Agglutinating Activity Research Articles

Antimicrobial Functions of Galectins from Fish, Mollusks, and Crustaceans: A Review.

Galectins are a member of the β-galactoside binding protein family, which play a pivotal role in the immune defense of vertebrates as a pattern recognition receptor and occupy an important position in the innate immune system of invertebrates. The study of galectins in aquatic organisms has only recently emerged. Galectins in aquatic animals exhibit agglutination activity toward bacteria, inhibit bacterial growth, and enhance phagocytosis of immune cells. Additionally, some galectins contribute to the antiviral immune defenses of aquatic animals. This review aims to review recent advancements in the antimicrobial mechanisms, molecular structures, and evolution of galectins from fish, mollusks, and crustaceans. The antimicrobial galectins, as crucial components in the innate immune defense, pave new avenues for developing innovative disease control strategies in aquaculture.

MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica

BackgroundSingle-domain von Willebrand factor type C (SVWC) constitute a protein family predominantly identified in arthropods, characterized by a SVWC domain and involved in diverse physiological processes such as host defense, stress resistance, and nutrient metabolism. Nevertheless, the physiological mechanisms underlying these functions remain inadequately comprehended.ResultsA massive expansion of the SVWC gene family in Musca domestica (MdSVWC) was discovered, with a count of 35. MdSVWC1 was selected as the representative of the SVWC family for functional analysis, which led to the identification of the immune function of MdSVWC1 as a novel pattern recognition receptor. MdSVWC1 is highly expressed in both the fat body and intestines and displays acute induction upon bacterial infection. Recombinant MdSVWC1 binds to surfaces of both bacteria and yeast through the recognition of multiple pathogen-associated molecular patterns and exhibits Ca2+-dependent agglutination activity. MdSVWC1 mutant flies exhibited elevated mortality and hindered bacterial elimination following bacterial infection as a result of reduced hemocyte phagocytic capability and weakened expression of antimicrobial peptide (AMP) genes. In contrast, administration of recombinant MdSVWC1 provided protection to flies from bacterial challenges by promoting phagocytosis and AMP genes expression, thereby preventing bacterial colonization. MdSPN16, a serine protease inhibitor, was identified as a target protein of MdSVWC1. It was postulated that the interaction of MdSVWC1 with MdSPN16 would result in the activation of an extracellular proteolytic cascade, which would then initiate the Toll signaling pathway and facilitate the expression of AMP genes.ConclusionsMdSVWC1 displays activity as a soluble pattern recognition receptor that regulates cellular and humoral immunity by recognizing microbial components and facilitating host defense.

A Novel Trichinella spiralis Galectin Strengthens the Macrophage ADCC Killing of Larvae via Driving M1 Polarization.

Galectin recognizes β-galactosides through its carbohydrate recognition domains (CRDs). This study aimed to determine the biological features of a novel Trichinella spiralis galectin (galactoside-binding lectin family protein, TsGLFP) and its role in driving macrophage M1 polarization and enhancing ADCC killing of larvae. TsGLFP belongs to the galectin family and has two CRDs. The complete TsGLFP cDNA sequence was cloned and then expressed in Escherichia coli BL21. The results of qPCR, Western blot, and indirect immunofluorescence tests (IIFTs) revealed that TsGLFP was expressed in various stages of T. spiralis worms and principally localized at the cuticle and around the female embryos of the nematode. rTsGLFP had the function of agglutinating mouse erythrocytes, and this agglutination activity could be inhibited by lactose. After the mouse macrophage RAW264.7 was incubated with rTsGLFP, the expression level of the M1 genes (iNOS, IL-6, and TNF-α) and NO production were obviously increased. After incubating macrophages with rTsGLFP, there was a noticeable rise in the expression levels of p-IκB-α and p-NF-κB p65. Additionally, rTsGLFP enhanced the macrophage's ability to kill newborn larvae by ADCC cytotoxicity. When the macrophages were pretreated with the specific p-NF-κB p65 inhibitor PDTC, and then stimulated with rTsGLFP, the expression levels of iNOS, NO, and p-NF-κB p65 and the macrophages' ADCC cytotoxicity were distinctly decreased. These findings indicated that rTsGLFP enhanced the macrophage ADCC killing of larvae by driving M1 polarization through activating the NF-κB pathway.

