Molecular and Biological Characterization of a Mannan-Binding Lectin from the Holothurian Apostichopus Japonicus

Abstract

To elucidate the origin and evolution of mannan-binding lectins (MBL), a new C-type lectin (CTL) specific for high-mannose glycans (MBL-AJ) was isolated from the coelomic plasma of the holothurian Apostichopus japonicus. MBL-AJ has oligomeric forms with identical 17-kDa subunits on SDS-PAGE. Among natural ligands, lectin hemagglutination activity was competitively inhibited by extracellular low-branched, but not high-branched, alpha-D-mannans isolated from marine halophilic bacteria and composed of alpha-1,2 and alpha-1,6 linked D-mannose residues. This suggests that the lectin interacts with backbone or inner side chain mannose residues, but not with terminal ones. The activity of the lectin was Ca(2+)-, pH-, and temperature-dependent. MBL-AJ cDNA was cloned from a holothurian coelomocyte cDNA library. The subunit of the mature protein has 159 amino acids and a single carbohydrate-recognition domain (CRD) of CTL. CRD contains a Glu-Pro-Asp amino acid sequence (EPN-motif) conserved for all known MBLs. A monospecific polyclonal antibody against MBL-AJ was obtained using the 34-kDa lectin dimer as an immunogen. The MBL-AJ has demonstrated immunochemical identity to the earlier isolated mannan-binding CTL from another holothurian, Cucumaria japonica. But a more interesting finding was cross-reactivity of MBL-AJ and human serum MBL detected by the antibody against MBL-AJ. Taking into consideration such MBL-AJ peculiarities as its carbohydrate specificity, the presence of a conserved region forming the mannose-binding site, common antigenic determinants with human MBL, and participation in defense reactions, it is possible that MBL-AJ belongs to the family of evolutionary conserved mannan-binding proteins.

Critical roles of sea cucumber C-type lectin in non-self recognition and bacterial clearance

“…A growing number of CTLs have recently been discovered from marine invertebrates, especially crustaceans and molluscs, including those from F. chinensis [16,21,46,47], L. vannamei [15,19,45,48], Penaeus monodon [49,50], C. farreri [18,23,24,26], A. irradians [22,25] and Crassostrea gigas [51], Crassostrea virginica [52], and Haliotis discus discus [17]. Relatively little information is available on CTLs in echinoderms, and a handful of sea cucumber CTLs have been reported, such as MBL-C (Cucumaria japonica) [53], SJL-I (S. japonicus) [35,36], MBL-AJ and AJCTL (A. japonicus) [37,38]. Although their mRNA expression patterns and biological characteristics have been described, the function of sea cucumber CTLs remains unclear.…”

“…Whlie in sea cucumbers, SJL-I was purified from Stichopus japonicus by affinity chromatography and its complete amino acid sequence was determined by peptide sequence analysis [35,36]. Later, MBL-AJ was isolated from the coelomic plasma of A. japonicus [37]. More recently, AJCTL has also been identified from A. japonicus and its expression profile was characterized [37,38].…”

“…Later, MBL-AJ was isolated from the coelomic plasma of A. japonicus [37]. More recently, AJCTL has also been identified from A. japonicus and its expression profile was characterized [37,38]. Most of these reports have focused on the purification of CTLs, characterization of amino acid sequences and their expression pattern in echinoderms.…”

A review of the immune molecules in the sea cucumber

“…The functional differences between echinoderm and mammalian MBL can be seen in the structural differences of the lectin molecules. According to the data, MBL-AJ contains the conservative carbohydrate-recognizing domain but does not have the collagen-like domain, which is responsible for complement activation [39].…”

Isolation and general characteristics of lectin from the mussel Mytilus trossulus

“…Both metal-independent [8,9] and metal-dependent lectins were found among lectins of marine bivalve mollusks. The activity of the latter was manifested only in the presence of divalent metals [11,12]. MTL is a Ca 2+ -independent lectin because it is capable of causing agglutination of erythrocytes regardless of the presence of Ca 2+ or EDTA.…”