Part:BBa_K4586000

cyclic citrullinated peptide (CCP1)

Part Description

This part codes for cyclic citrullinated vimentin which is a synthetic peptide designed to detect and measure specific antibodies that are elevated in autoimmune diseases, especially rheumatoid arthritis.

Usage

This part is implicated within our design in two units: first, the external domain (sensor) of our SYNNOTCH receptor that binds to the BCR of autoreactive B-cells that secrete ACPA to assist in identifying the presence of them within the media in order to tune and control the level of expression of our therapeutic agent according to the condition of the patient as reflected by the amount of the autoreactive B-cells. Second, it is expressed on the surface of exosomes conjugated to lamp2b to direct them toward auto-reactive B-cells that secrete ACPA and aid in their fusion with BCR.Thus, the possibility of delivering our therapeutic agent to the target cells is markedly improved as shown in figure 1 and figure 2.

Figure 1. illustrates the structure of the citrullinated vimentin (ccp1) within the extracellular domain of the Syn notch receptor expressed on the surface of the engineered MSC

Figure 1: This figure illustrates the construction of our engineered exosomes that express citrullinated vimentin on their membranes conjugated to a transmembrane protein known as lysosome-associated membrane glycoprotein 2b (lamp2b), which is unique to the membrane of exosomes.

Literature Characterization

Using sera from rheumatoid arthritis patients and healthy patients The study used gel electrophoresis and scatter diagrams to demonstrate the reactions of wild-type and mutant citrullinated vimentin.

A, There are 5 lanes containing blue staining of dodecyl sulfate-polyacrylamide gel of wild-type vimentin without peptidyl arginine deaminase treatment (lane 1), wild-type vimentin in the citrullinated form (lane 2), mutant vimentin without peptidyl arginine deaminase treatment (lane 3), and mutant vimentin in the citrullinated form (lane 4), while markers are located in lane 5. B, this is a scatter gram that demonstrates antibodies reactions against wild-type (wt), mutant (mv), and in vitro analogs (cwt,mcv). OD = optical density; NS = not significant.

characterization by mathematical modeling

This model is to simulate kinetics of the binding between CCP1 of the synthetic notch on the surface of the stem cell to BCR of autoreactive B-cell. When stem cells are injected into the body, the amount of free CCP1 of the synthetic notch receptor increases, and as BCR binds to it, the amount of free CCP1 portion decreases, forming a binding state of both that activates the internal domain ZF21.16VP64 of the synthetic notch

A, Graph(1) shows an increase in the free portion of CCP1 of synthetic notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line).

We made a comparison between the types of external domains of the syn-notch receptor according to their binding to BCR. Based on the assumption that higher binding affinity implies lower dissociation rate.

1)We modeled the kinetics of the CV external domain of the syn-notch receptor to explain the binding affinity between it and BCR, depending on the result of the docking score, It’s concluded that it wasn’t stable and the dissociation occurs spontaneously after the binding

Graph(1) shows an increase in the free portion of CV of synthetic notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line). that dissociates after binding as it is not stable.

2)We modeled the kinetics of the VIM external domain of the syn-notch receptor to explain the binding affinity between it and BCR, depending on the result of the docking score, It’s concluded that it wasn’t stable and the dissociation occurs spontaneously after the binding.

Graph(1) shows an increase in the free portion of VIM of synthetic notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line). that dissociates after binding as it is not stable.

3)We modeled the kinetics of the CCP1 external domain of the syn-notch receptor to explain the binding affinity between it and BCR. Depending on the result of the docking score, It’s concluded that it was stable and reached steady state after binding. Which is the most suitable type in our comparison.

Graph(1) shows an increase in the free portion of CCP1 of synthetic notch (representedas blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line). that dissociates after binding as it is not stable.

References

Bang, H., Egerer, K., Gauliard, A., et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum. 56(8), 2503-2511 (2007). Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 44
  Illegal NotI site found at 53
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]