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Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.
- Source :
-
Fish & shellfish immunology [Fish Shellfish Immunol] 2013 Jun; Vol. 34 (6), pp. 1505-13. Date of Electronic Publication: 2013 Mar 22. - Publication Year :
- 2013
-
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.<br /> (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Bivalvia immunology
Cloning, Molecular
Cystatin B chemistry
Cystatin B immunology
Cystatin B metabolism
Electrophoresis, Polyacrylamide Gel
Lipopolysaccharides
Molecular Sequence Data
Organ Specificity
Phylogeny
Poly I-C
Real-Time Polymerase Chain Reaction
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins immunology
Recombinant Proteins metabolism
Sequence Alignment
Vibrio
Bivalvia genetics
Cystatin B genetics
Gene Expression Regulation
Subjects
Details
- Language :
- English
- ISSN :
- 1095-9947
- Volume :
- 34
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Fish & shellfish immunology
- Publication Type :
- Academic Journal
- Accession number :
- 23528873
- Full Text :
- https://doi.org/10.1016/j.fsi.2013.03.349