Novel C-type lectin from razor clam Sinonovacula constricta agglutinates bacteria and erythrocytes in a Ca2+-dependent manner.

Among its other physiological roles, C-type lectins functioned as pattern recognition receptors (PRR) in innate immunity received much attention. In the present study, a novel C-type lectin was identified and characterized from the invertebrate razor clam Sinonovacula constrict and designated as ScCTL. The complete cDNA sequence of ScCTL was 828 bp in length and coded a secreted polypeptide of 158 amino acids with a typical CRD domain. Multiple sequence alignments combined with phylogenetic analysis both collectively confirmed that ScCTL was a novel member belong to lectin family. Spatial expression distribution analysis revealed that ScCTL was extensively expressed in all of the examined tissues, and the highest expression was detected in the hepatopancreas. After 1 × 107 CFU/mL Vibrio parahaemolyticus challenge by immersion infection, the ScCTL transcript in hepatopancreas and gill were markedly upregulated and arrived the maximum levels at 24 or 12 h after challenge, respectively. Recombinant ScCTL could agglutinate not only all tested bacteria but sheep and mouse erythrocyte in the presence of Ca2+. All of our studies suggested that ScCTL performed important roles in protecting cells from pathogenic infection in S. constrict.

FmLC6: An ultimate dual-CRD C-type lectin from Fenneropenaeus merguiensis mediated its roles in shrimp defense immunity towards bacteria and virus.

C-type lectins are a member of pattern recognition receptors (PRRs) that can interact with pathogen-associated molecular patterns of invading microorganisms by using their conserved motifs in carbohydrate recognition domain (CRD). The binding can trigger various immune responses in both direct and indirect mechanisms. Hereby, an ultimate C-type lectin with dual CRDs each of which containing a different motif was identified from hepatopancreas of Fenneropenaeus merguiensis (mentioned as FmLC6). The full-length cDNA of FmLC6 consisted of 1148 bp comprising one 1005 bp open reading frame (ORF) encoding a signal peptide and a mature protein of 317 residues. FmLC6 was composed of two CRDs with a highly conserved QPD (Gln-Pro-Asp) motif and one variant EPQ (Glu-Pro-Gln) motif for illustrating the carbohydrate binding affinity. The transcription of FmLC6 was detected only in hepatopancreas of normal shrimp. After injection with pathogens or immunostimulants, the expression of FmLC6 was significantly up-regulated and reached the highest level at 12 h post-injection except with lipoteichoic acid challenge. The FmLC6 expression was severely suppressed by knockdown based-silencing. This gene silencing with co-injection by Vibrio parahaemolyticus caused increasing in cumulative mortality and reduction of the median lethal time. Purified recombinant proteins of an entire ORF and two individual CRDs of FmLC6 produced in Escherichia coli could induce a broad spectrum of microbial agglutination with calcium dependence. The agglutination induced by rFmLC6, rCRD1 and rCRD2 was suppressed by galactose plus mannose, galactose and mannose, respectively which this event was confirmed by the inhibition of hemagglutination. All three recombinant proteins possessed ability to inhibit the bacterial growth with a dose-response. Purified rFmLC6 could bind directly to white spot syndrome virus particles and also its recombinant proteins including VP15, VP39A and VP28 with different affinity. Altogether, these results indicate that FmLC6 acts as a PRR to recognize invading microorganisms and leads to mediating the immune response to cooperation in pathogenic elimination via the binding, agglutination and antimicrobial activity.

Characterization and functional analysis of a novel C-type lectin from the swimming crab Portunus trituberculatus.

C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full-length cDNA of PtCTL1 was of 702 bp, containing a 5' untranslated region (UTR) of 91 bp, a 3' UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca2+-dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca2+-dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate.

Characterization and expression analysis of an intelectin gene from Megalobrama amblycephala with excellent bacterial binding and agglutination activity.

Intelectin is a recently discovered lectin that plays vital roles in the innate immune response, iron metabolism and early embryogenesis. The structure, expression pattern and function of intelectin in mammals and amphibians have been well studied, while not well known in fish. In this study, we cloned a intelectin (MamINTL) gene from blunt snout bream (Megalobrama amblycephala), examined its expression patterns and explored its roles in innate immune response. The MamINTL cDNA encoded 312 amino acids, with a pro-protein of 34 kDa. Sequence analysis revealed the presence of a fibrinogen-related domain and eight conserved cysteine residues in the MamINTL. The MamINTL mRNA was detectable at various developmental stages, while it increased significantly post hatching. In healthy adult M. amblycephala, MamINTL was detected in various tissues with the highest expression in the liver. Upon challenge with Aeromonas hydrophila, significantly up-regulated expression of the MamINTL mRNA was observed in the liver, spleen, kidney, intestine and gill. In addition, increased level of MamINTL protein detected by Western Blotting was also observed in the liver, kidney and spleen, indicating the participation of MamINTL in the immune response. Immunohistochemistry analysis of the M. amblycephala liver sections showed significant changes in expression and location post infection. In addition, the recombinant MamINTL showed excellent binding and agglutination activity against GFP-expressed E. coli in a Ca2+-dependent manner. Generally, the present study provides clues for a better understanding of the characterization, expression patterns and functions of fish intelectins.

Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides.

Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids.

Agglutination of intravenously administered phosphatidylcholine-containing lipid emulsions with serum C-reactive protein.

BACKGROUND: C-reactive protein (CRP) is able to bind phospholipids in the presence of calcium. We wanted to investigate the reaction of CRP with various commercial fat emulsions and to explore the impact of CRP agglutination on serum CRP levels. MATERIALS AND METHODS: Serum specimens were mixed with Intralipid 20% (soybean oil-based fat emulsion), Structolipid (structured oil-based fat emulsion), Omegaven (fish oil-based fat emulsion), or SMOFlipid (mixed soybean oil-, olive oil-, and fish oil-based emulsion) in Tris-calcium buffer (pH 7.5). After 30 minutes of incubation at 37°C, CRP-phospholipid complexes were turbidimetrically quantified and flow cytometric analysis was performed. Similarly, CRP complexes were monitored in vivo, following administration of fat emulsion. RESULTS: CRP was able to agglutinate phospholipid-containing lipid droplets present in the soybean oil-based fat emulsion and the structured oil-based fat emulsion. To a lesser extent, agglutination was observed for fish oil-containing fat emulsions, whereas no agglutination was noticed for the mixed soybean oil-, olive oil-, and fish oil-based emulsion. Results for propofol-containing emulsions were comparable. Agglutination correlated with phospholipid content of the emulsions. When in vivo agglutination occurred, plasma CRP values dropped due to consumption of CRP by phospholipid-induced agglutination. CONCLUSIONS: In this in vitro experiment, we demonstrated agglutination of CRP with phospholipids in various fat emulsions. Research studies are required in patients to determine which effects occur with various intravenous fat emulsions.

Immunomodulatory and hemagglutinating activities of acidic polysaccharides isolated from Combretum racemosum.

Extracts of leaves of different species of the genus Combretum have been used historically to treat a variety of medicinal problems. However, little is known about the active components conferring therapeutic properties to these extracts. In the present studies, we evaluated biochemical properties and immunomodulatory activity of polysaccharides isolated from the leaves of Combretum racemosum. Water-soluble polysaccharides from leaves of C. racemosum were extracted and fractionated by DEAE-cellulose and Diaion HP-20 to obtain a Diaion-bound fraction, designated Combretum polysaccharide-acidic bound or CP-AB, which was eluted with methanol, and an unbound fraction, designated as CP-AU. Molecular weight determination, sugar analysis, and other physical and chemical characterization of the fractions were performed. Fraction CP-AU (mol. weight 5.0 kDa) contained type II arabinogalactan and had potent immunomodulatory activity, inducing the production of interleukin (IL)-1ß, -6, -10, and tumor necrosis factor-α (TNF-α) by human peripheral blood mononuclear cells (PBMC) and MonoMac-6 monocytic cells. Likewise, intraperitoneal administration of CP-AU increased in vivo serum levels of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in mice. CP-AU-induced secretion of TNF-α in PBMC was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS. Treatment with CP-AU induced phosphorylation of Akt2, Akt3, GSK-3ß, HSP27, mTOR, and all p38 MAPK isoforms (α, ß, δ, and γ), as well as stimulation of AP-1/NF-κB transcriptional activity. In addition, CP-AU effectively agglutinated erythrocytes from several species, including human, mouse, and rabbit. In contrast, fraction CP-AB was inactive in all biological tests, including cytokine production and hemagglutination. These data suggest that at least part of the beneficial therapeutic effects reported for the water extracts of leaves from C. racemosum are due to modulation of leukocyte functions.

Efficacy of Baptisia tinctoria in the treatment of typhoid: its possible role in inducing antibody formation.

Typhoid is one of the most serious infectious bacterial diseases in third world countries. It is usually treated by traditional antibiotics but due to the appearance of antibiotic resistant strains physicians opt for phyto products and other alternative medicines for the treatment of typhoid. Baptisia, an extract from indigo plant root, has been proved to be highly effective ultradilute medicine for the treatment of typhoid; however, the mode of action of the ultradilute extract is uncertain. Due to the antigenic variations of Salmonella it seems to induce immuno system by activating both T and B cells by the formation of antibodies. This principle seems to be highly effective for the development of typhoid vaccine. The present studies found that Baptisia administration possibly caused a salmonella-like reaction in the body as this extract produces an endogenous antibody similar to salmonella reaction. Thus, this study suggests that Baptisia tinctoria extract can be used for the prevention and treatment of typhoid.

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss).

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.

Double liposomes mediated dual drug targeting for treatment of Helicobacter pylori infections.

