Sambucus Nigra Agglutinin Research Articles

Correlation between the sialylation of cell surface Thomsen-Friedenreich antigen and the metastatic potential of colon carcinoma cells in a mouse model.

The cell surface glycosylation profiles of a liver metastatic colon carcinoma variant cell line, SL4 cells previously selected from colon 38 cells in vivo for liver colonization were investigated. Flowcytometric analysis was performed with 7 plant lectins and 10 carbohydrate specific monoclonal antibodies. The results showed that peanut agglutinin (PNA), Sambucus nigra agglutinin, Ulex europeus agglutinin-I, anti-LeX, anti-LeY, and anti-Le(b) antibodies bound to the parental colon 38 cells but not to SL4 cells. Another variant cell line was selected in vitro for the paucity of cell surface PNA-binding sites using a magnetic cell sorter and was designated as 38-N4 cells. The binding profiles of plant lectins and carbohydrate-specific antibodies to 38-N4 cells were very similar to those of SL4 cells. After intrasplenic injections, metastatic ability of 38-N4 cells was higher than that of colon 38 cells. PNA binding to SL4 cells and 38-N4 cells was detected after sialidase treatment of these cells, indicating increased sialylation of Thomsen-Friedenreich antigen in these cells. The mRNA levels of sialyltransferases, ST3Gal I, ST3Gal II, ST6GalNAc I, and ST6GalNAc II, were compared. The level of ST3Gal II mRNA was elevated in both SL4 cells and 38-N4 cells, whereas the level of ST6GalNAc II mRNA was elevated in 38-N4 cells compared with colon 38 cells. According to the expression array analysis, there are other glycosyltransferase genes differentially expressed between SL4 and colon 38 cells, yet their involvement in the altered glycosylation in these cells is unclear.

N-acetylgalactosamine, N-acetylglucosamine and sialic acid expression in primary breast cancers

Binding of the lectin from Helix pomatia (HPA), which recognises N-acetylgalactosamine and N-acetylglucosamine glycans, is a predictor of metastasis and poor prognosis in a number of human adenocarcinomas, including breast cancer. The glycoproteins to which it binds in these tumours have been only partially characterised, and the mechanisms underlying their biosynthesis remain unknown. In this study, 111 primary breast cancers were assessed for binding of HPA and labelling characteristics were compared directly with those of Dolichos biflorus agglutinin and soybean agglutinin, both of which also recognise N-acetylgalactosamine, Griffonia simplicifolia agglutinin II, which recognises N-acetylglucosamine, and Limax flavus agglutinin, Sambucus nigra agglutinin and Maackia amurensis lectin I, all of which recognise sialic acids. Results indicate that the HPA-binding partners expressed by cancer cells are predominantly N-acetylgalactosamine glycans, but some recognition of N-acetylglucosamine species is also likely. There was no evidence to support the hypothesis that overexpression of these moieties results from failure in sialylation. Alternative mechanisms, for example alterations in levels of activity of appropriate glycosyl transferases or disruption in transport and processing mechanisms leading to failure of normal chain extension of glycans may be responsible, and these are areas that warrant further investigation.

Glycohistochemical characteristics of nasal polyps from patients with and without cystic fibrosis.

