Elsevier

Fish & Shellfish Immunology

Volume 59, December 2016, Pages 57-65
Fish & Shellfish Immunology

Short communication
Mitogen–activated protein kinase 1 from disk abalone (Haliotis discus discus): Roles in early development and immunity-related transcriptional responses

https://doi.org/10.1016/j.fsi.2016.10.031Get rights and content

Highlights

  • A MAPK1 from disk abalone (AbMAPK1) was identified and characterized.

  • Expression profile of abalone developmental stages was analyzed.

  • The expression profile of healthy tissues of adult abalones was investigated.

  • Transcriptional level of AbMAPK1 was modulated by both viral and bacterial exposure.

Abstract

Mitogen–activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5′ untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3′ UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism.

Introduction

Abalones are economically important gastropods belonging to the family Haliotidae (and the genus Haliotis). In the past few decades, frequent disease outbreaks have been reported in the aquaculture of abalones [1], [2], [3]. Similar with other invertebrates, the defense mechanism of abalone relies exclusively on the innate immunity. Therefore, understanding of their innate immune mechanism is important towards the disease control and successful culture.

Mitogen activated protein kinases (MAPKs), are categorized into the family of Ser/Thr protein kinases, present in a variety of tissues [4]. The MAPK signaling cascade contains three main and distinct signaling pathways, namely extracellular-signal regulated kinase (ERK) pathway, c-Jun N-terminal kinases (JNKs) pathway, and p38 family of MAPKs pathway [5]. Amongst these three, the ERK mediated pathway serves as the prototypical MAPK pathway. It plays crucial roles in multiple cell physiological events; like cell proliferation, differentiation, and development through responding to growth factors, mitogens, and environmental stressors [6], [7]. Moreover, ERK is also pivotal in the modulation of both innate and adaptive immunity. It has been found that ERK is involved in the development of T-cells by regulating Th17 and Treg-cell development [8]. In Caenorhabditis elegans, the ERK cascade was shown to have a protective response to rectal infection caused by a gram positive bacterial pathogen, Microbacterium nematophilum [9]. ERK signaling pathway is also involved in the largemouth bass virus-induced apoptosis [10]. The ERKs in Litopenaeus vannamei and Fenneropenaeus chinensis were increased at the early stages of white spot syndrome viral infection [11], revealing its involvement in the immune response.

MAPK1 (also known as ERK2) is the second member in the ERK subfamily, which contains a highly conserved serine/threonine kinase domain. To date, MAPK1 homologs have already been found in human [12], mice [13], chicken [14], frog [14], and zebrafish [15] genomes. Functional studies of the MAPK1 gene have been carried out extensively in human cells and it was found that MAPK1 contributed to the proliferation of human epithelioid malignant mesotheliomas [12]. MAPK1 has also been found to possess a specific function in normal trophoblast development in the mouse via regulating the proliferation of polar trophectoderm cells [16]. In Xenopus embryos, the modulation of MAPK1 was shown to affect the growth factor-stimulated mesoderm induction [17]. Additionally, ERK activation in zebrafish has the potential to induce heat shock proteins in thermal stress [7]. However, extensive information regarding the roles of MAPK1 in immune response is lacking, especially in the mollusks. Therefore, in the present study, we aimed to characterize the molecular features of MAPK1 gene from Haliotis discus discus and further analyze its mRNA expression in early developmental stages, and in various tissues of the mature abalone in healthy and pathophysiological scenarios.

Section snippets

Experimental organisms, chemicals, and reagents

Healthy disk abalones were obtained from the Youngsoo abalone farm (Jeju Island, Republic of Korea). Molecular biology grade chemicals were purchased from Sigma (USA) for the entire experiment, unless otherwise mentioned. SYBR® Premix Ex Taq™ and molecular markers were obtained from TaKaRa Bio, Japan or Thermo Scientific, Inc., USA. Trizol Reagent™ (Sigma-Aldrich, Missouri, USA) was used for the total RNA extraction. As a stimulant in the immune challenge experiment, polyinosinic:polycytidylic

Molecular features of AbMAPK1 cDNA and its amino acid sequence

The amino acid sequence of AbMAPK1 (GenBank accession No: KU351558) was deduced from the abalone cDNA database. It contained an open reading frame (ORF) of 1101 nucleotides, a 5′ untranslated region (UTR) of 23 nucleotides, and 3′ UTR of 448 nucleotides. The length of the AbMAPK1 amino acid sequence (367 aa) is similar to the other molluscan homologs, including those of Crassostrea gigas and Tegillarca granosa. It was predicted that the AbMAPK1 protein has a molecular mass of 42.20 kDa, a

Acknowledgments

This research was supported by Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS).

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