Elsevier

Fish & Shellfish Immunology

Volume 35, Issue 3, September 2013, Pages 1039-1043
Fish & Shellfish Immunology

Short communication
Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): Molecular characterization and transcriptional response upon immune stimulation

https://doi.org/10.1016/j.fsi.2013.07.005Get rights and content

Highlights

  • Kazal-type serine proteinase inhibitor (Ab-KPI) from disk abalone was characterized at transcriptional level.

  • In silico analysis reveals the presence of two Kazal domains.

  • Ab-KPI showed highly hepatopancreas-specific expression.

  • Ab-KPI showed significant induction upon bacterial, viral hemorrhagic septicemia virus challenge and tissue injury.

Abstract

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone.

Introduction

Endogenous proteinases present in the multicellular organisms regulate diverse and critically important physiological and immunological reactions [1]. Enzymatic regulation, which is mediated by different types of proteinase inhibitors (PIs), is important for the maintenance of all metabolic reactions in living cells; for example, PIs are involved in blood coagulation, activation of the complement system, inflammatory reactions, immune responses, and development [2]. Kazal-type proteinase inhibitors (KPIs) are widely identified in multicellular organisms, and mostly responsible for inhibition of serine proteinase [3].

Among serine PIs, KPIs are well characterized. KPI was originally isolated from bovine pancreas [4] in which the inhibitor controls excessive proteolytic activities in the alimentary organs. Related inhibitor genes were subsequently identified from invertebrates such as the arthropod, Pacifastacus leniusculus, and in blood sucking animals such as the insect, Rhodnius prolixus, in which KPIs assist in preventing blood coagulation, and thus facilitate insect to acquire more blood [5]. However, knowledge of kazal inhibitors in mollusks innate immunity is limited. To date, KPIs have been characterized from zhikong scallops, bay scallops, and eastern oyster. Several studies have shown that KPIs inhibit the activity of bacterial compounds like subtilisin, and exhibit bacteriostatic activity against Bacullius subtilis [6], [7] and Staphylococcus aurious [8]. Furthermore, a potent role for KPIs in invertebrate immunity has been demonstrated by investigations of their involvement in the response against pathogenic microbial challenge [9].

A typical or canonical kazal domain comprises 40–60 amino acid residues, including six cysteine residues that form a 1–5, 2–4, 3–6 disulphide bond pattern. A number of kazal domains have been shown to evolve at different evolutionary rates [9]. For example, certain amino acid residues that reside between two consecutive cysteines can vary between kazal domains within the same individual, and sometimes lack certain disulphide bridges.

Disk abalone is an economically important gastropod in Korean aquaculture. However, abalone farming has recently experienced outbreaks of mass mortality due to bacterial diseases [10]. A few pathogenic Vibrio species have been already identified for abalone diseases during last two decades [11]. Therefore, understanding of genes involved in anti-bacteria defense system of abalone is important for effective disease control. In this study, we characterized one of KPIs in disk abalone, Haliotis discus discus, designated as Ab-KPI. Our methods include cDNA cloning, sequence characterization, tissue-specific expression profiling, and transcription response in hemocytes, and mantle tissue following stimulation with bacteria, viral hemorrhagic septicemia virus (VHSV), and wound healing. Finally, we assessed the phylogenetics of Ab-KPI in the context of KPIs derived from other invertebrate species.

Section snippets

Isolation of kazal-type proteinase inhibitor cDNA in disk abalone

We constructed a normalized cDNA library from disk abalone using RNA isolated from whole tissues (gills, mantle, digestive tract, hepatopancreas, head, and muscle). Briefly, total RNA was isolated from pooled tissues of three healthy abalones using the Tri Reagent™ (Sigma, USA), according to the manufacturer's instructions. Polyadenylated messenger RNA (mRNA) was then purified using an mRNA isolation kit (FastTract® 2.0; Invitrogen, USA). First strand cDNA synthesis and normalization were

Results and discussion

Based on BLAST-X analysis of EST data from the normalized cDNA library, we have identified the full-length cDNA sequence of the kazal-type proteinase inhibitor from disk abalone (H. discus discus), designated as Ab-KPI. The sequence was deposited in NCBI under the accession number ADQ43244. The complete cDNA sequence is 600 bp in length, composed of a 26 bp 5′-UTR, 432 bp open reading frame (ORF) encoding 143 amino acids, and a 142 bp 3′-UTR including a polyadenylation (470AATAAA 475) signal

Acknowledgments

This research was supported by the MSIP (Ministry of Science, ICT & Future Planning), Korea, under the ITRC (Information Technology Research Center) support program supervised by the NIPA National IT Industry Promotion Agency (NIPA-2013-H0301-13-2009).

References (34)

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