Characterization and Expression Analysis of the C-Type Lectin Ladderlectin in Litopenaeus vannamei Post-WSSV Infection.

C-type lectins are known for agglutination activity and play crucial roles in regulating the prophenoloxidase (proPO) activation system, enhancing phagocytosis and encapsulation, synthesizing antimicrobial peptides, and mediating antiviral immune responses. This work cloned a C-type lectin, ladderlectin (LvLL), from Litopenaeus vannamei. LvLL comprised a 531 bp open reading frame (ORF) that encoded 176 amino acids. The predicted LvLL protein included a signal peptide and a CLECT domain. LvLL was predicted to feature a transmembrane region, suggesting it may be a transmembrane protein. LvLL was predominantly expressed in the shrimp's hepatopancreas. After WSSV infection, LvLL expression in the hepatopancreas increased significantly by 11.35-fold after 228 h, indicating a general upregulation. Knockdown of LvLL resulted in a significant decrease in WSSV viral load and a notable increase in shrimp survival rates. Additionally, knockdown of LvLL led to a significant downregulation of apoptosis-related genes Bcl-2 and caspase 8 and a significant upregulation of p53 and proPO in WSSV-infected shrimp. This study showed that LvLL played a vital role in the interaction between L. vannamei and WSSV.

Siglec7 functions as an inhibitory receptor of non-specific cytotoxic cells and can regulate the innate immune responses in a primitive vertebrate (Oreochromis niloticus)

In mammals, siglec7, an integral component of the siglecs, is principally found on the surface of natural killer (NK) cells, macrophages, and monocytes, where it interacts with various pathogens to perform immunological regulatory activities. Nonetheless, the immune defense and mechanism of siglec7 in early vertebrates remain unknown. In this study, we identified siglec7 from Oreochromis niloticus (OnSiglec7) and revealed its immune functions. Specifically, OnSiglec7 was abundantly expressed in immune-related tissues of healthy tilapia and its transcription level was strongly activated after being challenged with A. hydrophila, S. agalactiae, and Poly: IC. Meanwhile, OnSiglec7 protein was purified and analyzed, which could recognize multiple pathogens through binding and agglutinating activity. Moreover, OnSiglec7-positive cells were mainly distributed in non-specific cytotoxic cells (NCC) of tilapia HKLs and showed cell membrane localization. Furthermore, OnSiglec7 blockage affected multiple innate immune responses (inflammation, apoptosis, and pyroptosis process) by regulating the activation of MAPK, NF-κB, TLR, and JAK-STAT pathways. Finally, OnSiglec7 blockage also greatly enhanced the cytotoxic effect of tilapia NCC. Summarily, this study uncovers immune functions and mechanisms of siglec7 in primitive vertebrates, thereby enhancing our understanding of the systemic evolution and ancient functions of other siglecs within the host's innate immune system (to our knowledge).

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Dietary Psidium guajava, guava leaf extract protects Oreochromis niloticus, Nile tilapia from Pseudomonas aeruginosa infection and enhances growth

