Immunological and biological identification of tumour necrosis-like factor in sponges: endotoxin that mediates necrosis formation in xenografts.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.     

Sandwich enzyme-linked immunosorbent assay system for micro-detection of the wheat allergen, Tri a Bd 17 K

Seven monoclonal antibodies (mAbs) against the wheat allergen, Tri a Bd 17 K, were prepared to obtain mAbs suitable for a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for determination of the allergen. Two of the mAbs strongly immunoblotted the allergen purified from wheat flour. However, only one (1G11) of them was found to be suitable for sandwich ELISA. Epitope mapping against mAb-1G11 on the allergen showed that the mAb recognized the peptide containing Lys-38 and Gln-39 of the allergen. We developed a sandwich ELISA method consisting of Aleuria aurantia lectin for fixing the allergen and 1G11 as the first antibody that enabled 4-4,000 ng/well of the allergen to be determined.     

Sandwich Enzyme-linked Immunosorbent Assay System for Micro-detection of the Wheat Allergen,Tri a Bd 17 K

Seven monoclonal antibodies (mAbs) against the wheat allergen, Tri a Bd 17 K, were prepared to obtain mAbs suitable for a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for determination of the allergen. Two of the mAbs strongly immunoblotted the allergen purified from wheat flour. However, only one (1G11) of them was found to be suitable for sandwich ELISA. Epitope mapping against mAb-1G11 on the allergen showed that the mAb recognized the peptide containing Lys-38 and Gln-39 of the allergen. We developed a sandwich ELISA method consisting of Aleuria aurantia lectin for fixing the allergen and 1G11 as the first antibody that enabled 4-4,000 ng/well of the allergen to be determined.     

Directed migration of cells from the sponge Geodia cydonium

The migration behavior of cells from the sponge Geodia cydonium was studied in vitro, applying the 'Tissue Culture Slide Chamber' technique. The homologous lectin caused a directed cell migration with a maximal locomotory rate of 1.6 mum/min. Competition experiments using the solubilized lectin receptor (= antiaggregation receptor) revealed that the chemotactic ligand (= lectin) interacts directly with the lectin receptor which-in consequence-functions as the chemotactic receptor. The ability of the lectin to promote cell migration is abolished by coincubation with purified leucine aminopeptidase. Biochemical and immunochemical data revealed that this enzyme is present also on the surface of sponge cells. Furthermore, we present evidence that the chemotactic receptor (= anti-aggregation receptor) on the cell surface is, in an hitherto unknown manner, coupled with the intracellularly present actin filaments. From these data we conclude that the directed migration of Geodia cells is mediated by the interaction between the lectin (= chemotactic ligand) and the lectin receptor (= chemotactic receptor); it is very likely that also intracellular structural elements operate simultaneously and coordinately during cell migration.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Production and characterization of a monoclonal antibody against the seed lectin of the Dolichos biflorus plant

Spleen cells from mice immunized with the Dolichos biflorus seed lectin were fused with cells from the mouse myeloma Sp2/O-Ag14 cell line to form hybridomas. Those hybridomas producing antibodies against the seed lectin were cloned at least four times and the monoclonal antibodies from clone C11/64-56.28 were characterized and found to be specific for Subunit I of the lectin; they do not react with the structurally similar Subunit II. In previous studies, we have shown that although these two subunits appear to differ only at their COOH-terminal ends, only Subunit I has carbohydrate binding activity. Using a solid phase enzyme immunoassay, the antigenic determinant fr the monoclonal antibody was found to be located on the COOH-terminal cyanogen bromide fragment of this subunit. The monoclonal antibody inhibits the ability of the lectin to agglutinate erythrocytes and N-acetyl-D-galactosamine, the specific hapten for the lectin, inhibits the ability of the antibody to combine with the lectin. These results suggest that the monoclonal antibody recognizes a determinant that is located either at or near the active site of the lectin or that is conformationally interdependent with the active site.     

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.     

Sponge aggregation factor: in situ localization by fluorescent monoclonal antibody techniques

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction.     

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.     

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.     

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24 600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids (aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Identification and characterization of Tetrahymena myosin

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K⁺-/NH₄⁺-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain.     

Domain-specific distribution of carbohydrates in human fibronectins and the transformation-dependent translocation of branched type 2 chain defined by monoclonal antibody C6

Fibronectins purified from human plasma (termed pFN), spent culture media of human fibroblasts WI38 (termed cFN), and SV40 virus-transformed WI38/VA13 cells (termed tFN) and their cleavage fragments were compared with respect to their binding activities to lectins and anti-carbohydrate antibodies reacting with chemically well-defined structures. The following findings were of particular interest. About 25-35% of cFN and tFN carried a binary sialosyl type 2 chain (NeuAc alpha 2----3/or 6Gal beta 1----4GlcNAc) linked beta 1----3/beta 1----6 to the galactose residue and defined by monoclonal antibody C6. This structure was not detected in pFN. In cFN, the C6-defined structure was localized within the gelatin-binding domain, whereas in tFN the same structure was absent from this domain but was located at the NH2-terminal region of the central domain. Other carbohydrate determinants, defined by Ricinus communis lectin and concanavalin A before and after sialidase treatment, showed essentially identical domain distribution patterns among cFN, tFN, and pFN and were all located at the gelatin-binding domain (44 kDa), its precursor (60 kDa), and the Cell/Hep-2 domain (155/145 kDa). Although both cFN and tFN were reactive with lentil lectin, pFN was not. Fibronectin from transformed cells (tFN) showed much greater reactivity than cFN and pFN with wheat germ lectin before sialidase treatment and showed enhanced reactivity with R. communis lectin and peanut lectin after sialidase treatment, indicating that tFN is more highly sialylated than cFN and pFN. All fibronectins examined were strongly reactive with monoclonal antibody AH8-28, which binds to Gal beta 1----3GalNAc residues, and this reactivity was localized to both the NH2-terminal half and COOH-terminal half of the S-cyanylation-cleaved fibronectin molecule.     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Quantification of lectin in freshwater prawn (Macrobrachium rosenbergii ) hemolymph by ELISA

An enzyme-linked immunoabsorbent assay was developed to quantify the lectin present in the hemolymph of the freshwater prawn Macrobrachium rosenbergii. This method involves the use of murine monoclonal IgG1 with kappa light chain (designated as 3G1) antibodies raised against the purified lectin, the assay that we developed recognized as little as 30 ng/ml of lectin, and was used to measure the lectin concentration in animals at different maturation stages. The highest concentration of lectin was identified in the hemolymph from post-larval prawns and the lowest in molt stage adult animals. The hemagglutination activity of the lectin was four-fold higher in adult than in juvenile specimens, although in all cases N-acetylated sugar residues, such as N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetyl-D-neuraminic acid were inhibitors of the lectin activity, suggesting that lectin plays a role in the transport of N-acetylated sugar in juvenile prawns. Our results indicate that lectin concentration and hemagglutinating activity could be influenced by developmental conditions of the freshwater prawn.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.