Isolation, characterization, and extra-embryonic secretion of the Xenopus laevis embryonic epidermal lectin, XEEL

The Xenopus laevis embryonic epidermal lectin (XEEL) is a novel member of a group of lectins including mammalian intelectins, frog oocyte cortical granule lectins, and plasma lectins in lower vertebrates and ascidians. We isolated the XEEL protein from the extract of tailbud embryos by affinity chromatography on a galactose-Sepharose column. The XEEL protein is a homohexamer of 43-kDa N-glycosylated peptide subunits linked by disulfide bonds. It requires Ca(2+) for saccharide binding and shows a higher affinity to pentoses than hexoses and disaccharides. HEK-293T cells transfected with an expression vector containing the XEEL cDNA secrete into the culture medium the recombinant XEEL (rXEEL) that is similar to the purified XEEL in its molecular nature and saccharide-binding properties. Substitution of Asn-192 to Gln removed the N-linked carbohydrate and inhibited secretion of rXEEL but did not abolish the activity to bind to galactose-Sepharose. The embryo's XEEL content, as estimated by western blot analyses, increases during neurula/tailbud stages and declines after 1 week postfertilization. Immunofluorescence and immuno-electron microscopic analyses showed localization of the XEEL protein in a typical secretory granule pathway of nonciliated epidermal cells. When tailbud embryos were cultured in the standard medium, XEEL was accumulated in the medium, indicating secretion of XEEL into the environmental water. The rate of XEEL secretion greatly increased at around the hatching stage and stayed at a high level during the first week after hatching. XEEL may have a role in innate immunity to protect embryos and larvae against pathogenic microorganisms in the environmental water.     

肺表面活性物质相关蛋白A与肺部免疫防御研究进展

肺表面活性物质相关蛋白A（SP—A）是一种高度保守的亲水性糖蛋白,属于C-型凝素家族成员,相对分子质量为29～36kDa。SP—A是肺部重要的天然免疫防御分子,在肺的局部防御和天然免疫反应中起着十分重要的作用。它不仅可调节局部免疫和炎症反应、调理吞噬作用,还可凝集病原微生物、影响趋化作用及促进杀菌作用等。     

Characterization of Multisugar-Binding C-Type Lectin (SpliLec) from a Bacterial-Challenged Cotton Leafworm, Spodoptera littoralis

Background

Various proteins that display carbohydrate-binding activity in a Ca²⁺-dependent manner are classified into the C-type lectin family. They have one or two C-type carbohydrate-recognition domains (CRDs) composed of 110–130 amino acid residues in common. C-type lectins mediate cell adhesion, non-self recognition, and immuno-protection processes in immune responses and thus play significant roles in clearance of invaders, either as cell surface receptors for microbial carbohydrates or as soluble proteins existing in tissue fluids. The lectin of Spodoptera littoralis is still uncharacterized.

Methodology

A single orf encoding a deduced polypeptide consisting of an 18-residue signal peptide and a 291-residue mature peptide, termed SpliLec, was isolated from the haemolymph of the cotton leafworm, S. littoralis, after bacterial challenge using RACE-PCR. Sequence analyses of the data revealed that SpliLec consists of two CRDs. Short-form CRD₁ and long-form CRD₂ are stabilized by two and three highly conserved disulfide bonds, respectively. SpliLec shares homology with some dipteran lectins suggesting possible common ancestor. The purified SpliLec exhibited a 140-kDa molecular mass with a subunit molecular mass of 35 kDa. The hemagglutination assays of the SpliLec confirmed a thermally stable, multisugar-binding C-type lectin that binds different erythrocytes. The purified SpliLec agglutinated microorganisms and exhibited comparable antimicrobial activity against gram (+) and gram (−) bacteria too.

Conclusions

Our results suggested an important role of the SpliLec gene in cell adhesion and non-self recognition. It may cooperate with other AMPs in clearance of invaders of Spodoptera littoralis.     

Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewis^b and Man₃. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.     

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

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

Regulatory role of nitric oxide in lipopolysaccharides-triggered plant innate immunity

Recent studies have suggested that lipopolysaccharides (LPS) induce nitric oxide (NO) production and defense gene expression in plants. Our current work investigated the signaling mechanism of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) in LPS-induced innate immunity of Arabidopsis (Arabidopsis thaliana). We have provided evidence that LPS-elicited NO generation as well as increased antioxidant enzyme activities capable of maintaining the redox state could be important to protect plants against oxidative damage from pathogen attack. In addition, LPS-activated defense responses, including callose deposition and defense-related gene expression, are regulated through an NPR1-dependent signaling pathway. Our results contribute to elucidation of the signaling mechanism of NO and highlight an important role of NPR1 in modulating LPS-triggered innate immunity in plants. However, further research is necessary to clarify the cross-talk between mitochondria and NO on activating LPS-induced defense responses, and the regulatory mechanism of NO in LPS-induced innate immunity needs further improvement.     

Nuclear Hormone Receptor Regulation of MicroRNAs Controls Innate Immune Responses in C. elegans

Nuclear hormone receptors respond to small molecules such as retinoids or steroids and regulate development. Signaling in the conserved p38/PMK-1 MAP kinase pathway regulates innate immunity. In this study, we show that the Caenorhabditis elegans nuclear receptor DAF-12 negatively regulates the defense against pathogens via the downstream let-7 family of microRNAs, which directly target SKN-1, a gene downstream of PMK-1. These findings identify nuclear hormone receptors as components of innate immunity that crosstalk with the p38/PMK-1 MAP kinase pathway.     

Histochemistry of glycoconjugates in ovarian follicles of the adult house musk shrew, Suncus murinus

In ovarian follicles of the adult house musk shrew, Suncus murinus, glycoconjugates have been studied by means of light- and electron-microscopic histochemistry. The results obtained are that: (1) glycoconjugates of the zona pellucida of oocytes are provided with vicinal diol and acidic groupings, and sialic acid-galactose dimer, alpha-D-mannose, alpha-D-glucose, beta-D-galactose, N-acetyl-D-glucosamine and alpha-L-fucose residues; (2) glycoconjugates of the intercellular matrix of the granulosa and theca folliculi are comparable in histochemical properties to those of the zona pellucida, except for the relatively smaller amount of vicinal diol and acidic groupings, and (3) the zona pellucida can be divided into outer and inner layers by the binding degrees of the lectins used. The possible histophysiological significances of all these glycoconjugates are discussed with special reference to the particular ovarian follicular structures.     

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 β1-2 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.     

Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas.     

Lectin-mediated resistance impairs plant virus infection at the cellular level

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.     

Two Chitotriose-Specific Lectins Show Anti-Angiogenesis,Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle

The antiproliferative activity of two chito- specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI₅₀ values were approximately 8.4 μg ml^-1 (0.247 μM) and 142 μg ml^-1(14.8 μM) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G₀/G₁ arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.     

Ancient origin of mast cells

The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin·serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D₂ and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500 million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.     

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

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

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H₂O₂ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.     

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties

Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.     

Seeing strangers or announcing "danger": galectin-3 in two models of innate immunity

Recent investigations on the molecular mechanisms by which our immune system recognizes infections and initiates defense against those infections have led to the proposition of two models explaining the way our innate immunity system functions; the self-nonself model and the Danger model. In this review, the roles of galectin-3 in innate immunity against infections--host-pathogen interactions--will be discussed. We will shed light on the potential contribution of a beta-galactoside binding mammalian lectin, galectin-3 as a molecule implicated in innate immunity from the angle of both the self-nonself model and the Danger model.