Strategy for the characterization of our BioBricks

As described in the sections enzymes, affinity tags and synthetic peptides our system consists of several components. The general functionality of most components has already been shown, e.g. ability of the glycosidase NAGA to convert the erythrocytes of the A type to 0 [1] or the usability of the SNAP-Tag for the immobilizing of proteins [2]. However they have never been used in this context. and we need three events to take place to get a functional system:

1. Covalent binding of the peptides and the SNAP-substrate to the solid support
2. Covalent binding of the fusion proteins to the solid support
3. Efficient conversion of erythrocytes

Consequently we need to verify every single step to ensure that our approach might be successful and identify components which need optimization. Furthermore we can prove the function of the single domains of our fusion proteins independently: The glycosidases on the one hand and the immobilization tags on the other. These will be available as separate BioBricks and can be used in a independently by future teams. In the following we will provide a concept that enables us to answer these questions.

1. Do our fusion proteins show glycosidase activity? (Enzyme and substrate in solution)

   First we need to prove that the fusion proteins consisting of glycosidase domain and immobilization-tag are properly folded and show enzyme activity. Therefore we would use soluble polysaccharides mimicking the blood antigens present on the erythrocyte surface. These are commercially available (e.g. by Elicityl). There are several techniques on hand to monitor the cleavage of these substrates and the formation of the corresponding monosaccharides (galactose and/or N-acetylgalactosamine) e.g. thin-layer chromatography (TLC)[3], high-performance liquid chromatography (HPLC)[4] or NMR. Liu et al. reported that smaller substrate analogues such as GalNAcalpha-pNPP which are typically used to assay glycosidase activity spectrometrically might fail in detecting the activity of NAGA.[1]

FIGURE:Characterization1 Scheme illustrating the detection of glycosidase activity by TLC and HPLC of a hexasaccharide.
2. Can we efficiently convert erythrocytes to type 0? (Enzyme in solution, substrate on solid phase)

   However the isolated oligosaccharides can only serve as a rough model. If we regard the red blood cells as solid particles with an average size of 6-8uM we have to consider that the diffusion of our substrates (the oligosaccharide on the cell surface) is limited. Furthermore other biomolecules on the cell surface might inhibit the reaction. There are two possibilities to monitor the conversion of red blood cells to blood group 0. First we can use do cross-matching to observe whether our converted erythrocytes show hemagglutination if they are mixed with blood of the group 0 or the Bombay type in the case of the EABase. Second fluorescently labelled monoclonal antibodies against the blood antigens are commercially available and can be used to analyse the erythrocytes in FACS-analysis.

FIGURE:Characterization2 Scheme illustrating the detection of blood antigens on red blood cells by antibody detection and FACS.
3. Do our enzymes retain activity upon immobilization (Enzyme on solid phase, substrate in solution)

   The loading of the SulfoLink resin with our peptides can be roughly estimated by the fluorescence of the carboxyfluorescein coupled to the peptides. We plan to quantify the binding of the fusion proteins to the matrix by comparing the protein concentration in solution before and after incubation with the beads. At this step we can demonstrate the functionality of our immobilization domains.

   FIGURE:Characterization3 Loading of the SulphoLink resin with fluorescently labelled peptides and fusion protein.

   Subsequently we can perform a glycosidase assay using isolated oligosaccharides as detailed above. By normalizing the activity to the amount of bound protein and comparison with the enzyme in solution we can investigate whether immobilization has any impact on enzyme activity.

4. Do our immobilized enzymes (Enzyme on solid phase, substrate on solid phase) Again we cannot transfer the results obtained from the soluble substrate analogues to the red blood cells. There are two effects which might reduce the activity of our enzymes acting on the red blood cells. First protein which has not bound to the surface but diffused into the beads will not be available for the reaction as the mesh of the beads is too small to allow the erythrocytes to enter the bead. Furthermore both components enzyme and substrate are now bound to a solid phase reducing their degrees of freedom. The efficiency of red blood cell conversion can be monitored as described above. This is the final test our system has to pass!

[1] Liu, Q.P., Sulzenbacher, G., Yuan, H., Bennett, E.P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S.B., White, T., Neveu, J.M., Lane, W.S., Bourne, Y., Olsson, M.L., Henrissat, B. & Clausen, H. (2007). Bacterial glycosidases for the production of universal red blood cells. Nature biotechnology 25, 454-464.

[2] Engin, S., Fichtner D., Wedlich D., Fruk L. (2013). SNAP-tag as a tool for surface immobilization. Curr. Pharm. Des. 19, 5443-5448

[3] Anderson K.M., Ashida H., Maskos K., Dell A., Li S.-C., Li Y.-T. (2005). A Clostridial Endo-beta-galactosidase That Cleaves Both Blood Group A and B Glycotopes. J. Biol. Chem., 280:7720-7728

[4] Hayes B.K., Varki A. (1995). Fractionation and Analysis of Neutral Oligosaccharides by HPLC. Current Protocols in Molecular Biology, 17.21.9-17.13