This study investigated the impact of dietary guava leaf extract (GLE) on immunity, resistance to Pseudomonas aeruginosa infection, and growth in Oreochromis niloticus (Nile tilapia). Four groups of fish (28.6 g.fish−1) were fed diets supplemented with ethanolic GLE at 0 (control), 3, 5, or 7 g.fish−1 diet (GLE0diet, GLE3diet, GLE5diet, GLE7diet) at 28 °C. Fish were fed thrice daily at 15 g.kg−0.8.d−1 for 42 days. Innate immune parameters, including mucus-bactericidal activity (MBA), serum-bactericidal activity (SBA), bacterial agglutination activity (BAA), and phagocytic activity (PA), were assessed on days 14, 28, and 42. Weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were measured on day 42. Post-feeding, fish were injected with P. aeruginosa, and mortality was recorded for 30 days. Results showed that GLE7diet significantly increased MBA, SBA, BAA, and PA by 1.8-, 3.3-, 4.2-, and 3.5-fold, respectively, compared to control. GLE7diet also showed the highest significant reduction in post-challenge mortality (21 %) compared to the control group (83 %). Growth metrics for GLE7diet showed WG of 43.8 g, SGR of 2.21%.d−1, and FCR of 1.19, compared to WG of 37.1 g, SGR of 1.98%.d−1, and FCR of 1.41 in the control group. SGR showed an exponential increase (R² = 0.935) and FCR a decrease (R² = 0.958) with increasing GLE levels. GLE5diet and GLE7diet significantly increased MBA at all-time points compared to control. GLE7diet significantly increased SBA on days 14, 28, and 42 compared to control. GLE7diet significantly enhanced BAA at all-time points. GLE5diet and GLE7diet significantly increased PA at all time points compared to control. GLE7diet led to increased expression of IL-1, IL-6, IFN-α, IFN-β, and TNF-α genes, particularly from 10 to 30 days post-infection. Dietary GLE at 7 g.kg−1 significantly enhanced innate and acquired immune responses, growth performance, and resistance to P. aeruginosa in Nile tilapia. This study recommended incorporating 7 g.kg−1 GLE in Nile tilapia diets to improve disease resistance and growth performance.

Sgabd-2 plays specific role in immune response against biopesticide Metarhizium anisopliae in Aphis citricola

Metarhizium anisopliae is an effective biopesticide for controlling Aphis citricola, which has developed resistance to many chemical pesticides. However, the powerful immune system of A. citricola has limited the insecticidal efficacy of M. anisopliae. The co-evolution between insects and entomogenous fungi has led to emergence of new antifungal immune genes, which remain incompletely understood. In this study, an important immune gene Sgabd-2 was identified from A. citricola through transcriptome analysis. Sgabd-2 gene showed high expression in the 4th instar nymph and adult stages, and was mainly distributed in the abdominal region of A. citricola. The recombinant protein (rSgabd-2) exhibited no antifungal activity but demonstrated clear agglutination activity towards the conidia of M. anisopliae. RNA interference of Sgabd-2 by dsRNA feeding resulted in decreased phenoloxidase (PO) activity and weakened defense for A. citricola against M. anisopliae. Simultaneous silence of GNBP-1 and Sgabd-2 effectively reduced the immunity of A. citricola against M. anisopliae more than the individual RNAi of GNBP-1 or Sgabd-2. Furthermore, a genetically engineered M. anisopliae expressing double-stranded RNA (dsSgabd-2) targeting Sgabd-2 in A. citricola successfully suppressed the expression of Sgabd-2 and demonstrated increased virulence against A. citricola. Our findings elucidated Sgabd-2 as a critical new antifungal immune gene and proposed a genetic engineering strategy to enhance the insecticidal virulence of entomogenous fungi through RNAi-mediated inhibition of pest immune genes.

