Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

Abstract

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.

Introduction

Invertebrates lack antibody-mediated humoral immune systems. However, they are believed to possess efficient host defense mechanisms by virtue of their humoral defense molecules, which function similarly to antibodies (Arason, 1996). Lectins are protein complexes with carbohydrate-specific binding properties that have been widely expressed in invertebrates including bivalve molluscs. Regarding their physiological functions, bivalve lectins and other invertebrate humoral lectins reportedly act as opsonins for phagocytosis by the hemocytes and non-self molecule recognition receptors (Vasta et al., 1994, Olafsen, 1995, Wang et al., 2007).

Lectins have been isolated and characterized from the hemolymph of the following bivalve mollusc species: two lectins, gigalin E and H, purified from the Pacific oyster, Crassostrea gigas (Hardy et al., 1977, Olafsen et al., 1992); a galactose-specific lectin from the pearl oyster, Pinctada fucata martensii (Suzuki and Mori, 1989); a calcium-dependent lectin (C-type lectin) from the Chilean oyster, Ostrea chilensis (Minamikawa et al., 2004) and the mussel, Mytilus edulis (Renwrantz and Stahmer, 1983); a galactan-reactive agglutinin from the clam, Tridacna maxima (Baldo et al., 1978); a galactosyl-binding lectin from the blood clam, Anadara granosa (Dam et al., 1992); and two lectins, modiolin E and H isolated from the horse mussel, Modiolus modiolus (Tunkijjanukij et al., 1997). In addition, analyses of lectin genes in bivalve molluscs have been further developed in recent years. The C-type lectin (CFLec-1) was cloned from the Zhinkong scallop, Chlamys farreri (Wang et al., 2007). Furthermore, for the C-type lectin (CgCLec-1), the full coding sequence from C. gigas was identified and tissue expression analyses conducted (Yamaura et al., 2008). Some among these bivalve lectins act as opsonins (Renwrantz and Stahmer, 1983, Olafsen et al., 1992) and agglutinate various bacteria (Fisher and DiNuzzo, 1991, Tunkijjanukij et al., 1997). Therefore, lectins appear to play a critical role in the host-defense mechanisms of bivalve molluscs, both by recognizing and binding to pathogenic microorganisms and by opsonizing for phagocytic hemocytes.

The Manila clam, Ruditapes philippinarum, is an economically important bivalve for Southeast Asia and Japan. In recent years, incidents of mass mortality of clams caused by widely various agents have become a serious problem. Both cultured and natural production in Japan have decreased considerably: a pressing need exists to clarify the causes of these incidents and to develop research into host defense mechanisms.

In clams cultured in Korea, Bulgakov et al. (2004) reported that a lectin with a calcium-dependent carbohydrate-binding activity was purified from body extracts and was characterized for its biological and biochemical properties: molecular mass and the subunit structure of the lectin, in addition to the optimal pH, temperature dependence, and sugar specificity of lectin activity, etc. The lectin (designated Manila clam lectin, MCL) was bound to the surface of hypnospores from Perkinsus sp. or P. olseni, which is a protozoan parasite of R. philippinarum. In addition, MCL is reportedly synthesized in the hemocytes of the Manila clams and is expected to function as an opsonin (Kim et al., 2006). For that reason, MCL is thought to serve as a pathogen-recognition receptor (PRR) and/or opsonin in host defense mechanisms against infectious microorganisms or parasites via its multivalent carbohydrate-binding capability. Furthermore, expression sequence tag (EST) sequences have been collected and analyzed from the hemocytes of the Manila clams infected with P. olseni (Kang et al., 2006). Seven lectin clones were identified in the Manila clam cDNA library; among the lectin clones, two full-length cDNAs of the lectins were cloned. One is a simple C-type lectin, MCL-3; the other is a sialic acid-binding lectin. However, the biological and biochemical properties of the other lectins in the Manila clam remain to be clarified.

We report the purification and characterization of a novel isoform of the Manila clam Lectin (MCL-4) from the hemolymph plasma of R. philippinarum, with the intention of elucidating its host defense-related roles for marine bacteria, including a pathogenic Vibrio species.