Difference between revisions of "Part:BBa K3195003"

Latest revision as of 15:32, 20 October 2019

His-tagged Cathepsin B

This Cathepsin B from Branchiostoma belcheri tsingtauense^[1] is originally a kind of hydrolase in lysosome.In our project,it has been used to biodegrade microcystin,a kind of biotoxin synthesized by cyanobacteria.

Usage and Biology

Our BioBrick encodes a kind of cathepsin from Branchiostoma diverticulum epithelial cells. Cathepsin is a class of proteins found in the cells of various animal tissues, especially lysosome parts. Based on the results of bioinformatics analysis, the protein we found out belongs to cathepsin B. In Branchiostoma diverticulum epithelial cells, we speculate this kind of protein plays an important role in degrading cyanobacteria and microcystin. In fact, according to our experimental result, Cathepsin B protein has high efficiency in degradation of microcystin LR. So we construct a composite part so that other teams can easily use it.
This BioBrick can be implemented in any host expression system by cloning it into an appropriate vector.

Figure 1.The abscissa represent 28 enzymes in our project and the unit of ordinate value is mg/L.The sample of MC-LR/RR degraded respectively by 28 enzymes was measured by HPLC-MS-MS and the results are as shown. The ordinate value represents the remained microcystin in the degraded sample.The sample 2 was degraded by cathepsin B It shows that cathepsin B is the most effective enzyme among the 28 proteins in degradation of MC-LR.

Characterization

Expression

Figure 3. Expression tests of the target protein.
Annotations:
MW. Molecular weight marker.
Ø. Non-induced bacteria culture (negative control).
16 and 37. Incubation temperature (°C) during induction with IPTG.Induction with IPTG 1mM during 16h at 16°C, or during 4h for other temperatures.
NPE.native protein extract. DPE.denatured protein extract.

Figure 4. Final purification result in denatured condition Coomassie blue staining.Reducing-PAGE analysis. 2µg of sample loaded.
Annotations:1. Before dialysis 2. After dialysis

Activity assay

The BioBrick was characterized by measuring cathepsin B activity using Cathepsin B Activity Assay Kit. BioVision's Cathepsin B Activity Assay Kit is a fluorescence-based detection technique. Using AFC (7-amino-4-trifluoromethyl coumarin) labeled cathepsin-B priority substrate sequence RR. Cell solutes or other samples containing Cathepsin-B can digest RR-AFC and release free AFC. Free AFC can be easily quantified by fluorometer or fluorescence microtitrator plate.

We determined Km and Vmax for our cathepsin B by performing non-linear regression using Michaelis-Menten model as below. The two parameters were: Vmax=3749.56222 ± 87.82359 RFU/h Km=0.22768 ± 0.02604 mol/L

Figure 5.The non-linear fitting of Michaelis-Menten model

Molecular docking

In our project, we conducted homology modeling for better understanding its function. Then MOE Dock was used for molecular docking of cathepsin B with the small molecule(microcystin) and predicting the binding affinity. Small molecule was defined as ligand and cathepsin B as target. The binding site was identified by superpose cathepsin B with the original template structure, the position where the ligand in template structure was defined as binding site of cathepsin B. The docking workflow followed the “induced fit” protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. The weight used for tethering side chain atoms to their original positions was 10. The best ranked pose was selected as the final binding mode.
To investigate the binding affinity of cathepsin B with microcystin LR, docking simulation studies were carried out. The docking scores are shown in table 1.

The binding model between cathepsin B and microcystin LR are shown in Figure 3. The nitrogen atoms of guanidine group of mcirocystin LR, regarded as hydrogen bond donor, forms hydrogen bonds with the backbone oxygen atom of Gly90 and Cys92, and with the side-chain chlorine atom of Cys184 in cathepsin B respectively. The nitrogen atom of microcystin LR, regarded as hydrogen bond donor, forms a hydrogen bond with the backbone oxygen atom of Leu261 in cathepsin B.

Figure 6.Complex of microcystin LR and cathepsin B. (A) The 2D binding mode of Ciprofloxacin and cathepsin B. (B) The binding model of microcystin on molecular surface of cathepsin B. Microcystin LR is colored in cyan, the molecular surface of cathepsin B is colored in lightblue. (C) The 3D binding mode of microcystin LR and cathepsin B. Microcystin LR is colored in cyan, the surrounding residues in the binding pockets are colored in yellow, the backbone of the receptor is depicted as white cartoon with transparency.

The results of molecular docking show that the interaction sites between cathepsin B and microcystin LR were GLY-90, CYX-92, CYX-184, and LEU-261, respectively. Three of them were highly conserved in the evolution of species, and the amino acid LEU-261 was conserved in the alignment results. Though they were different amino acids, they still belonged to the same class. Although the action sites are highly conserved, the alignment results show that there are differences in the amino acid sequence environment around the action sites. Compared with other species, there are some unique amino acid fragments around the cathepsin B site of lancelet, which are labeled with grey background. We infer that it is these fragments that influence the spatial structure around the action sites that enable cathepsin B in amphioxus to interact with algae toxin, capture and complete the digestion of algae toxin. At the same time, by comparing the structures of microcystin LR and microcystin RR, the structural differences between the two kinds of microcystins are only one branch far from benzene ring. The results of molecular docking show that the amino acid sites of cathepsin B interacting with algae toxin generally concentrate around the common branched chains of the two algae toxins, so we judged that the structural differences between the two algae toxins had no decisive effect on the recognition and interaction of the two enzymes and algae toxins. It is also consistent with our experimental results that cathepsin B can independently decompose microcystin LR and microcystin RR.

Reference

[1]He Chunpeng, Han Tingyu, Liao Xin, Zhou Yuxin, Wang Xiuqiang, Guan Rui, Tian Tian, Li Yixin, Bi Changwei, Lu Na, He Ziyi, Hu Bing, Zhou Qiang, Hu Yue, Lu Zuhong and Chen J.-Y. Phagocytic intracellular digestion in amphioxus (Branchiostoma)285Proc. R. Soc. B http://doi.org/10.1098/rspb.2018.0438

Sequence and Features