Part:BBa_K3064014

hsaGlucagon->GGGGS->hsaIgG-Fc->P2A->HA->hsaTrim21

This part is used to degrade glucagon receptor (GCGR) endogenously.

Usage and Biology

Our composite part BBa_K3064014 play a necessary effect in our whole GCGR Predator system. This part is designed to recognised and bind with GCGR protein,and then recruit the proteasome to deplete the target protein through the ubiquitin-proteasome pathway. In this part, hsaGlucagon interacts with GCGR in a ligand-receptor binding manner. Trim21, the E3 ubiquitin ligase, plays the most important role in the degradation system. The C-terminal B30.2 domain on trim21 offers a site for the conservative Fc region of human lgG[1]. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.

Special Design

As a key functional element, special designs are taken for to optimize the applicability and adaptive of such parts . Our part is mainly composed of two important genes, and the efficient expression of these two genes and how to connect them become the focus of our design. Firstly, we used a connection element named GGGGS(BBa K3064025) to connect hsaGlucagon to hsalgG-fc. One of them is the specific binding site of the target receptor--GCGR. The other is the binding part of Trim21(BBa_K2653000). Second, we used self-cleaved sequence named P2A(BBa_K2653003), which allowed us to express two important genes at the same time. Make our parts more efficient. We also integrated HA tag(BBa_K2653004) into the Trim21 gene, which allows easy detection of Trim21 expression by Western Blotting analysis.

Figure 1.Representation of the function of the composite part.

Sequence and Features

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1830
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2499
  Illegal BamHI site found at 3037
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1787
  Illegal AgeI site found at 632
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1142
  Illegal BsaI site found at 2877
  Illegal SapI.rc site found at 1490
  

Characterization

This composite part can induce glucose concentration and achieve ubiquitination of GCGR. To verify whether it worked, we did a test of it.

Methods

HepG2 cells were transfected with Gcg-Fc expressing cassette or GCGR Predator (GCGR Pr) expressing cassette. Immunoprecipitation was performed with IgG-Fc targeting Protein A/G beads 48 h posttranscription followed up by Western blotting analysis probing GCGR and the Gcg-Fc domain of the GCGR Predator. Western Blotting is also performed to determine the degradation of GCGR.

Results

First, we used the method of immunoprecipitation to determine whether lgG-Fc will influence glucagon's function. And the experimental result shows that glucagon still binds with GCGR very well, which proved the integrity of this functional element. Then we have a function experiment to compared it to a control group. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.

Figure 2. Functional evaluation of the GCGR degradation system in HepG2 cells after 12h of liposome transfection. (A) Co-immunoprecipitation showing binding ability of glucagon-humanIgG Fc fusion protein with glucagon receptor(GCGR). (B) Western Blot showing the result of glucagon receptor(GCGR) degradation.

Method

To further validate that intracellular glucagon can be degraded by GCGR Predator, HepG2 cells were transfected with Trim21 expression cassette, Glucagon-Fc expressing cassette(this part) or GCGR Predator (GCGR Pre.) expressing cassette, and Western blotting analysis was performed to detect the protein level of GCGR(Fig 3A).

Result

We performed Western Blotting to further prove that such interaction is capable of triggering GCGR degradation. Results indicated that GCGR Pre. transfected cells showed a ~40% lower GCGR level compared to the vector control (Fig 3A and B) and this degradation relies on proteasome (Data not shown). Besides, there is no significant difference between the control group and groups that only overexpressed Glucagon-linker-IgG Fc (Gcg-Fc) or Trim21. Also, we found that both in four groups, Glycosylated GCGR (Glu-GCGR, ~64kDa) showed no difference in expression level.

Figure 3. (A) Western blotting assay showing the degradation of GCGR in the GCGR Predator transfected group and its quantified results (B).