Novel mannose-specific lectins found in torafugu, Takifugu rubripes: A review

In earlier work, we identified a novel mannose-specific lectin, termed pufflectin-s, from the skin mucus of torafugu, Takifugu rubripes. We here make a brief review on the lectin. The amino acid sequence of pufflectin-s shares sequence homology with mannose-binding lectins of monocotyledonous plants, and has conserved two of three carbohydrate recognition domains, QDNVY motifs, of these plant lectins. By site-directed mutagenesis, we verified that the QDNVY motif in the N-terminal region was critical to the mannose-binding function of pufflectin-s. RT-PCR and Northern blot analyses indicated that the pufflectin-s gene is expressed in the gill, oral cavity wall, esophagus, and skin. In addition, an isoform, pufflectin-i, which shares 91.4% amino acid identity with pufflectin-s, was isolated from the intestine. Using immunohistochemistry, pufflectin-s could be detected exclusively in the epithelial cells of the skin, gill, oral cavity wall and esophagus, whereas pufflectin-i was observed in both mucous and epithelial cells in the intestine. Nevertheless, mRNAs for both pufflectins were detected only in epithelial cells of these tissues with in situ hybridization. Pufflectin-s agglutinated some bacteria isolated from rearing water and from fish skin. This lectin also bound to a parasite, Heterobothrium okamotoi, suggesting that it may play an important role in the self-defense system of fugu.     

Expression of genes for two C-type lectins during budding of the ascidian Polyandrocarpa misakiensis

Two closely related cDNA fragments, named pTC14-1 and pTC14-2, encoding C-type lectins were cloned from the budding ascidian Polyandrocarpa misakiensis by means of the polymerase chain reaction. The amino acid sequence deduced from pTC14-1 was identical to that of a 14-kDa calcium-dependent galactose-binding lectin, TC-14, that had been purified from this species. Between the two clones, nucleotide sequence similarity was 90%, whilst that of the deduced amino acid sequences was 82%. The cDNA inserts of these clones hybridized weakly with each other. Antisense RNA probes prepared from these clones gave intense hybridization signals on Northern blots of the W strain, but very weak signals on those of the other strains. Therefore, both clones were suggested to originate from the W strain, but from two separate genes, since the base substitution was scattered throughout the entire translated region. The amount of TC14-1 mRNA increased during bud development, and peaked at 36 h after separation of the bud from the parental body wall. At this stage, extracellular matrix containing TC-14 lectin developed in the mesenchymal space around the morphogenetic region of the bud. There was much less TC14-2, than TC14-1 mRNA at every stage of bud development. TC14-1 and TC14-2 mRNAs were detected on Northern blots of RNAs from adults and growing buds, suggesting that these genes can be used as the earliest markers of budding in this species.     

In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus

The selection of probiotics for aquaculture is usually based on their antagonism towards pathogens. However, other criteria such as growth, attachment to intestinal mucus and production of beneficial compounds should also be considered. We suggest a protocol for the isolation and selection of potential probiotic bacteria based on their in vitro growth characteristics and propose a ranking index (RI) to screen potential aquaculture probionts. We suggest that the lag period and doubling time are the most important criteria for the comparison of growth curves, hence the RI is based on the doubling time (t(d)) and lag period (lambda) obtained from the growth profile of each bacterium. Bacteria were isolated from the gut of the common clownfish, Amphiprion percula, and screened for antagonistic activity towards seven aquatic pathogens. All five candidate probiotics showed antagonism to various aquatic pathogens. When grown in intestinal fish mucus no probiotic had a RI higher than the two tested pathogens (Aeromonas hydrophila and Vibrio alginolyticus). However, candidate probiont AP1 had a faster specific growth rate (micro) (0.05) than the pathogens (0.049 and 0.047 respectively), while AP5 grown in marine broth had a shorter lag period than the pathogens. Strategies to increase probiotic concentration include the inoculation of high concentrations and the preconditioning of these bacteria to reduce the lag period. It should be tested whether or not such strategies will allow the probiotic bacteria to dominate initially and thereby gain a competitive advantage. This could become an important aspect under in vivo conditions where both attachment and nutrient supply differ from that found in in vitro studies.     

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.     

Drag reduction of fish skin mucus: Relationship to mode of swimming and size

Drag reduction by skin mucus was measured from Gulf of Eilat fish. Activity of burst swimmers was greater than manoeuvre swimming fish. Large fish from the same species had higher mucus drag reducing activity than smaller specimens.     

