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P.D.S.U. Wickramasinghe (P.D.S.U. Wickramasinghe)
조회 296  인쇄하기 주소복사 트위터 공유 페이스북 공유 
Identification and characterization of cystatin B from black rockfish, Sebastes schlegelii, indicating its potent immunological importance

P.D.S.U. Wickramasinghea,b,1, Hyukjae Kwona,c,1, Don Anushka Sandaruwan Elvitigalad,*, Qiang Wana,c, Jehee Leea,c,*

aDepartment of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea
bDepartment of Chemistry, Faculty of Science, University of Colombo, Colombo-03, Sri Lanka
cMarine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
dDept. of Basic Science and Social Sciences for Nursing, Faculty of Nursing, University of Colombo, Thalapathpitiya, Nugegoda, 10250, Sri Lanka

1These authors contributed equally to this work.

*Corresponding author

Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine (8GG9) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5′-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3′-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts.

- 형식: Research article
- 게재일: 2020년 06월 (BRIC 등록일 2020-06-19)
- 연구진: 국내(교신)+국외 연구진 태극기
- 분야: Marine_Biology
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