Part:BBa_K1391104:Experience

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Applications of BBa_K1391104

B-cell receptors (BCRs) are multiprotein immune receptors found exclusively on the surface of B cells. The BCR multiprotein complex is centered around a membrane-bound IgM antibody. When the antibody binds to an extracellular antigen, receptors dimerize resulting in the phosphorylation of the intracellular tails of CD79A and CD79B by the tyrosine-protein kinase Lyn. In response, another cofactor, spleen tyrosine kinase (Syk), is recruited to the receptor and phosphorylated, initiating a signalling cascade that results in the proliferation of the activated B cells. This receptor is important in clonal selection of B cells during human immune response.

For this project, we engineered a BCR to respond to beta-amyloid plaques, the hallmark of Alzheimer's disease. This task was accomplished by using a beta-amyloid specific variable region [derived from Gantenerumab] in the membrane-bound IgM antibody. Our design was based on that of the Tango system [1], which capitalizes on the interaction between TEV protease (TEVp) and its cleavage site (TCS), an amino acid sequence for which the protease has a high affinity. A TEV cleavage site was used to link a transcriptional activator (Gal4VP16) to the intracellular tails of BCR accessory proteins CD79A and CD79B, and the receptor’s cofactor, Syk, was fused to TEV protease. Thus, when the modified receptor activates upon binding its antigen, beta-amyloid, Syk-TEVp fusion protein is recruited, bringing TEVp in close proximity to its cleavage site. This proximity of TEVp to TCS results in the cleavage of the transcriptional activator from the receptor releasing it to activate downstream gene circuits.

The engineered BCR we developed binds beta amyloid with high specificity and releases a transcriptional activator upon binding, making it an extremely valuable tool in the detection of Alzheimer’s Disease. More importantly, the IgM antibody that determines what the receptor binds can be easily swapped out as can the transcription factor the receptor releases. This means that the receptor we developed can bind to any molecule that an antibody can be produced against and it can release any transcription factor in response to the binding of the target molecule. This modularity allows this receptor to be generalized to almost any extracellular sensing making it an invaluable part of any synthetic biologists toolkit.

This is a protein found natively in B cells. CD79 is a heterodimer of CD79A and CD79B that is part of signal transduction in the B-cell receptor. The IgM Heavy chain, IgM light chain, CD79A subunit, and CD79B subunit require processing in the ER and must all be expressed in order to membrane localize instead of being retained in the ER by quality checking mechanisms. Binding to the antigen (beta-amyloid) causes two BCR complexes to dimerize. This allowsLyn to phosphorylate the CD79 heterodimer which causes the recruitment of Syk. As a warning, recruited Syk can cause cross talk with native cell signaling pathways as can other proteins. Care must be taken to check protein interactions and point mutate Syk if needed. We attach a protease (TEV protease) to Syk and a transcriptional activator (Gal4-VP16) to CD79 using a cleavage site (TEV cleavage site). When The BCR is activated by our antigen, Syk-TEVp cleaves the cleavage sit and releases Gal4-VP16 which can activate a synthetic system.

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