Part:BBa_K2976013

TLR2-P2A-TLR1-T2A-CD14

Toll-like receptor (TLR) is a family of transmembrane glycoprotein that participates in the innate immune response to microbial agents [1]. TLR1 and TLR2 could form active heterodimers when exposed to some pathogen-associated molecular pattern molecules (PAMPs). CD14 is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, which preferentially expresses on monocytes and macrophages. It cooperates with TLR1/2 heterodimers and acts as a co-receptor for recognizing plenty of substances in lipoarabinomannan (LAM) biosynthesis [2]. 2A peptides, or 2A-like sequences, is a peptide that could mediate protein self-cleavage and be applied in expression of more than one gene in cells [3].

Usage

In 2019 CPU_CHINA project, TLR1, TLR2 and CD14 were co-expressed to form the TLR2:TLR1:CD14 cluster on the designer cell membrane by using 2A self-cleavage peptides. As a M.tuberculosis sensor, the complex could recognize the substances of M.tuberculosis and then stimulate the downstream signaling pathway. Then, activated NF-κB initiates transcription of the gene circuits to express other proteins in our project.

Biology

After being activated with Mtb, the activation cluster TLR1:TLR2:CD14 triggers NF-κB signaling pathways via MYD88 and TRAF6. NF-κB proteins exist in the cytoplasm in an inactive form because of their combination with the IκB proteins. IκB proteins mask the nuclear-localization sequences (NLSs) of NF-κB subunits and retain it in the cytoplasm [4]. Activation of TLR1:TLR2:CD14 cluster cause the degradation of IκB proteins by proteasomes. Then, NF-κB subunits could pass through the nuclear pore complex (NPC) and cause the expression of an array of pro-inflammatory cytokines and chemokines [5]. Similarly, NF-κB subunits also can bind the NF-κB induced promoter and initiate transcription of the downstream genes behind these promoters.

Characterization

This year, we 2019 CPU_CHINA attempted to develop a novel strategy for treating tuberculosis based on immune-like cells. Since our immune-like cells should recognize M.tuberculosis, TLR2:TLR1:CD14 cluster is required on the designer cell membrane. Thus, we conducted some researches on this composite part and obtained valuable results.

Plasmid (TLR2-P2A-TLR1-T2A-CD14) was transfected into HEK293T cells. In order to determine whether TLR1:TLR2:CD14 cluster was successfully expressed on the membrane of the designer cells, we performed Western blotting assay and Flow cytometry analysis.

Figure 1.Western blot analysis of TLR1, TLR2 and CD14 expression and activation of TLR signaling pathway.

Western blot (Figure 1) result shows that TLR1, TLR2 and CD14 were successfully expressed in HEK293T cells after 48h transfection (A). In addition, we also investigated whether NF-κB signaling pathway could be activated in a TLR1/2 dependent pathway. Transfected cells were treated with Pam3Cys-Ser-(Lys)4, a TLR1/TLR2 agonist. According to the results, phosphorylation of IκB was elevated and IκB was down-regulated, which indicate the activation of NF-κB signaling pathway in TLR1/TLR2/CD14 transfected HEK 293T cells, and the successful expression of TLR1 and TLR2 on the cell membrane.

Figure 2.Flow cytometry analysis of TLR1, TLR2 and CD14 expression.

Flow cytometry results (Figure 2) show that CD14 was significantly expressed on the membrane of artificial HEK293 cells, while strong fluorescence intensity was observed in neither TLR1 nor TLR2. Combined with the result of successful activation, it is inferred that TLR1/2 heterodimer formation caused the fluorescence disappearance.

For more details, please check out our demonstrate page.

Reference

[1] Lim, K. H., & Staudt, L. M. (2013). Signaling in innate immunity and inflammation. Cold Spring Harbor Perspectives in Biology, 5(1):a011247.

[2] Zanoni, I. , & Granucci, F. . (2013). Role of cd14 in host protection against infections and in metabolism regulation. Frontiers in Cellular and Infection Microbiology, 3, 32.

[3] Ikegami, T. , Lee, B. , Smith, J. K. , Hill, T. E. , & Park, A. . (2016). Optimized p2a for reporter gene insertion into nipah virus results in efficient ribosomal skipping and wild-type lethality. Journal of General Virology, 97(4), 839-843.

[4] Li, Q. , & Verma, I. M. . (2002). NF-kappaB regulation in the immune system. Nature Reviews Immunology, 2(10), 725.

[5] Zhang, Q. , Lenardo, M. J. , & Baltimore, D. . (2017). 30 years of nf-κb: a blossoming of relevance to human pathobiology. Cell, 168(1-2), 37-57.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 5212
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 552
  Illegal BamHI site found at 3226
  Illegal XhoI site found at 5243
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5057
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2541
  Illegal BsaI site found at 5063