To investigate whether cystic fibrosis (CF)-related nasal polyps exhibit significantly distinct glycohistochemical characteristics when compared with single vs massive nasal polyps obtained from patients without CF. Glycohistochemical characteristics were identified by means of 8 histochemical probes, including 5 plant lectins (peanut, gorse seed, wheat germ, Maackia amurensis, and Sambucus nigra agglutinins), 2 animal lectins (14- and 16-kd galectins), and 1 neoglycoprotein (exposing the Thomsen-Friedenreich antigen). The binding of the 8 glycohistochemical markers was determined by means of computer-assisted microscopy. For each probe, 3 quantitative parameters were computed: the labeling index, which describes the percentage of tissue area specifically stained by a given marker; the mean optical density, which reflects the staining intensity; and the concentrational heterogeneity, which characterizes the level of heterogeneity of the staining intensity. A series of 61 nasal mucosa specimens was analyzed, including 6 normal cases, 23 single and 18 massive polyposis cases without CF, and 14 nasal polyps associated with CF. Normal and polyposal nasal mucosa differed in terms of the amounts and linkage types of sialic acids (revealed by the wheat germ, M amurensis, and S nigra agglutinins) rather than the characteristics of galactoside expression (monitored with the peanut agglutinin and 2 animal galectins). In contrast, nasal polyps markedly differed between patients with and without CF with respect to galactoside expression (revealed by the peanut agglutinin and the 14-kd galectin) and the display of binding site(s) for the neoglycoprotein. Normal and polyposal nasal mucosa differ essentially in sialic acid presentation, while nasal polyps from patients with CF have a higher level of various lectin-reactive galactoside residues than nasal polyps from those without CF.

Identification of sialic acids on the cell surface of Candida albicans

The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1.6×10 6 to 2.8×10 6. The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein- Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)- Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated β-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are α2,6- and α2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The α2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly- L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.

Increased N-linked glycosylation leading to oversialylation of monomeric immunoglobulin A1 from patients with Sjögren's syndrome.

Increased serum immunoglobulin A (IgA) level is a common finding in primary Sjögren's syndrome (pSS). IgA might not be properly eliminated because of an abnormal glycosylation. We reported previously that IgA1 from patients with pSS was oversialylated. We extend this finding by showing that monomeric IgA1 contains more sialic acid (SA) in patients than in controls, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot with Sambucus nigra agglutinin (SNA), a lectin specific for SA. To localize this excess of SA on the N- and/or O-linked oligosaccharides, we analysed them separately, using N- and O-linked oligosaccharide profiling kits based on fluorophore-assisted carbohydrate electrophoresis. N-linked, but not O-linked, oligosaccharides of patients' IgA1 were oversialylated, and this seemed to be linked to an excess of SA on the same number of polysaccharides as normal IgA1. To localize the abnormality to the Fab and/or Fc fragments, monomeric IgA1 was digested with protease, separated and transferred to nitrocellulose, where SA was identified by SNA. Both Fab and Fc fragments appeared to be oversialylated. Oversialylation of N-linked oligosaccharides of IgA1 from patients with pSS might prevent the recognition of IgA by receptors that are responsible for their clearance, resulting in an excess of serum IgA and related immune complexes.

Enhanced sialyltransferase activity in B lymphocytes from patients with primary Sjögren's syndrome.

Despite the indisputable role of immunoglobulin (Ig)A in the pathogenesis of primary Sjögren syndrome (pSS), the causative abnormality remains largely unknown. As an extension of our report that IgA is oversialylated in this disease, the thrust of the present study was to measure the sialyltransferase (ST) activity in B lymphocytes. ST containing lysates of B cells from 17 pSS patients and 10 controls, were obtained using a combination of detergents, and incubated with affinity purified IgA that had been previously desialylated. The deposition of cytidine 5' monophosphate sialic acid (SA) by ST from B cells onto IgA was detected by two ELISA based upon the use of biotinylated lectins (Sambucus nigra agglutinin which is specific for alpha2-6 SA and Maackia amurensis which is specific for alpha2-3 SA). In parallel, the amount of SA on IgA from ten of the 17 patients and eight of the 10 controls was assayed using the same method. An excess of alpha2-3 and alpha2-6 SA on IgA was found in those patients with excessive activity of alpha2-3 and alpha2-6 ST. Thus, IgA hypersialylation in pSS patients may result from undue activity of ST.

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver.

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.