Extraction and Hemagglutination Activity Study of Lectins from Hygrophorus lucorum Kalchbr

Edible fungus lectin hasanti-tumor, anti-virus, immune regulation and other functions. In this experiment, the wild Hygrophorus lucorum Kalchbr was used as the material, and the lectin was extracted by leaching method. The lectin was treated with physical and chemical factors, and the hemagglutination method was used to detect the change rule of lectin agglutination activity. The extraction process of lectin was optimized by single factor experiment and orthogonal experiment. The study of agglutination activity showed that: glycerol, lactose and maltose had significant inhibitory effects; when treated at 80℃, the agglutination activity was lost; pH<5.0 or pH>9.0, the agglutination activity was greatly inhibited; AI3+, Fe3+ had obvious promoting effects. Lectin extraction single factor test results: PBS (0.50mol/L, pH6.5) was used as the extraction reagent, the ratio of solid to liquid(g/mL) was1:10, and the fruiting bodies were extracted for 5 hours at 30℃. 40% saturation ammonium sulfate salting-out treatment, the salting-out product has the highest agglutination activity. The optimal process combination obtained by the or thogonal test is: using PBS (0.50mol/L, pH 6.5) as the extractant, according to the solid-liquid ratio (g/mL) 1:10, 40℃, leaching for 4h, the quantitative agglutination activity of lectin was 0.9609.

A fungal-binding agglutinin in the skin slime of Japanese flounder (Paralichthys olivaceus) is glyceraldehyde 3-phosphate dehydrogenase.

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-β in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. These results indicated that Smcys10 is involved in the host antibacterial immune response.

Reduction of hemagglutination induced by a SARS-CoV-2 spike protein fragment using an amyloid-binding benzothiazole amphiphile

COVID-19 infection is associated with a variety of vascular occlusive morbidities. However, a comprehensive understanding of how this virus can induce vascular complications remains lacking. Here, we show that a peptide fragment of SARS-CoV-2 spike protein, S192 (sequence 192-211), is capable of forming amyloid-like aggregates that can induce agglutination of red blood cells, which was not observed with low- and non-aggregated S192 peptide. We subsequently screened eight amyloid-binding molecules and identified BAM1-EG6, a benzothiazole amphiphile, as a promising candidate capable of binding to aggregated S192 and partially inhibiting its agglutination activity. These results provide new insight into a potential molecular mechanism for the capability of spike protein metabolites to contribute to COVID-19-related blood complications and suggest a new therapeutic approach for combating microvascular morbidities in COVID-19 patients.

Galectin-8-like isoform X1 mediates antibacterial, antiviral, and antioxidant responses in red-lip mullet (Planiliza haematocheilus) through positive modulation of pro-inflammatory cytokine, chemokine, and enzymatic antioxidant activity

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H2O2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Hemostatic activity &lt;i&gt;in vitro&lt;/i&gt; of lectin-containing substances of bryathypes on the example of some Antarctic and Belarus representatives

BACKGROUND: The modern market of medical devices in Belarus and Russia is represented by a wide range of hemostatic agents, of which the most popular are local hemostatics of plant origin possessing the significant technological potential for renewal and improvement. A promising reserve for this may be biologically active compounds of mosses, which are characterized by anti-inflammatory, antibacterial and antifungal effects. However, their hemostatic effect is almost not studied, which determines the relevance of this work.&#x0D; AIM: The aim of this work is to study the effect of lectin-containing substances from mosses of three species collected in East Antarctica and Belarus on the parameters of human blood hemostasis in vitro.&#x0D; MATERIALS AND METHODS: We studied mosses of the genera Bryum, Ceratodon, and Coscinodon, collected in the area of the Belarusian Antarctic station Gora Vechernyaya in East Antarctica and in Belarus. Lectin-containing substances of mosses were obtained by extracting shoots in 0.05 M tris-HCl buffer (pH 8.0), centrifugation, filtration. The assessment of the biological activity of lectin-containing substances in mosses was carried out by the agglutination reaction of rabbit erythrocytes, as well as the effect on human platelet aggregation and in the test for activated partial thromboplastin time.&#x0D; RESULTS: It was established that lectin-containing substances of the studied moss species had agglutinating activity against erythrocytes in the range from 11708.28 (Belarusian samples) to 1333979.59 U/mg of protein (Antarctic samples) depending on the species and localization; initiated the aggregation of human platelets (25–80% of the effect of thrombin) regardless of blood group, Rh and gender of donors; influenced the plasma link of hemostasis, reducing activated partial thromboplastin time (by 15–18%).&#x0D; CONCLUSIONS: It was found that some species of mosses of the genera Bryum, Ceratodon and Coscinodon of Antarctica and Belarus had an agglutinating and hemostatic effect on erythrocytes and platelets, with the greatest activity noted for Antarctic species. A hypothesis has been put forward that the observed phenomenon is due to the structural features of proteins, including lectins. It is assumed that lectins are possible inducers of erythrocyte agglutination and platelet aggregation in mosses. It is shown that the moss species Bryum pseudotriquetrum and Ceratodon purpureus have a certain resource potential in Belarus for their annual harvest. The results obtained expand the list of moss species with hemostatic activity, and can be used to develop new hemostatics of plant origin for local use from Belarusian plant materials.

OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

Antitumor Activity of a Lectin Purified from Punica granatum Pulps against Ehrlich Ascites Carcinoma (EAC) Cells.

Lectins are carbohydrate-binding proteins with various pharmacological activities, such as antimicrobial, antidiabetic, antioxidant, and anticancer. Punica granatum fruit extract has traditional uses, however, the anti-cancer activity of purified lectin isolated from P. granatum pulp is yet to be reported. The goals of this study are purification, characterization of the lectin from P. granatum, and examination of the purified lectin's anticancer potential. Diethylaminoethyl (DEAE) ion-exchange chromatography was used to purify the lectin, and SDSPAGE was used to check the purity and homogeneity of the lectin. Spectrometric and chemical analysis were used to characterize the lectin. The anticancer activity of the lectin was examined using in vivo and in vitro functional assays. A lectin, designated as PgL of 28.0 ± 1.0 kDa molecular mass, was isolated and purified from the pulps of P. granatum and the lectin contains 40% sugar. Also, it is a bivalent ion-dependent lectin and lost its 75% activity in the presence of urea (8M). The lectin agglutinated blood cells of humans and rats, and sugar molecules such as 4-nitrophenyl-α-D-manopyranoside and 2- nitrophenyl -β- D-glucopyranoside inhibited PgL's hemagglutination activity. At pH ranges of 6.0-8.0 and temperature ranges of 30°C -80°C, PgL exhibited the highest agglutination activity. In vitro MTT assay showed that PgL inhibited Ehrlich ascites carcinoma (EAC) cell growth in a dose-dependent manner. PgL exhibited 39 % and 58.52 % growth inhibition of EAC cells in the mice model at 1.5 and 3.0 mg/kg/day (i.p.), respectively. In addition, PgL significantly increased the survival time (32.0 % and 49.3 %) of EAC-bearing mice at 1.5 and 3.0 mg/kg/day doses (i.p.), respectively, in comparison to untreated EAC-bearing animals (p < 0.01). Also, PgL reduced the tumor weight of EAC-bearing mice (66.6 versus 39.13%; p < 0.01) at the dose of 3.0 mg/kg/day treatment. Furthermore, supplementation of PgL restored the haematological parameters toward normal levels deteriorated in EAC-bearing animals by the toxicity of EAC cells. The results indicated that the purified lectin has anticancer activity and has the potential to be developed as an effective chemotherapy agent.

PvMR1, a novel C-type lectin plays a crucial role in the antibacterial immune response of Pacific white shrimp, Penaeus vannamei

