Part:BBa_K3504007

Gly-Ala repeats

NOTICE: Parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.

Part Description

Glycine-Alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis.

Usage

EBNA1 glycine-alanine repeat (GAR) domain when inserted in cis with CTL epitope can inhibit antigen presentation through preventing cytotoxic T-lymphocyte-epitope generation. GAR appears to inhibit the proteosomal degradation of indicator proteins into which the gly-ala repeat is introduced, therefore evades the recognition by CTLs. previous findings show that the Gly-Ala repeat prevents presentation of MHC class I-restricted epitopes through the inhibiting the ubiquitin/proteasome pathway.

Characterization

We have made simulations using mathematical modelling techniques which showed lower levels of CTLs degredation when adding the glycine-alanine repeats to our circuit compared to its degredation ratio without the repeats.

Figure 1. CTLs degredation ratio between it with the inhibitory effect of Gly-Ala repeat on CTLs attack on the left compared to traditionally without it on the right.

Improvements

This part was improved by changes in its sequence that allowed it to further protect the circuit from CTLs immune attacks

Figure 1. Optimized Gly-Ala repeats.

References

1-Ossevoort, M., et al. “Creation of Immune ‘Stealth’ Genes for Gene Therapy through Fusion with the Gly-Ala Repeat of EBNA-1.” Gene Therapy, vol. 10, no. 24, 1 Nov. 2003, pp. 2020–2028, www.nature.com/articles/3302098, 10.1038/sj.gt.3302098. Accessed 21 Oct. 2020.

2-Blake, Neil W., et al. “Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1.” Journal of Virology, vol. 73, no. 9, 1 Sept. 1999, pp. 7381–7389, www.ncbi.nlm.nih.gov/pmc/articles/PMC104265/. Accessed 21 Oct. 2020.

3-Rumpold, H., et al. “The Glycine-Alanine Repeating Region Is the Major Epitope of the Epstein-Barr Nuclear Antigen-1 (EBNA-1).” The Journal of Immunology, vol. 138, no. 2, 15 Jan. 1987, pp. 593–599, www.jimmunol.org/content/138/2/593. Accessed 21 Oct. 2020.

4-“Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing.” Immunity, vol. 7, no. 6, 1 Dec. 1997, pp. 791–802, www.sciencedirect.com/science/article/pii/S1074761300803970?via%3Dihub, 10.1016/S1074-7613(00)80397-0. Accessed 21 Oct. 2020.

5-Levitskaya, Jelena, et al. “Inhibition of Ubiquitin/Proteasome-Dependent Protein Degradation by the Gly-Ala Repeat Domain of the Epstein–Barr Virus Nuclear Antigen 1.” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 23, 11 Nov. 1997, pp. 12616–12621, www.ncbi.nlm.nih.gov/pmc/articles/PMC25057/. Accessed 21 Oct. 2020. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 40
  Illegal NgoMIV site found at 64
  Illegal NgoMIV site found at 262
  Illegal NgoMIV site found at 403
  Illegal NgoMIV site found at 442
  Illegal NgoMIV site found at 505
  Illegal NgoMIV site found at 571
  
• 1000
  COMPATIBLE WITH RFC[1000]