Disk abalone (Haliotis discus discus) expresses a novel antistasin-like serine protease inhibitor: Molecular cloning and immune response against bacterial infection.
A novel antistasin-like cDNA homologue named as Ab-Antistasin was isolated from the disk abalone Haliotis discus discus normalized cDNA library. The Ab-Antistasin (1398-bp) consisted of an 1185-bp open reading frame encoding 395 amino acid (aa) residues. The predicted molecular mass and isoelectric point of Ab-Antistasin was 44 kDa and 8.5, respectively, and showed highest identity (23.1%) to Hydra magnipapillata antistasin. The most striking feature of Ab-Antistasin is the 12-fold internal repeats (IR) of an antistasin-like domain. Ten of the 12 IR domains (26-27 aa) are highly conserved, with 6 cysteines and 1 glycine. Ab-Antistasin was comprised of three Bowman-Birk serine protease inhibitor family motifs. The recombinant Ab-Antistasin (rAb-Antistasin) was over-expressed in Escherichia coli and purified using a pMAL system. rAb-Antistasin (10 microM) was able to inhibit trypsin activity by 66% in a dose-dependent manner. Moreover, it exhibited low prolongation activity for coagulation in an APTT assay (86.0 s compared to control 42.0 s) with human blood. Endogenous Ab-Antistasin mRNA was found to be expressed in digestive tract, hepatopancreas, hemocytes, abductor muscle and mantle, with highest expression levels in digestive tract followed by hepatopancreas and hemocytes. Quantitative real time PCR results revealed that Ab-Antistasin transcription was significantly induced at 3 h post-infection (p.i.) after challenged by a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) in the abalone digestive tract; in the hemocytes, induction occurred at 6 and 12 h. The results indicated that Ab-Antistasin could play an important role in the immune responses of mollusks.
Nikapitiya
Chamilani
C
Department of Marine Life Sciences, College of Ocean Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
De Zoysa
Mahanama
M
Oh
Chulhong
C
Lee
Youngdeuk
Y
Ekanayake
Prashani Mudika
PM
Whang
Ilson
I
Choi
Cheol Young
CY
Lee
Jae-Seong
JS
Lee
Jehee
J
D000595:Amino Acid Sequence; D000818:Animals; D018407:Bacterial Physiological Phenomena; D001483:Base Sequence; D003001:Cloning, Molecular; D049851:Gastropoda / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000382:microbiology*; D005786:Gene Expression Regulation; D007447:Invertebrate Hormones / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007447:Invertebrate Hormones; C053155:antistasin