Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber.

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Development of Antihuman Killer Cell Lectin-Like Receptor Subfamily G Member 1 Monoclonal Antibodies for Flow Cytometry.

Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, was identified as an inhibitory receptor expressed on natural killer (NK) cells and certain T cells. The protein regulates effector functions and developmental processes in these cells. In this study, we established a specific and sensitive monoclonal antibody (mAb) for human KLRG1 (hKLRG1), which is useful for flow cytometry, using a Cell-Based Immunization and Screening (CBIS) method. The established anti-hKLRG1 mAb, KLMab-1 (mouse IgG1, kappa), reacted with overexpressed hKLRG1 in Chinese hamster ovary-K1 (CHO/hKLRG1) and human NK cells, which also expressed endogenous hKLRG1 as confirmed by flow cytometry. KLMab-1, which was established by the CBIS method, could be useful for elucidating the hKLRG1-related biological response by flow cytometry.

Molecular characterization and biological effect of a C-type lectin receptor in Qihe crucian carp, Carassius auratus.

C-type lectin receptors, as the important members of pattern-recognition receptors, play the crucial roles in the innate immune system, which discriminate self and non-self by recognizing and binding the carbohydrates on the surface of microorganism. In this study, we identified a C-type lectin receptor gene in Qihe crucian carp Carassius auratus (named as CaCLR). The full-length cDNA of CaCLR was composed of 1130 bp, with a 226 bp 5'-untranslated region (UTR), a 792 bp ORF encoding a 263aa protein, and a 112 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of CaCLR is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. With regard to the mRNA transcript of CaCLR, it was ubiquitously detected in the tested tissues, among which it was the most abundant in head kidney. The temporal expressions of CaCLR were obviously up-regulated in liver, spleen, kidney, and head kidney after Aeromonas hydrophila and poly I: C challenge, respectively, and the patterns of expression changes were in a time-depended manner. The recombinant CaCLR (rCaCLR) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with lipopolysaccharide (LPS), peptidoglycan (PGN), ß-Glucan, and Mannan, as well as five microorganisms including fungus (Saccharomyces cerevisiae), Gram-negative bacteria (A. hydrophila, E. coli and Vibrio anguillarum), and Gram-positive bacteria (Micrococcus lysodeikticus). In the presence of rCaCLR, the eliminating capacity against A. hydrophila could be enhanced in C. auratus. Taken together, CaCLR is involved in the antibacterial defense in C. auratus.

Purification and partial characterization of carbohydrate-recognition protein C-type lectin from Hemifusus pugilinus.

A mannose binding lectin (C-type lectin) was detected in a molluscan snail Hemifusus pugilinus, this lectin molecule was isolated and purified from the plasma using mannose-fixed sepharose CL-4B column affinity chromatography. The purified protein corresponds to the molecular weight of 118 kDa on an SDS-PAGE gel. The divalent cation-dependent nature of the H. pugilinus lectin (Hp-Lec) evidenced through pH and thermal stability analysis using Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) respectively. Functional investigations of the Hp-Lec reveal a broad spectrum of bacterial agglutination activity against wide range of Gram-positive and Gram-negative bacterial strains. Furthermore, Hp-Lec displayed the haemo agglutination activity against vertebrate red blood cells (RBCs) and its titers were recorded. Excitingly, microbial virulent pathogens such as fungal strains tested against the purified Hp-Lec (25 and 50 µg/ml), which exhibits the effective antifungal activity against tested fungal pathogens such as Aspergillus niger and A. flavus.

Potential Role of Platelet-Activating C-Type Lectin-Like Proteins in Viper Envenomation Induced Thrombotic Microangiopathy Symptom.

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

Micrurus surinamensis Peruvian snake venom: Cytotoxic activity and purification of a C-type lectin protein (Ms-CTL) highly toxic to cardiomyoblast-derived H9c2 cells.

Micrurus surinamensis (Cuvier, 1817), popularly known as aquatic coral snake, has a broad geographic distribution in the Rainforest of South America. The purpose of this study was to investigate the cytotoxic effect caused by M. surinamensis venom in H9c2 cardiomyoblast cells and to identify protein components involved in cardiotoxic processes. Venom cardiotoxic potential is evidenced by cell viability reduction in a concentration-dependent manner. We have purified one of venom components responsible for this effect after three chromatographic steps: a cytotoxic 23.461 kDa protein, as determined by mass spectrometry. A 19-residue sequence (DCPSGWSSYEGSCYNFFQR) of the purified protein was deduced by MS/MS and exhibited high homology with N-terminal region of C-type lectin from snake venoms. This protein was named Ms-CTL. Morphologically, H9c2 incubation with Ms-CTL led to a significant cellular retraction and formation of cellular aggregates, as observed by microscopy phase-contrast images. Our results indicate that M. surinamensis venom is highly toxic to H9c2 cardiomyoblast cell and less or not cytotoxic to other cell lines, such as HaCat, VERO and U373. Results presented herein will help understanding the mechanisms that underlie cellular damage and tissue destruction, being useful in the development of alternative therapies against these coral snake bites.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Snake C-Type Lectins Potentially Contribute to the Prey Immobilization in Protobothrops mucrosquamatus and Trimeresurus stejnegeri Venoms.

