Identification and molecular characterization of a mucosal lectin (MeML) from the blue mussel Mytilus edulis and its potential role in particle capture

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Abstract

Molecular recognition of food particles has been suspected to play an important role in particle selection in suspension feeding bivalves. Lectins are a group of sugar-binding proteins that are widely involved in biological recognition. They have been reported in mucus covering bivalves feeding organs and were recently shown to mediate particle sorting in these animals. In this study, we report a novel putative C-type lectin from the blue mussel Mytilus edulis. The cDNA of this lectin (hereby designated MeML for M. edulis mucocyte lectin) is 459 bp long encoding a 152-residue protein. MeML presents a signal peptide and a single carbohydrate recognition domain (CRD) which contains a QPS (Gln, Pro, and Ser) motif and two putative conserved sites, WND and ENC, for calcium binding. MeML was expressed in mucocytes lining the epithelium of pallial organs (gills, labial palps and mantle) and intestine, and its expression was significantly up-regulated following starvation. MeML transcript was not detected in other tissues including hemocytes. MeML is suspected to play a role in the capture of food particle which further support the involvement of this lectin in particle selection mechanism.

Introduction

In the aquatic environment, communication between organisms is often based on molecular language. In numerous physiological processes (defense, reproduction, and predation) marine organisms interact with each other using thousands of organic metabolites belonging to diverse chemical groups (Faulkner, 2002, Hay, 1996 and references therein). Among molecules known for non-self recognition and cell-to-cell interactions, lectins are a large and diverse group of sugar-binding proteins that specifically and reversibly bind to glycans covering living cells (Sharon and Lis, 2004, Vasta, 2009). Their functions are diverse and they have been described to play a role in the defense mechanism (Kenjo et al., 2001, Tasumi and Vasta, 2007), in reproduction (Springer et al., 2008), in parasitism (Hager and Carruthers, 2008, Stevens et al., 2006), and in symbiosis (Bulgheresi et al., 2006, Nyholm and McFall-Ngai, 2004, Wood-Charlson et al., 2006). They can assist the organism by immobilizing particles through agglutination (Fisher and Dinuzzo, 1991, Pales Espinosa et al., 2009) and encapsulation (Koizumi et al., 1999) or can initiate a cascade of events leading, for example, to host colonization (Nyholm and McFall-Ngai, 2004) or to limit pathogen infection (Holmskov et al., 2003).

Lectins are ubiquitously distributed in nature as they are found in viruses, bacteria, fungi, plants, invertebrates and vertebrates (Sharon and Lis, 2004, Vasta and Ahmed, 2008). In bivalves, lectins have been mostly described in hemolymph (Zhang et al., 2009), associated or not with hemocyte membranes (Tasumi and Vasta, 2007), and linked to the defense mechanism (Fisher and Dinuzzo, 1991, Minamikawa et al., 2004, Tripp, 1992, Zhang et al., 2009). In some rare cases, bivalve lectins have been found to be potentially involved in other functions. For example, in Codakia orbicularis, a clam known for its symbiotic relationship with a sulfide-oxidizing chemoautotrophic bacteria (Frenkiel and Moueza, 1995), the lectin “codakine” has been found to be the predominant protein in the gill (Gourdine and Smith-Ravin, 2002) leading Gourdine et al. (2007) to propose its involvement in the mediation of symbiosis. Additionally, the presence of lectins have been suspected (Fisher, 1992) and recently demonstrated (Pales Espinosa et al., 2008) in mucus covering pallial organs (gills, labial palps) in the oyster Crassostrea virginica (Pales Espinosa et al., 2009, Pales Espinosa et al., 2010) and the mussel Mytilus edulis (Pales Espinosa et al., submitted).

Suspension feeding bivalves are well known to be able to select among particles (Cognie et al., 2003, Newell and Jordan, 1983, Pales Espinosa et al., 2008, Ward and Shumway, 2004). Although some aspects of the selection process have been elucidated, the actual mechanism(s) by which particles of poor quality are rejected into pseudofeces while those of higher quality are ingested remain unclear. Among theories advanced in the literature, some studies support the idea that bivalves use chemical cues to discriminate among particles (Beninger and Decottignies, 2005, Beninger et al., 2004, Kiorboe and Mohlenberg, 1981, Newell and Jordan, 1983, Pales Espinosa et al., 2007, Shumway et al., 1985, Ward and Targett, 1989). More recently, our results in oysters (Pales Espinosa et al., 2009, Pales Espinosa et al., 2010) and mussels (Pales Espinosa et al., submitted) showed that particle selection in bivalves is mediated by interactions between lectins present in mucus covering feeding organs and carbohydrates associated with the surface of suspended food particles. Although our previous studies represent, to the best of our knowledge, the first indications for the involvement of lectins in the feeding mechanism of metazoans, carbohydrate–lectin interactions have already been shown to be involved in the feeding mechanisms of predatory protozoans. For example, previous studies have demonstrated the involvement of mannose-binding lectins as a feeding receptor for recognizing preys in the marine dinoflagellate Oxyrrhis marina (Wootton et al., 2007) and in the amoeba Acanthamoebe castellanii (Allen and Dawidowicz, 1990).

In this study, we screened public EST (Expressed Sequence Tag) databases and used a diverse set of molecular techniques to identify lectin candidates that are produced in the feeding organs of the blue mussel, M. edulis. These investigations allowed the identification of a secretory lectin (hereby designated MeML for M. edulis mucocyte lectin) that is specifically produced in mucocytes lining mussel feeding organs (gills, labial palps). The full lectin sequence is presented and the expression of this molecule in response to starvation was investigated. Results highlight the potential involvement of this lectin in particle capture processes.

Section snippets

Animals

Adult (60 to 70 mm in length) blue mussels, M. edulis, were collected from Long Island Sound (Port Jefferson, NY, USA). Their external shell surface was scrubbed to remove mud and marine life. Mussels were then randomly subdivided into 3 different groups. The first 2 groups were immediately used for RNA extraction/cDNA amplification or in situ hybridization analysis. The last group was acclimated in the lab before being used in the starvation study (see below).

RNA extraction

Eight mussels were bled from the

Identification of MeML

Among the 13 tested ESTs, 3 candidates were not detected in any of the tested organs and 9 were homogenously expressed in all tissues including hemocytes. Only one candidate (EST 11) was expressed in the labial palps, gills and the digestive gland (weak signal) but not in hemocytes (Fig. 1). Further analysis of this EST revealed that it codes for a complete protein, hereby designated MeML [GenBank accession no. HM049926]. The complete sequence consisted of a 459 bp encoding for a predicted

Discussion

Most previously described lectins in marine invertebrates were identified in hemolymph or hemocytes and were suspected or found to be involved in the defense system against pathogens (Tasumi and Vasta, 2007, Vasta et al., 1984, Zheng et al., 2008). Given their diverse molecular structures and their specific interactions with carbohydrate moieties, lectins were also found in organs and tissues other than blood, and have been shown to play roles as diverse as the establishment of symbiosis (

Acknowledgments

This work was funded in part by a grant from the National Science Foundation to EPE and BA (IOS-0718453).

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