title: Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.
abstract: Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
authors.forename: H K A
authors.initials: HK
authors.lastname: Premachandra
authors.forename: Don Anushka Sandaruwan
authors.initials: DA
authors.lastname: Elvitigala
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Eunmi
authors.initials: E
authors.lastname: Kim
authors.forename: Mahanama
authors.initials: M
authors.lastname: De Zoysa
authors.forename: Bong-Soo
authors.initials: BS
authors.lastname: Lim
authors.forename: Sang-Yeob
authors.initials: SY
authors.lastname: Yeo
authors.forename: Seokryel
authors.initials: S
authors.lastname: Kim
authors.forename: Myoung-Ae
authors.initials: MA
authors.lastname: Park
authors.forename: Hae-Chul
authors.initials: HC
authors.lastname: Park
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D055313:Cystatin B / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D008070:Lipopolysaccharides; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D011070:Poly I-C; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D014733:Vibrio
chemical_list: D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D055313:Cystatin B; D011070:Poly I-C