Snake venoms contain components selected to immobilize prey. The venoms from Elapidae mainly contain neurotoxins, which are critical for rapid prey paralysis, while the venoms from Viperidae and Colubridae may contain fewer neurotoxins but are likely to induce circulatory disorders. Here, we show that the venoms from Protobothrops mucrosquamatus and Trimeresurus stejnegeri are comparable to those of Naja atra in prey immobilization. Further studies indicate that snake C-type lectin-like proteins (snaclecs), which are one of the main nonenzymatic components in viper venoms, are responsible for rapid prey immobilization. Snaclecs (mucetin and stejnulxin) from the venoms of P. mucrosquamatus and T. stejnegeri induce the aggregation of both mammalian platelets and avian thrombocytes, leading to acute cerebral ischemia, and reduced animal locomotor activity and exploration in the open field test. Viper venoms in the absence of snaclecs fail to aggregate platelets and thrombocytes, and thus show an attenuated ability to cause cerebral ischemia and immobilization of their prey. This work provides novel insights into the prey immobilization mechanism of Viperidae snakes and the understanding of viper envenomation-induced cerebral infarction.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

A Hint of Primitive Mucosal Immunity in Shrimp through Marsupenaeus japonicus Gill C-Type Lectin.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is Marsupenaeus japonicus gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from M. japonicus displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and Vibrio counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.

Carbohydrate-independent antibiofilm effect of Bothrops jararacussu lectin BJcuL on Staphylococcus aureus.

The antivirulence approach to fighting biofilm-based infections caused by Staphylococcus aureus is a promising therapy that has been studied extensively. Here, we compare the antibiofilm activity of a purified lectin from Bothrops jararacussu venom (BJcuL) and commercial lectins obtained from Triticum vulgaris (Wheat Germ Agglutinin, WGA), Bandeiraea simplicifolia BS-II, and Maclura pomifera. Only WGA had antibiofilm activity, although no effect was seen on pre-formed biofilms. The pre-incubation of WGA and BJcuL with their preferential sugars inhibited the biological activity of WGA, but not that of BJcuL, suggesting that biofilm disruption does not involve carbohydrate-recognition domains (CRDs). Quantitative real-time PCR showed that BJcuL promotes modulation of expression of S. aureus genes involved in biofilm formation. Light microscopy revealed cocci and small cell clusters after biofilm formation in the presence of BJcuL, showing that the lectin treatment was unable to completely disrupt biofilm structure. Exposing the free cells to 50 times the minimum inhibitory concentration of gentamicin or ciprofloxacin did not prevent biofilm reestablishment, although inhibition was stronger than in the control (no lectin). This disruption of the biofilm architecture can expose the bacterial cell and may facilitate clearance by the immune system.

Lectin isolated from Bothrops jararacussu venom induces IL-10 release by TCD4+ cells and TNF-α release by monocytes and natural killer cells.

BjcuL is a C-type lectin isolated from Bothrops jararacussu snake venom with specificity for binding ß-d-galactose units. BjcuL is not toxic to human peripheral blood mononuclear cells (PBMCs), but it inhibits PBMC proliferation and stimulates these cells to produce superoxide anions and hydrogen peroxide primarily via lymphocyte stimulation; it does not stimulate the production of nitric oxide and PGE2 . The purpose of this study was to investigate the effect of BjcuL on PBMC activation with a focus on cytokine release modulating PBMC proliferation. The results showed for the first time that BjcuL coupled to FITC interacted with monocytes, B cells, natural killer (NK) cells, and with subpopulations of T cells. These cell-cell interactions can lead to cell activation and inflammatory cytokines release, such as IL-6 and TNF-α, as well as the anti-inflammatory cytokine IL-10. In addition, TNF-α release was attributed to NK cells and monocytes, whereas IL-10 was attributed to TCD4+ and Treg cells when stimulated by BjcuL. The temporal cytokines profile produced by cells when stimulated with this lectin allows us to assert that BjcuL has immunomodulatory activity in this context.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Identification and characterization of two novel C-type lectins from the larvae of housefly, Musca domestica L.

