considering the sheer amount of parameters and combinations, we concluded with Hossein that a Design of experiments combined with multiple fluid dynamics simulations could help us identify optimal parameters. In that regard, with the help of Hossein who generated the different model geometry projects, we simulated the flow of blood in different systems.
This helped to concretise our idea of implementing our enzymes into a fluidic system for blood antigen enzymatic removal. Hossein had a huge impact on defining the most relevant parameters to consider as variables, leaving the other parameters as constant depending on the selected variables.
In regard to the models, we simplified the simulation to 2-dimensional models in accordance with his suggestions to speed up the computation and to preliminarily identify the configuration area, leading to better results.
Additionally, as we wanted to investigate the efficiency of our different enzymes, Hossein suggested that simulation of enzymatic reaction could be coupled to the fluid dynamics simulation. Such analysis is still ongoing, as such simulation involves large computation resources.
In consideration of the need for universal and personalised blood transfusion, we designed our project to respond to such needs according to the recommendation of experts. Our project has the potential to relieve the disadvantages of past trials on enzymatic removal of ABO-type antigens and to expand the knowledge on human Rh complex structure.
As a result of the implication and suggestion of experts in their respective fields, we reshaped our project from the conceptualisation to specific modelling steps, to better fit the needs and to optimise our system for a realistic and efficient use in the clinical field.