Grading dysplasia in colorectal adenomas by means of the quantitative binding pattern determination of Arachis hypogaea, dolichos biflorus, Amaranthus caudatus, Maackia amurensis, and Sambucus nigra agglutinins

The current study deals with the setting up of a new tool that enables the benign versus the malignant nature of colorectal adenomas to be determined accurately. The 2 objectives are to determine (1) whether adenomas should, or should not, be included in a 2- or a 3-tier grading system, and (2) whether severe dysplasias and carcinomas in situ share common or different biological characteristics. The levels of expression of different types of glycoconjugates were characterized in a series of 166 colorectal specimens, including 14 normal, 90 dysplastic, and 62 cancerous cases. The glycoconjugate expressions were demonstrated for 5 lectins, namely, Arachis hypogaea (PNA), Dolichos biflorus (DBA), Amaranthus caudatus (ACA), Maackia amurensis (MAA) and Sambucus nigra (SNA). The glycoconjugates demonstrated by these 5 lectins belong to the family of the Thomsen-Friedenreich antigens. The binding patterns of the 5 lectins were quantitatively determined by means of computer-assisted microscopy. The quantitative data were submitted to discriminant analyses. Our results show that the specific glycochemical staining patterns could be identified unambiguously and without misclassification between benign (normal and low dysplasia) and malignant (ie, either as moderate/severe dysplasia, carcinoma in situ, or cancer) cases. The data also strongly suggested that (1) dysplasias seem to be distinguishable in 2 instead of 3 groups, that is, low versus moderate/severe (high); and (2) moderate/severe dysplasias are biologically distinct from carcinomas in situ. The methodology developed can be applied directly in routine diagnosis to identify moderate/severe dysplasia specimens already exhibiting features common to carcinomas, and which therefore should be treated consistently in view of the fact that our data strongly suggest that most moderate/severe dysplasias are still benign, whereas carcinomas in situ are real carcinomatous lesions.

Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils

Dietary lectins, present in beans and other edible plant products, pose a potential threat to consumers due to their capacity to induce histamine release from basophils. In this study, we analyzed the capacity of 16 common, in particular dietary, lectins to induce human basophils to secrete IL-4 and IL-13, the key promoters of Th2 responses and IgE synthesis. Several of the lectins, especially concanavalin A, lentil lectin, phytohemagglutinin, Pisum sativum agglutinin and Sambucus nigra agglutinin, triggered basophils to release IL-4 at concentrations of up to 1 ng / 106 basophils. Lectins with high IL-4-inducing capacity also stimulated the release of IL-13 and histamine. Lectin-induced IL-4 and IL-13 release reached a maximum after 4 – 6 h and more than 18 h, respectively. Affinoblotting revealed that lectins with the capacity to induce mediator release bind to IgE, suggesting IgE binding as initial step of signal generation. In conclusion, several dietary lectins can trigger human basophils to release IL-4 and IL-13. Since lectins can enter the circulation after oral uptake, they might play a role in inducing the so-called early IL-4 required to switch the immune response towards a Th2 response and type I allergy.

Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils.

Dietary lectins, present in beans and other edible plant products, pose a potential threat to consumers due to their capacity to induce histamine release from basophils. In this study, we analyzed the capacity of 16 common, in particular dietary, lectins to induce human basophils to secrete IL-4 and IL-13, the key promoters of Th2 responses and IgE synthesis. Several of the lectins, especially concanavalin A, lentil lectin, phytohemagglutinin, Pisum sativum agglutinin and Sambucus nigra agglutinin, triggered basophils to release IL-4 at concentrations of up to 1 ng/10(6) basophils. Lectins with high IL-4-inducing capacity also stimulated the release of IL-13 and histamine. Lectin-induced IL-4 and IL-13 release reached a maximum after 4-6 h and more than 18 h, respectively. Affinoblotting revealed that lectins with the capacity to induce mediator release bind to IgE, suggesting IgE binding as initial step of signal generation. In conclusion, several dietary lectins can trigger human basophils to release IL-4 and IL-13. Since lectins can enter the circulation after oral uptake, they might play a role in inducing the so-called early IL-4 required to switch the immune response towards a Th2 response and type I allergy.