C-type lectins (CTLs) are important immune molecules in innate immune, which participate in non-self recognition and clearance of pathogens. Here, a new CTL with two distinct C-type lectin domains (CTLDs) from Pacific white shrimp Penaeus vannamei, designated as PvMR1 was identified. The obtained PvMR1 coding sequence (CDS) was 1044 bp long encoding a protein with 347 amino acids. PvMR1 had two CTLD, a conserved mannose-specific EPN motif and a galactose-specific QPD motif, clustering into the same branch as the crustacean CTLs. PvMR1 was widely distributed in shrimp tissues with the highest transcription level in the hepatopancreas, with significantly induced mRNA expression on the hepatopancreas and intestines after immune challenge with Vibrio anguillarum. In vitro assays with recombinant PvMR1 (rPvMR1) protein revealed that it exhibited a wide range of antimicrobial activity, bacterial binding ability, and bacterial agglutination activity in a Ca2+-independent manner. Moreover, PvMR1 promoted bacterial phagocytosis in hemocytes. Furthermore, rPvMR1 treatment could significantly enhance the bacterial clearance in hemolymph and greatly improved the survival of shrimp under V. anguillarum infection in vivo. These results collectively suggest that PvMR1 plays an important role in antibacterial immune response of P. vannamei.

Largemouth bass galectin, MsGal-9: Mediating various functions as a pattern recognition receptor and a potential damage-associated molecular pattern

Galectins are lectins that bind to β-galactose and are widely expressed in immune system tissues, playing pivotal roles in innate immunity through their conserved carbohydrate-recognition domains (CRDs). In this present investigation, a tandem-repeat galectin was discovered in the largemouth bass, Micropterus salmoides (designated as MsGal-9). The open reading frame of MsGal-9 encodes two CRDs, each containing two consensus motifs that are essential for ligand binding. MsGal-9 is expressed in various tissues of the largemouth bass, with particularly high expression levels in the liver and spleen. The full-length form of MsGal-9, as well as the N-terminal (MsGal-9-N) and C-terminal (MsGal-9-C) CRDs, were individually recombined. Their ability for nonself recognition was studied. The three recombinant proteins were able to bind to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS), with MsGal-9 displaying the highest binding activity. Furthermore, rMsGal-9-N exhibited higher binding activity towards GLU in comparison to rMsGal-9-C. Further investigations revealed that the full-length rMsGal-9 could significantly bind to Gram-positive bacteria, Gram-negative bacteria, and fungi, while rMsGal-9-C specifically bound to Escherichia coli. However, rMsGal-9-N did not exhibit significant binding activity towards any microbes. These findings indicate that MsGal-9 requires both CRDs to cooperate in order to fulfill its nonself recognition function. All three recombinant proteins demonstrated agglutination activity towards various microbes, with MsGal-9 and MsGal-9-N displaying a similar broad binding spectrum, while MsGal-9-C agglutinated three types of bacteria. Moreover, both MsGal-9 and MsGal-9-N were capable of coagulating largemouth bass red blood cells, whereas MsGal-9-C lacked this ability. However, MsGal-9-C played a significant role in enhancing the encapsulation of leukocytes in comparison to MsGal-9-N. All three proteins acted as potential damage-associated molecular patterns (DAMPs), inducing apoptosis in leukocytes.

Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus)

Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could be useful in terms of protective antigens for vaccine development. This study aims to evaluate the potential use of Tdr protein as a novel subunit vaccine against A. veronii infection in Nile tilapia. The Tdr gene from A. veronii was cloned into the pET28b expression vector, and the recombinant protein was subsequently produced in Escherichia coli strain BL21 (DE3). Tdr was expressed as an insoluble protein and purified by affinity chromatography. Antigenicity test indicated that this protein was recognized by serum from A. veronii infected fish. When Nile tilapia were immunized with the Tdr protein, specific antibody levels increased significantly (p-value <0.05) at 7 days post-immunization (dpi), and peaked at 21 dpi compared to antibody levels at 0 dpi. Furthermore, bacterial agglutination activity was observed in the fish serum immunized with the Tdr protein, indicating that specific antibodies in the serum can detect Tdr on the bacterial cell surface. These results suggest that Tdr protein has potential as a vaccine candidate. However, challenging tests with A.veronii in Nile tilapia needs to be investigated to thoroughly evaluate its protective efficacy for future applications.