A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.     

The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.

C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.     

Budding-specific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates.

We have examined immunocytochemically the expression, localization and in vivo function of a calcium-dependent and galactose-binding 14 x 10(3) Mr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner epithelium of a double-walled bud vesicle. Soon after the bud entered the developmental phase, the granules were secreted into the mesenchymal space, where the lectin-positive extracellular matrix (ECM) developed. The lectin was also produced and secreted by granular leucocytes during budding. Hemoblasts, pluripotent stem cells in the blood, were often found in association with the ECM and they aggregated with epithelial cells to form organ rudiments. The lectin showed a high binding affinity for hemoblast precursors. The blockage of epithelial transformation of stem cells by galactose in in vivo bioassy was ineffective in the presence of the lectin. Polyclonal anti-lectin antibody prevented the hemoblasts spreading on the ECM and moving toward the epithelium, but it did not block the cell-cell adhesion of hemoblasts. By three days of bud development, lectin granules and ECM have almost disappeared from the developing bud together with a cessation of hemoblast aggregation. These results show that Polyandrocarpa lectin is a component of the ECM induced specifically in budding and suggest strongly that it plays a role in bud morphogenesis by directing the migration of pluripotent stem cells to the epithelium.     

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host,Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of approximately 14 and approximately 17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.     

How C-type lectins detect pathogens

Glycosylation of proteins has proven extremely important in a variety of cellular processes, including enzyme trafficking, tissue homing and immune functions. In the past decade, increasing interest in carbohydrate-mediated mechanisms has led to the identification of novel carbohydrate-recognizing receptors expressed on cells of the immune system. These non-enzymatic lectins contain one or more carbohydrate recognition domains (CRDs) that determine their specificity. In addition to their cell adhesion functions, lectins now also appear to play a major role in pathogen recognition. Depending on their structure and mode of action, lectins are subdivided in several groups. In this review, we focus on the calcium (Ca(2+))-dependent lectin group, known as C-type lectins, with the dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) as a prototype type II C-type lectin organized in microdomains, and their role as pathogen recognition receptors in sensing microbes. Moreover, the cross-talk of C-type lectins with other receptors, such as Toll-like receptors, will be discussed, highlighting the emerging model that microbial recognition is based on a complex network of interacting receptors.     

C-type lectin OCILRP2/Clr-g and its ligand NKRP1f costimulate T cell proliferation and IL-2 production

We are reporting the identification of a novel C-type lectin receptor-ligand pair that is involved in T cell costimulation. The receptor, OCILRP2/Clr-g, is rapidly induced following T cell activation and maintained at a substantial level of up to 72 h. The ligand, NKRP1f, is predominantly expressed on dendritic cells (DC). The soluble OCILRP2-Ig blocking protein significantly suppresses specific antigen-stimulated T cell proliferation as well as IL-2 secretion both in vitro and in vivo; conversely, NKRP1f-expressing antigen presenting cells (APC) enhance B7.1/CD28-mediated costimulation for T cell proliferation through interaction with OCILRP2/Clr-g. Our studies reveal a unique functional interaction between two C-type lectins, OCILRP2/Clr-g and NKRP1f, during APC-mediated T cell costimulation and suggest a role for C-type lectins in maintaining T cell response or memory in vivo.     

Molecular collaborations between serpins and trefoil factor promote endodermal cell growth and gastrointestinal differentiation in budding tunicates

We present evidence supporting novel collaborations between the serine protease inhibitor (serpin) and the trefoil factor during the budding stage of the tunicate Polyandrocarpa misakiensis. Using a maltose-binding protein/P-serpin fusion protein, two polypeptides of 40 kDa and 45 kDa were pulled down from Polyandrocarpa homogenates. Based on their partial amino acid sequence data, a single cDNA (928 bp) was cloned. It encodes a polypeptide that has five tandem repeats of a trefoil consensus motif. Thus, we termed the cDNA P-trefoil. Both P-trefoil and P-serpin were expressed exclusively by coelomic cells during budding. P-Trefoil was expressed mainly by coelomic cells throughout the asexual life cycle of Polyandrocarpa, while P-Serpin was localized particularly in coelomic cells and in the extracellular matrix in developing buds. The native P-Trefoil protein showed aminopeptidase activity. It induced cell growth in cultured Polyandrocarpa cells at a concentration of 8 microg/mL. P-Serpin reinforced this activity of P-Trefoil. Further, a mixture of P-Trefoil and P-Serpin exhibited the in vitro induction of a gut-specific alkaline phosphatase. These results show for the first time that a serpin can interact with a trefoil factor to play a role in the cellular growth and differentiation of the gastric epithelium.     

Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas

As an initial step in the functional analysis of lectins in the Pacific oyster, Crassostrea gigas, we attempted to obtain the full coding sequences of C. gigas lectins and conduct tissue expression analyses. To obtain lectin genes quickly, we identified C. gigas expressed sequence tags that coded for lectins in GenBank, and selected three encoding partial sequences of C-type lectin 1 (CgCLec-1), galectin (CgGal) and fucolectin. We obtained full open reading frames of CgCLec-1 and CgGal cDNAs by RACE-PCR. CgCLec-1 is a typical C-type lectin with a signal peptide and C-type lectin domain. CgCLec-1 mRNA was expressed only in specialized basophilic cells involved with digestive enzyme secretion in the digestive gland, suggesting that CgCLec-1 is secreted into the lumen of the digestive diverticula. CgGal is a prototype galectin with a single galactose-binding domain that was expressed in all of the tissues examined. As suggested for vertebrate galectin-1 (prototype galectin), CgGal may function in general cell activities such as cell adhesion. Fucolectin in C. gigas was expressed specifically in the gonads, indicating a possible function in gonadal development. CgCLec-1 and CgGal expression in hemocytes was not upregulated after injecting Vibrio tubiashii into adductor muscle, suggesting that bacterial infection does not induce synthesis of these lectins. Of the three lectins examined, CgCLec-1 is an interesting target for future investigations of innate immunity in the digestive system of C. gigas.     

ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures

Abstract C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca²⁺-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca²⁺-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis , which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors.
ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.     

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

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

Cell adhesive activity of two animal lectins through different recognition mechanisms

Cell adhesive activity of two animal lectins, frog (Rana catesbeiana) S-type 14K lectin and echinoidin (a C-type lectin from sea urchin plasma), was studied with human rhabdomyosarcoma (RD) cells. RD cells attached to and spread on plastic plates coated with each lectin. Cell adhesion by the frog lectin was completely inhibited by the addition of lactose or asialofetuin glycopeptide. Echinoidin-induced cell adhesion was only inhibited by peptide GRGDS. Since echinoidin is known to contain an RGD-sequence, our results clearly indicate that this sequence is active as the cell adhesive signal. These results suggest that some of the animal lectins may function as a cell adhesive molecule rather than using the carbohydrate-recognition mechanism.     

Dendritic cell-associated lectin-1: a novel dendritic cell-associated,C-type lectin-like molecule enhances T cell secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12-13 just 3' to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a(+) DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4(+) CD45RA(+) T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4(+) T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4(+) T cell proliferation or IL-4 and IFN-gamma secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4(+) T cells toward a Th2 response by enhancing their secretion of IL-4.     

A novel strategy for mammalian cell surface glycome profiling using lectin microarray

The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wild-type and beta1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Phospholipids and their derivatives as mitogen and motogen of budding tunicates

In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A(2) (PLA(2)) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.     

Different immunoreactivities of anti-soluble lactose lectin antisera to tissues from early chick embryos: a histochemical study

The location of soluble lactose-binding proteins (S-lac lectins) has been studied by immunohistochemical methods during morphogenesis of the chick embryo, when segregation and early differentiation of organ primordia was occurring. Using a panel of polyclonal antisera raised to various purified lectin preparations, we observed striking differences in the antigenic properties of these antisera, indicating that diverse versions of the lectins may be expressed during development. The antisera referred to as anti-L-16, anti-M-16, anti-S-14 and anti-I-14 were respectively raised to native or denatured 16 kDa lectins from adult liver and embryonic muscle and to 14 kDa lectins from embryonic skin and adult intestine. Having determined the optimal immunohistochemical conditions in the preparation of embryo sections (fixation, embedding, sectioning) we show that anti-L-16, anti-S-14 and anti-I-14 mostly bind the lectins expressed at the cell surface, in the extracellular matrix and in some released secretion. As previously shown, anti-L-16 and anti-S-14 are also able to recognize the cytoplasmic form of some migrative lectin-rich cells (primitive streak, neural crest cells, germ cells). Anti-M-16 was bound exclusively to the cytoplasmic form of the 16 kDa lectin in the same cell lines as above and also in some others, such as in the notochord, the myotomal part of the somites, the pharyngeal endoderm and the cardiac muscle. These different antigenic properties may be applied to the accurate mapping of various lectin isoforms and evaluation of the respective contribution of their intra-and extracellular variants during development and differentiation.