Elsevier

Fish & Shellfish Immunology

Volume 67, August 2017, Pages 675-683
Fish & Shellfish Immunology

Full length article
Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas

https://doi.org/10.1016/j.fsi.2017.06.058Get rights and content

Highlights

  • Antimicrobial effect of 60S ribosomal protein L29 (cgRPL29) was firstly identified from the Pacific oyster, crassostrea gigas.

  • cgRPL29 consists of 54 amino acids with 6484.6 Da and showed strong antimicrobial activity against bacteria.

  • cgRPL29 was constitutively expressed and might form unordered/non-amphipathic conformation.

  • cgRPL29 might contribute to innate defense function in the Pacific oyster, crassostrea gigas.

Abstract

We purified an ∼6.4-kDa antimicrobial peptide from an acidified gill extract of the Pacific oyster, Crassostrea gigas, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide was composed of 54 amino acids and had a molecular weight of 6484.6 Da. Comparison of the amino acid sequence and molecular weight with those of other known proteins or peptides revealed that the peptide had high identity with the 60S ribosomal protein L29, and so was named cgRPL29.

The full-length cgRPL29 cDNA of the Pacific oyster comprised 325-bp, including a 5'-untranslated region (UTR) of 100-bp, a 3′-UTR of 57-bp, and an open reading frame of 168-bp encoding 55 amino acids, with a Met residue at the N-terminus. The cgRPL29 mRNA tissue distribution suggested that it is constitutively expressed in a non-tissue-specific manner. Secondary structural prediction and homology modeling indicated cgRPL29 have an unordered structure containing two partial α-helical regions. This is to our knowledge the first report of the antimicrobial effect of the 60S ribosomal protein L29 from marine invertebrates.

Introduction

Oysters are sessile and filter-feeding marine invertebrates that live in shallow-water regions such as intertidal zones or estuaries. Their habitats and feeding styles result in tremendous environmental stress, which can increase the risk of infection by diverse pathogens including bacteria, viruses, fungi, and parasites; in addition, temperature/salinity fluctuation can affect the immune status of oysters [1]. To combat potential invaders, marine invertebrates have developed effective defense mechanisms. Marine invertebrates, including oyster species, lack acquired immunity based on T-lymphocyte subsets and clonally derived immunoglobulins from B-lymphocytes, and rely solely on an innate immune system with humoral and cellular branches to protect against pathogens [2]. Cellular immunity is mediated by immune cells (e.g., hemocytes) and involves phagocytosis, encapsulation, and respiratory burst; humoral immunity is mediated by lectins, agglutinins, antimicrobial proteins/peptides and lysosomal enzymes in plasma [2], [3]. There is increasing evidence that antimicrobial peptides (AMPs) play an important role in innate immunity [2].

AMPs are conserved components of the innate immune system of all organisms, are amphipathic, and comprise 12–50 amino acids with a positive net charge, forming an α-helix or β-sheet [4], [5]. Based upon their structure and composition, AMPs target the negatively charged bacterial membrane via ionic interactions, and undergo hydrophobic interactions with the acyl groups of phospholipids, resulting in pore formation and membrane disruption [6]. To date, several hundred AMPs have been identified from diverse organisms from fungi to human, and are considered key components of the host innate immune system [7].

Three categories of AMPs have been identified in oysters: i) AMPs, ii) antimicrobial proteins (>50 kDa), and iii) ubiquitous proteins with antimicrobial activity in addition to some other functions. To date, various intact antimicrobial peptides and proteins have been reported in oysters, the best-known of which is defensin [8]. The first oyster defensin was identified in the gill of the American oyster, Crassostrea virginica, using biological approaches [9]. Three oyster defensins have been identified in the Pacific oyster, Crassostrea gigas, using genomic approaches [8], [10]. Other AMPs including cgMolluscidin, big defensins, and cg-Prps have also been identified in Pacific oysters [11], [12], [13]. Furthermore, several antimicrobial proteins (>50 kDa) including cgBPI and cg-Mpeg1 have also been identified in Pacific oysters using genomic approaches [14], [15]. The third category includes AMPs derived from ubiquitous proteins (for example, histones, ubiquitin, and ribosomal proteins) not previously associated with immunity. Parasins were the first reported AMPs derived from a histone protein (H2A) of catfish [16]. The best-studied histone-derived AMPs are buforins, which exhibit 100% sequence similarity with the N-terminal region of histone H2A, which interacts directly with nucleic acids [17]. The potent AMP Rp S30 from rainbow trout is identical to the 40S ribosomal protein small subunit [18]. In invertebrates, oyster ubiquitin (cgUbiquitin) and oyster histones (CvH2Bs, CgH1/H5) have antimicrobial activity in addition to their biological functions [19], [20], [21]. However, relatively little is known about the roles of ubiquitous proteins in innate immunity.

In this investigation, we report the purification, cDNA cloning, tissue distribution and predicted structural information of a ubiquitous antimicrobial protein (60S ribosomal protein L29) from an acidified gill extract of the Pacific oyster, C. gigas.

Section snippets

Tissue extraction

Fifty adult oysters, 6.0–11.0 cm in shell length, were obtained from a local seafood market in Gunsan, Korea in January 2015. Each oyster was processed individually immediately upon receipt in the laboratory. The shell was gently washed with tap water and opened with a shucking knife. Gills were harvested and placed into a 15-mL sterile polypropylene tube on wet ice until 5 mL of each tissue had been harvested from a total of five or six oysters. The tissues were added to a 50-mL beaker

Antibacterial and hemolytic activities of the extract

We initially subjected acidified, crude extract from the Pacific oyster gill to URDA (Fig. 1A); the results indicated potent antimicrobial activity against B. subtilis KCTC1021 and E. coli D31, with greater activity against the former than the latter. Treatment of extract with trypsin significantly decreased its antimicrobial activity, suggesting the presence of a proteinaceous antimicrobial compound. Therefore, we selected B. subtilis KCTC1021 for use in subsequent assays of antimicrobial

Discussion

Invertebrates lack acquired immunity but have an innate immune system with both humoral and cellular branches [2]. Antimicrobial peptides or proteins are major first-line defense factors in the humoral immune systems of invertebrates. To date, three categories of antimicrobial peptides/proteins have been identified in oysters: AMPs, antimicrobial proteins (>50 kDa), and ubiquitous proteins with functions other than their antimicrobial activity. Various antimicrobial peptides or proteins

Acknowledgments

This research was supported by the project titled “Development of Biomaterials derived from the Western tideland organisms” (grant number: 20140447), funded by the Ministry of Oceans and Fisheries, Korea. This work was also supported by a grant from the National Institute of Fisheries Science in the Republic of Korea (grant number R2017022).

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1

Both authors contributed equally to this work.

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