Lectins and antimicrobial peptides (AMPs) are widely distributed in various insects and play crucial roles in primary host defense against pathogenic microorganisms. Two AMPs (cecropin and attacin) have been identified and characterized in the larvae of housefly. In this study, two novel C-type lectins (CTLs) were obtained from Musca domestica, while their agglutinating and antiviral properties were evaluated. Real-time PCR analysis showed that the mRNA levels of four immune genes (MdCTL1, MdCTL2, Cecropin, and Attacin) from M. domestica were significantly upregulated after injection with killed Gram-negative Escherichia coli. Moreover, purified MdCTL1-2 proteins can agglutinate E. coli and Staphylococcus aureus in the presence of calcium ions, suggesting their immune function is Ca2+ dependent. Sequence analysis indicated that typical WND and QPD motifs were found in the Ca2+ -binding site 2 of carbohydrate recognition domain from MdCTL1-2, which was consistent with their agglutinating activities. Subsequently, antiviral experiments indicated that MdCTL1-2 proteins could significantly reduce the infection rate of Spodoptera frugiperda 9 cells by the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, indicating they might play important roles in insect innate immunity against microbial pathogens. In addition, MdCTL1-2 proteins could effectively inhibit the replication of influenza H1 N1 virus, which was similar to the effect of ribavirin. These results suggested that two novel CTLs could be considered a promising drug candidate for the treatment of influenza. Moreover, it is believed that the discovery of the CTLs with antiviral effects in M. domestica will improve our understanding of the molecular mechanism of insect immune response against viruses.

C-Type Lectin Receptor (CLR)-Fc Fusion Proteins As Tools to Screen for Novel CLR/Bacteria Interactions: An Exemplary Study on Preselected Campylobacter jejuni Isolates.

C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This "Methods" paper describes applications of CLR-Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the here-described applications of CLR-Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.

Macrovipecetin, a C-type lectin from Macrovipera lebetina venom, inhibits proliferation migration and invasion of SK-MEL-28 human melanoma cells and enhances their sensitivity to cisplatin.

BACKGROUND: The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. METHODS: Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. RESULTS: Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced the expression of TRADD, p53, Bax, Bim and Bad and down-regulated the Bcl-2 expression and ROS levels in SK-MEL-28 cells. Interestingly, these treatments impaired SK-MEL-28 cell adhesion, migration and invasion through modulating the function and expression of αvß3 integrin along with regulating E-cadherin, vimentin, ß-catenin, c-Src and RhoA expression. In silico study suggested that only the α chain of macrovipecetin interacts with a region overlapping the RGD motif binding site on this integrin. CONCLUSIONS: We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells. GENERAL SIGNIFICANCE: The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.

Identification and preliminary characterization of a putative C-type lectin receptor-like protein in the T. cruzi tomato lectin endocytic-enriched proteome.

Trypanosoma cruzi, the etiological agent of the Chagas' disease in Latin America undergoes a complex life cycle involving two hosts, a mammalian host and a reduviid insect vector (triatomine). In the insect midgut the parasite multiplies as epimastigote forms, which rely on endocytosis for their energy requirement. We recently showed that posttranslational modification of endocytic N-glycoproteins by tomato lectin (TL) binding-N-glycans is crucial for receptor-mediated endocytosis (RME) in epimastigote forms. In an attempt to characterize the endocytic proteome we used a TL affinity chromatography, which significantly enriched glycoproteins of the trypanosomal endocytic pathway. In addition to various lysosomal hydrolases, we found an endosomal C-type lectin-like protein, which displays some structural and topological characteristics of the mammalian lectin receptor superfamily. This lectin encoding a large transmembrane protein of around 375kDa contained three putative extracellular N-terminal C-type lectin domains (CTLD) and located inside the flagellar pocket (FP)/cytostome and endosomal compartments of the insect stage of the parasite and on the surface of the plasma membrane of intracellular amastigote parasites. Noteworthy, this endogenous lectin displayed similar sugar-binding specificity to that of TL and therefore could be important in either the N-glycan mediated endocytosis or parasite adhesion to host cells. We postulated that during the evolution of trypanosomatids, genes encoding lectin harboring 3 CTDLs represent an old acquisition present in free-living, monoxenic and heteroxenic trypanosomatids, which would have been secondarily lost in extracellular parasites from the T. brucei clade.

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.