Relevance of sialoglycoconjugates in murine thymocytes during maturation and selection in the thymus

Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 coreceptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. In this work we evaluated the changes in the sialylation pattern in thymus sections from normal and dexamethasone treated mice. We used sialic acid specific lectins, such as Sambucus nigra agglutinin (SNA, NeuAcα2,6-Gal specific) and Maackia amurensis agglutinin (MAA, NeuAcα2,3-Gal specific). Our results indicate that the sialylation pattern was modified during the maturation process of thymic cells. The immature CD4-CD8– and CD4+CD8+ cortical thymocytes were recognized by SNA, whereas the mature single positive (CD4+ or CD8+) medullary cells, preferentially bound MAA lectin. However, in the corticomedullary region we found not only SNA+ cells, but also MAA+ cells. In the thymus of dexamethasone treated mice, the clusters of thymocytes undergoing apoptosis in the cortex were characteristically stained by SNA. These results suggest that in the initial stages of the differentiation pathway, a great number of thymocytes express an α2,6 linked sialic acid on their surface and as they progress to more mature stages there is a change in the sialylation pattern to α2,3 linked sialic acids probably due to a regulated expression of different sialyltransferases, which could be modulated by the thymic microenviroment. Abbreviations: SNA (Sambucus Nigra agglutinin); MAA (Maackia amurensis agglutination); NeuAc (N-acetyl-neuraminic acid); Gal (Galactose); GalNac (N-acetyl-galactosamine); GlcNAc (N-acetyl-glucosamine); Ser (Serine); Thr (Threonine); CMP (Cytidine monophosphate); LPA (Limulus polyphemus agglutinin); DAB (3,3' diaminobenzidine tetrahydrochloryde); HE (Hematoxilin-eosin); FITC (Fluorescein isothiocyante)

Lectin analysis of human immunoglobulin G N-glycan sialylation.

The lectins Sambucus nigra agglutinin (SNA) and Ricinus communis agglutinin (RCA), specific for alpha2,6 linked sialylation, and terminal galactose respectively were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation. SNA was shown to bind N-glycan alpha2,6-linked sialic acid only. Sialidase analysis confirmed that this is the dominant, if not exclusive linkage. Total IgG sialylation was estimated at 1.0 microg SA/mg IgG (or about 0.5 mole per mole) using a biochemical sialic acid assay. SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state. In contrast, SNA failed to bind the IgG Fc fragment in its native form, but displayed strong binding after the Fc was denatured. This allowed the construction of quantitative assays capable of measuring both IgG Fab and Fc alpha2,6-sialylation without the need for enzymatic peptide digestion.

Sialylation of E-cadherin does not change the spontaneous or ET-18-OMe-mediated aggregation of MCF-7 human breast cancer cells.

We have investigated the role of sialylation on cell-cell adhesion mediated by E-cadherin. Two MCF-7 human breast cancer cell variants were studied: MCF-7/AZ cells showed a spontaneous cell-cell adhesion in the fast and slow aggregation assay. whereas the adhesion deficient MCF-7/6 cell variant failed to form larger aggregates, suggesting that E-cadherin was not functional under the conditions of both assays. We measured the sialyltransferase activities using Galbeta1-3GalNAcalpha-O-benzyl and Galbeta1-4GlcNAcalpha-O-benzyl as acceptor substrates as well as mRNA levels of four sialyltransferases, ST3Gal I, ST3Gal III, ST3Gal IV, ST6Gal I, using multiplex RT-PCR in MCF-7 cell variants. The alpha2-6 and alpha2-3 sialylation of E-cadherin was investigated by immuno-blot using Sambucus nigra agglutinin and Maackia amurensis agglutinin. Compared to the adhesion-proficient MCF-7/AZ cells, the adhesion-deficient MCF-7/6 cell line apparently lacks ST6Gal I mRNA, has a lower ST3Gal I mRNA, a lower ST3Gal I sialyltransferase activity, and no alpha2-3 linked sialic acid moieties on E-cadherin. The potential anti-cancer drug 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe, 48 h, 25 microg/ml) belonging to the class of alkyllysophospholipids restored the E-cadherin function in the adhesion-deficient MCF-7/6 cells as evidenced by an increased aggregation. ET-18-OMe caused loss of ST6Gal I mRNA in MCF-7/AZ cells but no changes of sialyltransferase activities or sialic acid moieties on E-cadherin could be observed. We conclude that Ca2+-dependent, E-cadherin-specific homotypic adhesion of MCF-7/AZ or MCF-7/6 cells treated with ET-18-OMe was not affected by sialylation of E-cadherin.

