Short communicationKazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): Molecular characterization and transcriptional response upon immune stimulation
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)
- et al.
Disk abalone (Haliotis discus discus) expresses a novel antistasin-like serine protease inhibitor: molecular cloning and immune response against bacterial infection
Fish & Shellfish Immunology
(2010) - et al.
The physiological aspects of fibrinolysis
Thrombosis Research
(1994) - et al.
Three kazal-type serine proteinase inhibitors from the red swamp crayfish Procambarus clarkii and the characterization, function analysis of hcPcSPI2
Fish & Shellfish Immunology
(2010) - et al.
High sequence variability among hemocyte-specific kazal-type proteinase inhibitors in decapod crustaceans
Developmental and Comparative Immunology
(2010) - et al.
A five-domain kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities
Developmental and Comparative Immunology
(2006) - et al.
Domain inhibitory and bacteriostatic activities of the five-domain kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon
Developmental and Comparative Immunology
(2009) - et al.
Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra's innate immune system
Developmental and Comparative Immunology
(2009) - et al.
Structure and function of invertebrate kazal-type serine proteinase inhibitors
Developmental and Comparative Immunology
(2010) - et al.
Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis cracherodii
Journal of Invertebrate Pathology
(1995) - et al.
Basic local alignment search tool
Journal of Molecular Biology
(1990)
First molluscan TNF-alpha homologue of the TNF superfamily in disk abalone: molecular characterization and expression analysis
Fish & Shellfish Immunology
First molluscan transcription factor activator protein-1 (Ap-1) member from disk abalone and its expression profiling against immune challenge and tissue injury
Fish & Shellfish Immunology
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
A four-kazal domain protein in Litopenaeus vannamei hemocytes
Developmental and Comparative Immunology
A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus
Comparative Biochemistry and Physiology Part B, Biochemistry & Molecular Biology
Molecular cloning and expression of a novel kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain
Fish & Shellfish Immunology
Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)
Fish & Shellfish Immunology
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Antimicrobial activity of a newly identified Kazal-type serine proteinase inhibitor, CcKPI1, from the jellyfish Cyanea capillata
2018, International Journal of Biological MacromoleculesA homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury
2015, Fish and Shellfish ImmunologyCitation Excerpt :The present results are consistent with our previous studies performed on various rock bream SPIs [28,47,48]. In contrast, various reports have demonstrated upregulated transcription of SPIs following tissue injury [45,53]. Shigetomi et al. (2010) reported the consequence of KSPIs in response to tissue injury [7].
Three novel clade B serine protease inhibitors from disk abalone, Haliotis discus discus: Molecular perspectives and responses to immune challenges and tissue injury
2015, Fish and Shellfish ImmunologyCitation Excerpt :The current study examined the mRNA expression of each AbSERPIN. In previous studies, disk abalone Ab-Antistasin [30] and Ab-KPI [31] PIs showed the highest expression in the digestive tract and hepatopancreas, respectively. A recent study in octopus reported that the highest expression level of a SERPIN occurred in hepatopancreas [38].
Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response
2015, Fish and Shellfish ImmunologyCitation Excerpt :However, the existing research on Serpins is primarily focused on crustaceans and insects, especially on crab (E. sinensis), shrimp (Litopenaeus vannamei, P. monodon), moth (M. sexta) and silkworm (Bombyx mori) [20,21,23,25,26]. Although several studies have been reported on SPIs in molluscs, the emphasis was mainly laid on Kazal- [27–29] and Kunitz-SPIs [30]. Presently, the molecular characteristics, compositional properties, and structural-functional mechanisms of Serpins in molluscs are still poorly understood.
Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata
2014, Fish and Shellfish ImmunologyCitation Excerpt :The expression levels of the poKSPI mRNA were the highest in mantle and gonad, and the lowest in haemocyte and intestine (Fig. 2). Similarly, the expression level of MdSPI-1 mRNA in surf clam was also higher in mantle than in other tissue [22]. The high expression level of KSPI mRNA in mantle was possibly due to the direct contact of this tissue with the external medium and consequently with contagious pathogens.