Truncated and Mutated Form of Cathepsin B

EDIT: Cathepsin B is a lysosomal protease present in the cytosol of various cancer types. We overexpressed wild-type cathepsin B in HEK293T cells to investigate cathepsin B induced cleavage of different peptide linkers via a fluorescence readout assay. We successfully showed that the linker GFLG was efficiently cleaved by cathepsin B in vivo. Furthermore, we were able to demonstrate that wild-type cathepsin B matured into its active forms when overexpressed in HEK293T cells. Together, these findings enable the functionalization of our PICasSO system for a wide range of therapeutic and synthetic biology applications.

1. Sequence overview

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 566
Illegal BglII site found at 665
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 67
Illegal NgoMIV site found at 919
Illegal AgeI site found at 751
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Cathepsin B is a cysteine protease typically located in lysosomes or secreted outside the cell, where it degrades proteins of the extracellular matrix (Ruan et al., 2015). To facilitate lysosomal escape of cathepsin B, cells were treated with low concentrations of doxorubicin. As an alternative strategy, we created a cytosolic single-chain version of cathepsin B. Full-length human cathepsin B has an N-terminal signal peptide facilitating targeting and translation of cathepsin B into the rough endoplasmic reticulum (Ni et al., 2022). Procathepsin B is then transported into the lysosome where it matures into its active form by cleavage into a light and heavy chain (Szulc-Dąbrowska et al., 2020).

3. Assembly and part evolution

We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the transactivator VP64. VP64 was conjugated to the DNA-binding domain (DBD) of Gal4 through the GFLG linker (BBa_K5237020). Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the GFLG linker reduces mCherry expression (see Fig. 1).

Figure 1: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay. The DNA-binding domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and consequently reduces mCherry expression.

We transfected our genetic constructs into HEK293T cells. The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable peptide linker (see Fig. 2). For all linkers, we did not observe a large decrease in fluorescence intensity between the negative control and test conditions. In some conditions, the fluorescence intensity even increased between the negative control and test conditions.

Figure 2: Fluorescence Readout After 48 hours for Five Different Peptide Linkers and Three Different Conditions. The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected.

Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. The first modification we made to the gene encoding for human cathepsin B, was the deletion of the first twenty amino acids. This N-terminally truncated version of cathepsin B had previously been observed to have catalytic activity even in the absence of lysosomal proteases like pepsin (Müntener et al., 2005). Furthermore, we introduced three point mutations into the polypeptide chain of cathepsin B (D22A, H110A, and R116A). This has been shown to increase the activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate binding pocket of cathepsin B (Nägler et al., 1997).

4. Results

The Truncated and Mutated Form of Cathepsin B is not Catalytically Active in Vivo

We performed the same fluorescence readout assay in HEK293T cells that we also used for wild-type cathepsin B (BBa_K5237100). The fluorescence intensity of mCherry was measured 48 hours after transfection. However, we observed no decrease in fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not cleaved (see Fig. 3).

Figure 3: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B. The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected.

The Truncated and Mutated Form of Cathepsin B was Poorly Expressed in HEK293T Cells

Figure 4 shows a western blot of the wild-type (wt) version of cathepsin B as well as the truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression.

Figure 4: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin. From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein, β-tubulin, is visible in all samples at 55 kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42 kDa, mature single-chain cathepsin B at 33 kDa and mature double-chain cathepsin B at 26 kDa. The band for the truncated and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36 kDa.

Conclusion

Our results indicate that the truncated and mutated form of cathepsin B was poorly expressed in HEK293T cells. Therefore, we continued to use the wild-type form of cathepsin B for further experiments. After consulting the literature, we decided to treat cells with the cytostaticum doxorubicin to induce lysosomal escape of cathepsin B, as had been previously reported (Bien et al., 2004).

5. References

Gramespacher, J. A., Stevens, A. J., nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. https://doi.org/10.1021/jacs.7b02618

Jin, C., EI-Sagheer, A. H., Li, S., Vallis, K. A., Tan, W., & Brown, T. (2022). Engineering Enzyme-Cleavable Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers. Angewandte Chemie International Edition, 61(13), e202114016. https://doi.org/10.1002/anie.202114016

Ruan, H., Hao, S., Young, P., & Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer Res, 56, 23-40.

Shim, N., Jeon, S. I., Yang, S., Park, J. Y., Jo, M., Kim, J., Choi, J., Yun, W. S., Kim, J., Lee, Y., Shim, M. K., Kim, Y., & Kim, K. (2022). Comparative study of cathepsin B-cleavable linkers for the optimal design of cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy. Biomaterials, 289, 121806. https://doi.org/10.1016/j.biomaterials.2022.121806

Wang, J., Liu, M., Zhang, X., Wang, X., Xiong, M., & Luo, D. (2024). Stimuli-responsive linkers and their application in molecular imaging. Exploration, 4(4), 20230027. https://doi.org/10.1002/EXP.20230027

Zhong, Y.-J., Shao, L.-H., & Li, Y. (2013). Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy (Review). Int J Oncol, 42(2), 373-383. https://doi.org/10.3892/ijo.2012.1754

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