Differentiation elicits negative regulation of human β-galactoside α2,6-sialyltransferase at the mRNA level in the HL-60 cell line

We studied the regulation of the β-galactoside α2,6-sialyltransferase (hST6Gal I) gene during HL-60 cell differentiation induced with dimethyl sulfoxide (DMSO), all trans-retinoic acid (ATRA), and phorbol myristate acetate (PMA). During HL-60 cell line differentiation, cell surface levels of α2,6-sialic acids expression decreased, as measured by flow cytometric analysis using Sambucus nigra agglutinin (SNA). Activities of hST6Gal I and levels of hST6Gal I mRNA dramatically decreased after 1 day of stimulation. Using reverse transcription polymerase chain reaction (PT-PCR), we found the major hST6Gal I mRNA isoform in HL-60 cells contains 5′-untranslated exons Y and Z. These results suggest that the expression of cell surface α2,6-sialic acids is controlled at the mRNA level, which is regulated by a promoter located 5′-upstream of exon Y.

Role of sialic acid in the endocytosis of prosaposin by the nonciliated cells of the rat efferent ducts.

The present study examines the mechanism of endocytosis of testicular prosaposin by the nonciliated cells of the efferent ducts. Testicular prosaposin is secreted by Sertoli cells into the lumen of the seminiferous tubules as a 70 kDa isomer where it binds to the tail of spermatozoa. In the efferent ducts, after dissociating from the plasma membrane of the spermatozoa, prosaposin is endocytosed by the nonciliated cells, presumably by receptor-mediated endocytosis. The initial step of receptor-mediated endocytosis usually results from the binding of a ligand's terminal oligosaccharide to a receptor on the cell surface. Thus, in the present study, several monosaccharides were injected in the lumen of the efferent ducts to compete with the binding and endocytosis of prosaposin. A quantitative electron microscopic approach was utilized and the number of gold particles, indicating anti-prosaposin immunoreactive sites, were scored over the various cell compartments including the plasma membrane, endocytic vesicles, early endosomes, and late endosomes. The length of the plasma membrane and the areas of endocytic vesicles, early endosomes, and late endosomes were measured with an image analyzer and the number of grains expressed per microm (plasma membrane) and microm2 (endocytic vesicles/endosomes) respectively. The quantitative analysis was performed in untreated animals (controls) and animals treated with various sugars (i.e., glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine and N-acetylgalactosamine) injected into the lumen of the efferent ducts at a concentration of 20 mM. Sialic acid caused the greatest decrease in the labeling density of the endocytic elements. Mannose 6-phosphate also caused a decrease in labeling but to a lesser extent. Various amounts of sialic acid (0.02 mM, 0.2 mM, 2 mM, 20 mM, and 200 mM) showed that most of these concentrations produced a significant decrease in the labeling density of endocytic vesicles and endosomes. Moreover, Western blots of prosaposin isolated from seminiferous tubular fluids followed by glycan analysis with Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), revealed that this protein has sialic acid residues that are terminally linked to galactose and/or N-acetylgalactosamine (alpha-NeuNAc-[2->6]-Gal and alpha-NeuNAc-[2->6]-GalNAc). These data indicate that testicular prosaposin is removed from the lumen of the efferent ducts by the noncialiated cells via a receptor that recognizes prosaposin's terminal sialic acid residues.

