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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 100964009 Publication Model: Electronic Cited Medium: Internet ISSN: 1420-3049 (Electronic) Linking ISSN: 14203049 NLM ISO Abbreviation: Molecules Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, c1995-

Cloning, Molecular* ; Gene Expression* ; Recombinant Proteins*; Antineoplastic Agents/*pharmacology ; Metalloendopeptidases/*genetics ; Metalloendopeptidases/*pharmacology ; Sea Cucumbers/*genetics; Amino Acid Sequence ; Animals ; Apoptosis ; Carcinoma, Lewis Lung ; Cell Line, Tumor ; Cell Movement/drug effects ; Dose-Response Relationship, Drug ; Extracellular Matrix Proteins/metabolism ; Metalloendopeptidases/chemistry ; Metalloendopeptidases/metabolism ; Phylogeny ; Protein Interaction Domains and Motifs ; Sea Cucumbers/classification ; Sea Cucumbers/metabolism ; Solubility

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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Keywords: ADAMTS13-like; LLC cells; RGD; TSP 1; anti-tumoral activity; rAj-Tspin; sea cucumber

0 (Antineoplastic Agents)
0 (Extracellular Matrix Proteins)
0 (Recombinant Proteins)
EC 3.4.24.- (Metalloendopeptidases)

Date Created: 20220111 Date Completed: 20220214 Latest Revision: 20220214

20231215

PMC8746392

10.3390/molecules27010229

35011462