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

Towards functional glycomics by localization of binding sites for tissue lectins: lectin histochemical reactivity for galectins during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster

Endogenous lectins act as effectors of cellular activities such as growth regulation, migration, and adhesion. Following their immunohistochemical localization in our previous study (Saussez et al. in Histochem Cell Biol 123:29–41, 2005) we purified several galectins and used them as tools for monitoring accessible binding sites. Herein, we report the use of galectin histochemistry for the analysis of diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT). Sections of normal kidney and DES-treated kidney were analyzed with biotinylated galectins-1, -3 (full-length and truncated), and -7. Accessible binding sites were detected, localization was predominantly extracellular and confined to medium-sized and large tumors. Monitoring the SHKT-derived HKT-1097 line, processed in vitro or as xenograft material, cytoplasmic and nuclear staining for galectins-1, -3, and -3tr could be observed. Adaptation of SHKT cells to long-term growth in culture is thus associated with emergence of this signal. Our data set illustrates the feasibility to complement immunohistochemical data by application of the tissue lectins as probes, and to detect regulation of galectin reactivity with differential characteristics within tumor progression in vivo and unique features of the tumor cell line in vitro and in vivo.     

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways “Legionellosis” and “Complement and coagulation cascades” were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein–protein interaction network analysis and “Response to stress” GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.     

Antibacterial factors in skin mucus of rabbitfishes

Aqueous extracts from the skin mucus of two species of rabbitfishes Siganus fuscescens and S. guttatus showed antibacterial activity against gram-negative bacteria. Stability tests and chromatographic analyses suggested that the S. fuscescens antibacterial factor is an acidic glycoprotein with a molecular mass of 400 kDa.     

Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 g/g at 29 d then decreasing to 2.5 g/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.Abbreviations DAS double antibody sandwich - ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - PSL peanut seed lectin - SBL soybean lectin     

Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri

Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca²⁺. We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents.     

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue α helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as—no voltage dependence, only one unitary conductance, linear relation ship current-voltage—, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

Stability of spatial pattern of fish species diversity in the Strait of Sicily (central Mediterranean)

In this paper, species diversity of demersal fish communities was analysed over an area covering about 45,000 km² of the Italian side of the Strait of Sicily (central Mediterranean). Fish abundance data come from a 10-year series (1994–2003) of experimental bottom trawl surveys carried out within the framework of the international program MEDITS. A simple GIS-based method was proposed to identify areas supporting high or low values of diversity and evaluate their temporal stability. A well-defined spatio-temporal pattern in diversity emerged from the analysis, with some areas of great ecological relevance being identified. Importantly, the greatest diversity within the fish communities was consistently seen at the offshore bank on the western part of the south Sicilian shelf (Adventure Bank). The site also supports high total biomass of demersal resources and shows the presence of species of great concern to fisheries. Results suggest that Adventure Bank represents a priority site for investigating the possibility of innovative management of marine ecosystems and demersal fisheries in offshore zones.     

Molecular approaches to diversity of populations of apicomplexan parasites

Apicomplexan parasites include many parasites of importance either for livestock or as causative agents of human diseases. The importance of these parasites has been recognised by the European Commission and resulted in support of the COST (Cooperation in Science and Technology) Action 857 ‘Apicomplexan Biology in the Post-Genomic Era’. In this review we discuss the current understanding in ‘Biodiversity and Population Genetics’ of the major apicomplexan parasites, namely the Eimeria spp., Cryptosporidium spp., Toxoplasma gondii, Neosporacaninum, Theileria spp. and Plasmodium spp. During the past decade molecular tools for characterizing and monitoring parasite populations have been firmly established as an integral part of field studies and intervention trials. Analyses have been conducted for most apicomplexan pathogens to describe the extent of genetic diversity, infection dynamics or population structure. The underlying key question for all parasites is to understand how genetic diversity influences epidemiology and pathogenicity and its implication in therapeutic and vaccination strategies as well as disease control. Similarities in the basic biology and disease or transmission patterns among this order of parasites promote multifaceted discussions and comparison of epidemiological approaches and methodological tools. This fosters mutual learning and has the potential for cross-fertilisation of ideas and technical approaches.     

Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose

6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ～3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics.