Characterization of Thrombospondin Type 1 Repeat in Haliotis diversicolor and Its Possible Role in Osteoinduction

• Published in: Marine biotechnology (New York, N.Y.) N.Y.), 2021-08, Vol.23 (4), p.641-652
• Main Authors: Habuddha, Valainipha, Suwannasing, Chanyatip, Buddawong, Aticha, Seenprachawong, Kanokwan, Duangchan, Thitinat, Sombutkayasith, Chanyarak, Supokawej, Aungkura, Weerachatyanukul, Wattana, Asuvapongpatana, Somluk
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedical materials; Bone growth; Bone healing; Cbfa-1 protein; Cell differentiation; Cells; Collagen (type I); DNA; Engineering; Epithelium; Extracellular; Freshwater & Marine Ecology; Haliotis diversicolor; Life Sciences; Ligands; Localization; Mantle; Marine molluscs; Membrane proteins; Microbiology; Mineralization; Nucleotide sequence; Original Article; Osteoblasts; Osteogenesis; PCR; Proteins; Recombinants; Repair; Sequences; Signaling; Smad2 gene; Smad2 protein; Thrombospondin; Tissue; Transcription; Upper mantle; Zoology
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-021-10054-3

Thrombospondin repeats (TSR) are important peptide domains present in the sequences of many extracellular and transmembrane proteins with which a variety of ligands interact. In this study, we characterized Hd TSR domains in the ADAMTS3 protein of Thai abalone, Haliotis diversicolor , based on the transcriptomic analysis of its mantle tissues. PCR amplification and localization studies demonstrated the existence of Hd TSR transcript and protein in H. diversicolor tissues, particularly in both the inner and outer mantle epithelial folds. We, therefore, generated a short recombinant protein, termed Hd TSR1/2, based on the existence of the WxxWxxW or WxxxxW motif (which binds to TGF-β, a known signaling in bone formation/repair) in Hd TSR1 and Hd TSR2 sequences and used it to test the osteoinduction function in the pre-osteoblastic cell line, MC3T3-E1. This recombinant protein demonstrated the ability to induce the differentiation of MC3T3-E1 cells by the concentration- and time-dependent upregulation of many known osteogenic markers, including RUNX2, COL1A1, OCN, and OPN. We also demonstrated the upregulation of the SMAD2 gene after cell treatment with Hd TSR1/2 proteinindicating its possible interaction through TGF-β, which thus activates its downstream signaling cascade and triggers the biomineralization process in the differentiated osteoblastic cells. Together, Hd TSR domains existed in an extracellular ADAMTS3 protein in the mantle epithelium of H. diversicolor and played a role in osteoinduction as similar to the other nacreous proteins, opening up its possibility to be developed as an inducing agent of bone repair.