F-type Lectin Research Articles

Host Glycan Recognition by a Pore Forming Toxin

An exposed F-type lectin domain fused to the N-terminus of a cholesterol-dependent cytolysin scaffold allows Streptococcus mitis lectinolysin to cluster at fucose-rich sites on target cell membranes, thereby leading to increased pore-forming toxin activity. In this issue of Structure, Feil and coworkers define the structural basis for lectinolysin glycan-binding specificity.

Diversified Carbohydrate-Binding Lectins from Marine Resources

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.

Molecular cloning and expression analysis of two distinct F-type lectins from the rock bream, Oplegnathus fasciatus

Several lectin families characterized by distinct signature sequence motifs and structural folds, such as C-type, peptidoglycan recognition protein, ficolin, pentraxins, and most recently galectins, have been implicated in immune surveillance. In this study, two distinct F-type lectins RbFTL-1 and RbFTL-2, from the rock bream ( Oplegnathus fasciatus), were identified and their expression was analyzed. The full-length cDNA of RbFTL-1 was composed of 1204 bp with a 945-bp open reading frame (ORF) that encoded a 314 amino-acid protein, while that of RbFTL-2 consisted of 1614 bp with a 951-bp ORF encoding a 316 amino-acid protein. RbFTL-1 and RbFTL-2 mRNAs were predominately expressed in the head-kidney and in the liver, respectively. Levels of the RbFTL-1 mRNA transcript increased up to 5.0- and 2.8-fold in the head-kidney and trunk-kidney compared to the muscle, respectively, while those of the RbFTL-2 mRNA transcript increased up to 12.0-fold in liver. The expression of RbFTL-1 and RbFTL-2 were differentially up-regulated in rock bream challenged with Edwardsiella tarda, Streptococcus iniae, and RSIV, with significant increases at 1 and 3 h post-challenge compared to the controls.

F-type lectin involved in defense against bacterial infection in the pearl oyster ( Pinctada martensii)

In invertebrates and vertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. In the present study, we report the cloning of an F-type lectin (designated as PmF-lectin) from pearl oyster ( Pinctada martensii) using a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of PmF-lectin contains an open reading frame (ORF) of 579 bp coding for192 amino acids. The deduced polypeptide possesses six conserved residues of the F-lectin family critical for the formation of disulfide bonds (Cys 43-Cys 143, Cys 75-Cys 76 and Cys 102-Cys 119). Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) analyses in adult tissues showed that the PmF-lectin mRNA was abundantly expressed in haemocytes and gill, moderately expressed in the mantle, and rarely expressed in other tissues tested. After challenge with Vibrio alginolyticus, expression of PmF-lectin mRNA in haemocytes was dramatically up-regulated, reaching the highest level (13-fold higher than that of the control group) at 3 h post challenge, and then dropped gradually. These results suggest that PmF-lectin is a member of the F-lectin family and is involved in the innate immune response in pearl oyster.

Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (Lateolabrax japonicus)

The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5' untranslated region (UTR) of 39bp, an ORF of 933bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82kDa and a 3' UTR of 332bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2h later the expression level became up-regulated, and 6h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.

Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical 81-Å-long and 60-Å-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking “non-self” carbohydrate ligands and “self” carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1–3Gal/GalNAc-containing and Gal/GalNAcβ1–4GlcNAcβ1–6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1–3Gal, and Galβ1–3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity ( K d = 3 × 10 − 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.

F-type lectin from the sea bass ( Dicentrarchus labrax): Purification, cDNA cloning, tissue expression and localization, and opsonic activity

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel ( Anguilla anguilla) and striped bass ( Morone saxatilis) led to the identification of a novel lectin family (“F-type” lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass ( Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-recognition domains that display the F-type sequence motif. In situ hybridization and immunohistochemical analysis revealed that DlFBL is specifically expressed and localized in hepatocytes and intestinal cells. Exposure of formalin-killed Escherichia coli to DlFBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls, suggesting that DlFBL may function as an opsonin in plasma and intestinal mucus.

Isolation and characterization of a fish F-type lectin from gilt head bream ( Sparus aurata) serum

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose–agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 °C, retained partial activity by treatment at 70 °C, and was fully inactivated at 90 °C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass ( Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.

Characterization of a Binary Tandem Domain F-type Lectin from Striped Bass (Morone saxatilis)

Among other functions, lectins play an important role in the innate immune response of vertebrates and invertebrates by recognizing exposed glycans on the surface of potential pathogens. Despite the typically weak interaction of lectin domains with their carbohydrate ligands, they usually achieve high avidity through oligomeric structures or by the presence of tandem carbohydrate-binding domains along the polypeptide. The recently described structure of the fucose-binding European eel agglutinin revealed a novel lectin fold (the "F-type" fold), which is shared with other carbohydrate-binding proteins and apparently unrelated proteins from prokaryotes to vertebrates, and a unique fucose-binding sequence motif. Here we described the biochemical and molecular characterization of a unique fucose-binding lectin (MsaFBP32) isolated from serum of the striped bass (Morone saxatilis), composed of two tandem domains that exhibit the eel carbohydrate recognition sequence motif, which we designate F-type. We also described a novel lectin family ("F-type") constituted by a large number of proteins exhibiting greater multiples of the F-type motif, either tandemly arrayed or in mosaic combinations with other domains, including a putative transmembrane receptor, that suggests an extensive functional diversification of this lectin family. Among the tandem lectins, MsaFBP32 and other tandem binary homologues appear unique in that although their N-terminal domain shows close similarity to the fucose recognition domain of the eel agglutinin, their C-terminal domain exhibits changes that potentially could confer a distinct specificity for fucosylated ligands. In contrast with the amniotes, in which the F-type lectins appear conspicuously absent, the widespread gene duplication in the teleost fish suggests these F-type lectins acquired increasing evolutionary value within this taxon.