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 downregulated. 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