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis.

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O-glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1-3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.

Sialoglycoproteins and penultimate sugar expression pattern in developing murine olfactory and respiratory mucosa.

Sialic acid residues are constant constituents of the glycoproteins of the airways in all species. Sialoglycoproteins are the main acidic glycoprotein and their functions are to mediate cell adherence, to control the viscoelasticity of mucus and to serve as receptor sites for the binding of exogenous macromolecules. The purpose of this study was to investigate the differences in the distribution of sialoglycoproteins as a terminal sugar and in the composition of the penultimate sugar according to aging in the murine nasal respiratory and olfactory mucosa. Nasal cavities of mice (BALB/c) were fixed by intracardiac perfusion with 2.0% glutaraldehyde and embedded in Epon 812. First, the serial sections were stained with Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA). Then, the adjacent sections were stained with DBA and PNA before and after neuraminidase digestion in all experimental groups. Apical cell surfaces of olfactory mucosa and cilia on a few ciliated cells in the mucosa of the septum and nasal floor were labelled with MAA, but cell surfaces of respiratory mucosa, Bowman's glands and goblet cells were not labelled with MAA, irrespective of aging. Apical cell surfaces of both olfactory and respiratory mucosa and Bowman's glands were stained with SNA, however, goblet cells were not labelled with SNA. After neuraminidase digestion to remove terminal sialic acid residues of sialoglycoproteins, only cell surfaces of respiratory mucosa were labelled with PNA, but goblet cells, cell surfaces of olfactory mucosa and Bowman's glands were not labelled with PNA. Cell surfaces and Bowman's glands of olfactory mucosa were labelled with DBA after neuraminidase digestion, but cell surfaces of respiratory mucosa and goblet cells were not labelled with DBA. Our results indicate that there were different carbohydrate structures of sialoglycoconjugates in olfactory and respiratory mucosa, and it was not influenced by aging.

The major elderberry (Sambucus nigra) fruit protein is a lectin derived from a truncated type 2 ribosome-inactivating protein.

The major protein of elderberry (Sambucus nigra L.) fruits is a lectin, called Sambucus nigra agglutinin IVf or SNAIVf. This lectin is composed of subunits that strongly resemble the B chain of the type 2 ribosome-inactivating protein (RIP), called SNAVf, present in the same tissue. To corroborate the possible relationship between both proteins their corresponding cDNAs were cloned and compared. Alignment of the deduced amino acid sequences revealed that the cDNA encoding SNAIVf is almost identical to that of SNAVf except that its A chain is truncated. Northern blot analysis confirmed that the mRNA encoding SNAIVf is about 500 nucleotides shorter than the SNAVf mRNA. In addition, the occurrence of a truncated type 2 RIP gene was unambiguously demonstrated by the analysis of PCR amplified genomic sequences. These results not only demonstrate for the first time that a plant lectin is encoded by a truncated type 2 RIP gene but also address important questions with respect to the molecular evolution of RIP and lectins.

Interferon-gamma enhancement of E-selectin expression on endothelial cells is inhibited by monensin.

The expression of E-selectin reaches a maximum 4-6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-alpha (TNF-alpha) and then declines to basal level within 24 h. If interferon-gamma (IFN-gamma) is added to the cell culture medium together with TNF-alpha the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-gamma induced persistent surface expression of E-selectin. SDS-PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-gamma produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-gamma/TNF-alpha compared to TNF-alpha alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monesin, a potent inhibitor of late Golgi function, together with both TNF-alpha and IFN-gamma, the additive effect of IFN-gamma on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-gamma induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-gamma/TNF-alpha in the presence of several different inhibitors of N-glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.

CBP70, a glycosylated nuclear lectin.

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.