Qiao R et al. (2021), Cloning, Expression and Inhibitory Effects on L...

ECB-ART-50584

Molecules 2021 Dec 30;271:. doi: 10.3390/molecules27010229.

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Cloning, Expression and Inhibitory Effects on Lewis Lung Carcinoma Cells of rAj-Tspin from Sea Cucumber (Apostichopus japonicus).

Qiao R , Xiao R , Chen Z , Jiang J , Yuan C , Ning S , Wang J , Zhou Z .

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In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber (Apostichopus japonicus), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it's effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.

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	Figure A1. Nucleic-acid (upper lines) and amino-acid (lower lines) sequences of ADAMTS13-like from A. japonicus. Green color indicates the 10 TSP1 domains, except the third TSP1 domain, which is marked with red.
	Figure A2. Nucleic-acid (upper lines) and amino-acid (lower lines) sequences of the third TSP1 domain (Aj-Tspin) in the ADAMTS13-like sequence from A. japonicus. Green indicates cysteine residues (lower lines) and their nucleic-acid (upper lines). Red indicates the nucleic-acid (upper lines) and amino-acid (lower lines) of the RGD motif.
	Figure 1. Phylogenetic tree analysis of ADAMTS13-like from A. japonicus.
	Figure 2. Domain analysis of ADAMTS13-like from A. japonicus.
	Figure 3. Multiple-sequence alignment of disintegrins from snakes, leeches, ticks, gadflies and lampreys, as well as Aj-Tspin from sea cucumbers. Adinbitor, saxatilin, barbourin, decorsin, ornatin, variabilin, savignygrin, tablysin, Lj-RGD3 and Aj-Tspin are, respectively, from Agkistrodon halys brevicaudus stejneger, Gloydius saxatilis, Sistrurus miliarius barbouri, Macrobdella decora, Placobdella ornata, Dermacentor variabilis, Ornithodoros savignyi, Tabanus yao, Lampetra japonica and A. japonicus.
	Figure 4. Purified rAj-Tspin was detected on Tricine SDS-PAGE. Lane 1: 10 μL of purified rAj-Tspin; Lane 2: IPTG-induced expression of recombinant BL21 cells; Lane 3: non-induced expression of recombinant BL21 cells; Lane 4: spectra multicolor low range protein ladder (Thermo Scientific, Waltham MA, USA).
	Figure 5. CCK-8 assay detected the inhibitory effects of rAj-Tspin on the proliferation of LLC cells. , * and ***, respectively, indicate p < 0.05, p < 0.01, and p < 0.001 (compared with control group). Each assay was repeated three times.
	Figure 6. Wright-Giemsa staining assay detected the effect of rAj-Tspin on the morphology of LLC cells. The bar indicates 10 μm; (a) control; (b) 1.85 μM rAj-Tspin; (c) 2.20 μM rAj-Tspin; (d) 3.04 μM rAj-Tspin.
	Figure 7. The apoptosis of LLC cells induced by rAj-Tspin was detected by TUNEL assay (Zeiss laser-scanning confocal microscopy, 200×). The bar indicates 20 μm; (a) control; (b) 1.85 μM rAj-Tspin; (c) 2.20 μM rAj-Tspin; (d) 3.04 μM rAj-Tspin.
	Figure 8. Effect of rAj-Tspin on the cytoskeleton and nuclei of the LLC cells. Green indicates the cytoskeleton marked by the phalloidin-FITC. Blue indicates the nuclei marked by Hoechst 33258 (Zeiss laser-scanning confocal microscopy, 630×). The bar indicates 10 μm.
	Figure 9. rAj-Tspin inhibited the adhesion of LLC cells. , * and ***, respectively, indicate p < 0.05, p < 0.01, and p < 0.001 (compared with the empty-medium groups).
	Figure 10. rAj-Tspin inhibited the migration of LLC cells (OLYMPUS optical microscopy, 200×). and *, respectively, indicate p < 0.01 and p < 0.001 (compared with the empty-medium and bFGF treatment groups).
	Figure 11. rAj-Tspin inhibited the invasion of LLC cells (OLYMPUS optical microscopy, 200×). and *, respectively, indicate p < 0.01 and p < 0.001 (compared with the empty-medium and bFGF treatment groups).