Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties

H. K A Premachandra, Don Anushka Sandaruwan Elvitigala, Ilson Whang, Eunmi Kim, Mahanama De Zoysa, Bong Soo Lim, Sang Yeob Yeo, Seokryel Kim, Myoung Ae Park, Hae Chul Park, Jehee Lee

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 (297bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11kDa 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 invivo 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.

Original languageEnglish
Pages (from-to)1505-1513
Number of pages9
JournalFish and Shellfish Immunology
Volume34
Issue number6
DOIs
Publication statusPublished - 2013 Jun 1

Fingerprint

Cystatin B
Cystatins
Maltose-Binding Proteins
Ruditapes philippinarum
cystatins
Papain
Cysteine Proteases
Pathology
pathology
functional properties
gills
papain
cysteine proteinases
maltose
Recombinant Fusion Proteins
hemocytes
Cysteine Proteinase Inhibitors
Poly I-C
Peptides
protein

Keywords

  • Cystatin B
  • Manila clam
  • Papain inhibitory activity
  • Structural characterization
  • Transcriptional analysis

ASJC Scopus subject areas

  • Aquatic Science
  • Environmental Chemistry

Cite this

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties. / Premachandra, H. K A; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Kim, Eunmi; De Zoysa, Mahanama; Lim, Bong Soo; Yeo, Sang Yeob; Kim, Seokryel; Park, Myoung Ae; Park, Hae Chul; Lee, Jehee.

In: Fish and Shellfish Immunology, Vol. 34, No. 6, 01.06.2013, p. 1505-1513.

Research output: Contribution to journalArticle

Premachandra, H. K A ; Elvitigala, Don Anushka Sandaruwan ; Whang, Ilson ; Kim, Eunmi ; De Zoysa, Mahanama ; Lim, Bong Soo ; Yeo, Sang Yeob ; Kim, Seokryel ; Park, Myoung Ae ; Park, Hae Chul ; Lee, Jehee. / Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties. In: Fish and Shellfish Immunology. 2013 ; Vol. 34, No. 6. pp. 1505-1513.
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AU - Kim, Eunmi

AU - De Zoysa, Mahanama

AU - Lim, Bong Soo

AU - Yeo, Sang Yeob

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AB - 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 (297bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11kDa 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 invivo 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.

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