In the present study the potential of phosphatidylethanolamine (PE) lipid anchored double liposomes (DL) to incorporate two drugs in a single system is exploited as a tool to augment the H. pylori eradication rate. Preparation of DL involves two steps, first formation of primary (inner) liposomes by thin film hydration method containing one drug, then addition of suspension of inner liposomes on thin film of lipid containing the other drug. The success of formation of DL was characterized by optical and transmission electron microscopy. Quantitation of DL-bacterial interaction was evaluated in terms of percent growth inhibition (%GI) on reference strain of H. pylori ATCC 26695. To confirm specific binding efficacy of DL to H. pylori PE surface receptor we performed an agglutination assay. Agglutination in DL treated H. pylori suspension suggested selectivity of DL towards the PE surface receptor of H. pylori. Monotherapy is generally not recommended for treatment of a H. pylori infection due to the danger of development of resistance and unacceptably low eradication rates. Therefore combination therapy with amoxicillin trihydrate (AMOX) as anti-H. pylori agent and ranitidine bismuth citrate (RBC) as antisecretory agent were selected for the study with an expectation that this dual-drug delivery approach will exert acceptable anti-H. pylori activity.

[Development of anti-Alzheimer's disease drug based on beta-amyloid hypothesis].

Currently, there are five anti-Alzheimer's disease drugs approved. These are tacrine, donepezil, rivastigmine, galantamine, and memantine. The mechanism of the first four drugs is acetylcholinesterase inhibition, while memantine is an NMDA-receptor antagonist. However, these drugs do not cure Alzheimer's, but are only symptomatic treatments. Therefore, a cure for Alzheimer's disease is truly needed. Alzheimer's disease is a progressive neurodegenerative disease characterized by cognitive deficits. The cause of the disease is not well understood, but research indicates that the aggregation of beta-amyloid is the fundamental cause. This theory suggests that beta-amyloid aggregation causes neurotoxicity. Therefore, development of the next anti-Alzheimer's disease drug is based on the beta-amyloid theory. We are now studying natural products, such as mulberry leaf extracts and curcumin derivatives, as potential cure for Alzheimer's disease. In this report, we describe some data about these natural products and derivatives.

SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties.

Over expression of lectin genes in E. coli often gives inclusion bodies that are solubilised to characterize lectins. We made N-terminal fusion of the Allium sativum leaf agglutinin (ASAL) with SUMO (small ubiquitin related modifier) peptide. The SUMO peptide allowed expression of the recombinant lectin in E. coli, predominantly in soluble form. The soluble fusion protein could be purified by immobilized metal affinity column (IMAC), followed by size exclusion chromatography. The SUMO protease failed to cleave the SUMO peptide from ASAL. This may be due to steric hindrance caused by the homodimer structure of the chimeric ASAL. Some properties like dimerization, haemagglutination and insecticidal properties of the recombinant SUMO-ASAL fusion protein were comparable to the plant derived native lectin. However, glycan array analysis revealed that the carbohydrate binding specificity of the recombinant SUMO-ASAL was altered. Further, the fusion protein was not toxic to E. coli (native ASAL exhibited toxicity). The recombinant lectin was more thermo-labile as compared to the native lectin. Three important findings of this study are: (1) sugar specificity of ASAL can be altered by amino-terminal fusion; (2) anti-E. coli activity of ASAL can be eliminated by N-terminal SUMO fusion and (3) SUMO-ASAL may be a preferred candidate insecticidal protein for the development of transgenic plants.

Isolation and characterization of SAP and CRP, two pentraxins from Pangasianodon (Pangasius) hypophthalmus.

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 microg SAP and 56 microg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1-2 mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca(2+) ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.

A C-type lectin is involved in the innate immune response of Chinese white shrimp.

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

Purification and physicochemical characterization of two galactose-specific isolectins from the seeds of Trichosanthes cordata.

A galactose-specific lectin has been purified from the seeds of Trichosanthes cordata by affinity chromatography on crosslinked guar gum. The affinity-eluted lectin could be resolved into two isolectins, TCA-I and TCA-II by ion-exchange chromatography on DEAE cellulose. The molecular weights of the isolectins were determined as 59 and 52 kDa by SDS-PAGE. TCA-I is a heterodimer in which the two subunits with masses of 32 and 27 kDa, are covalently connected by disulfide bonds. TCA-I and TCA-II are glycoproteins with 6.2% and 6.8% covalently bound neutral sugar, respectively. CD spectroscopic studies indicate that the two isolectins are very similar in secondary structure and contain about 8 to 10% alpha-helix, 37-38% beta-sheet, 20% beta-turns, and 32-33% unordered structures. These isolectins have similar carbohydrate specificities as revealed by hemagglutination-inhibition assays. Carbohydrate specificity, subunit size and composition, and secondary structure of TCA isolectins suggest close similarity to type-II ribosome inactivating proteins. The agglutination activity of TCA-I was found to be highest in the pH range 7.0-8.0. The lectin activity was unaffected between 0 and 40 degrees C, but decreased dramatically above 40 degrees C. Association constant for the interaction of TCA-I with lactose was determined by monitoring ligand-induced changes in the protein intrinsic fluorescence characteristics as 7.42 x 10(3) M(-1) at 25 degrees C. The exposure and accessibility of the tryptophan residues of TCA-I and the effect of ligand binding on them have been probed by quenching studies employing neutral and ionic quenchers.

A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains.

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by D-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop.

Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity.

Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA(maize). Heterologous expression of GNA(maize) in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA(maize) is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA(maize) prefers complex glycans containing beta1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA(maize) are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.