Part:BBa_K2976003

Cluster of differentiation 14 (CD14)

CD14 is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, which preferentially expresses on monocytes and macrophages. It cooperates with other proteins to mediate the innate immune response to pathogen-associated molecular pattern molecules (PAMPs). For example, CD14 cooperates with TLR1/2 heterodimers and acts as a co-receptor for recognizing lipopolysaccharide.

Usage

In 2019 CPU_CHINA project, we express CD14 to form the TLR2:TLR1:CD14 cluster on the designer cell membrane. As a Mtb sensor, the complex could recognize the substances of Mtb 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 TLR2:TLR1:CD14 triggers NF-κB signaling pathways via MYD88 and TRAF6. NF-κB proteins exist in the cytoplasm in an inactive form because of their association with the IκB proteins. IκB proteins mask the nuclear-localization sequences (NLSs) of NF-κB subunits and retain it in the cytoplasm. Activation of TLR2:TLR1: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. 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. And CD14 cooperates with TLR1/2 heterodimers and act as a co-receptor for recognizing lipopolysaccharide. Thus, we conducted some researches on CD14, the basic part and obtained valuable results.

Plasmid (TLR2-P2A-TLR1-T2A-CD14) was transfected into HEK293T cells. In order to determine whether CD14 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 CD14 expression and activation of TLR signaling pathway.

Western blot (Figure 1) result shows that CD14 was 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. 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 CD14 on the cell membrane.

Flow cytometry results (Figure 2) show that CD14 was significantly expressed on the membrane of artificial HEK293 cells.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 336
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 367
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 187