One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions

Protein-carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove excess labeling reagent. After biotinylation, the structural and biological integrity of the glycans was intact as determined by HPLC, mass spectrometry, and a plant lectin assay. By using the human C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), we demonstrate that the biotinylated glycans can be used in a glycan array to determine binding specificities of lectins. Moreover, we show that fluorescent beads coated with selected biotinylated glycans bind to DC-SIGN-expressing dendritic cells in vitro. Finally, by using biotinylated high-mannose N-glycans, we could visualize DC-SIGN-expressing cells in lymph node tissue. The availability of easy biotinylation methods for oligosaccharides such as those described here greatly facilitates the functional analysis of lectins. In addition, the biotinylated glycans will be great tools for investigating functional lectin receptors in situ.     

Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant mutation in ConA-like lectins

The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides.     

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.     

AnimalLectinDb: An integrated animal lectin database

Lectins, a class of carbohydrate-binding proteins and widely recognized to play a range of crucial roles in many cell-cell recognition events triggering several important cellular processes encompass different members that are diverse in their protein structures, carbohydrate affinities and specificities, their larger biological roles and potential applications. To attain an effective use of all the diverse data initially an animal lectin database 'AnimalLectinDb' with information pertaining to taxonomic, structural, domain architecture, molecular sequence, carbohydrate structure and blood group specificity has been developed. It is expected to be of high value not only for basic study in lectin biology but also for advanced research in pursuing several applications in biotechnology, immunology, and clinical practice. AVAILABILITY: The database is available for free at http://www.research-bioinformatics.in.     

Development of cortical vesicles in Sicyonia ingentis ova: their heterogeneity and role in elaboration of the hatching envelope

In the marine shrimp Sicyonia ingentis, ova lack cortical vesicles at spawning. Previous ultrastructural studies suggested that two different populations of cortical vesicles (dense vesicles and the ring vesicles) appear within 30 min post-spawning. These vesicles undergo sequential exocytosis (exocytosis of the dense vesicles followed by exocytosis of the ring vesicles) that leads to the formation of a hatching envelope around the ovum (see Pillai and Clark: Tissue & Cell 20:941-52, 1988). In the present study, lectins were used as molecular probes to study the development of cortical vesicles subsequent to spawning and the role of these vesicles in formation and elaboration of the hatching envelope. Isolated envelopes were screened with 11 different lectins to determine what group(s) were specific to the envelope glycoconjugates; Concanavalin A (Con A), Griffonia simplicifolia (GS II), Lens culinaris (LCA), and wheat germ agglutinin (WGA) bound to the envelopes. FITC-lectin studies of sectioned ova (fixed at various time points after spawning) utilizing WGA and LCA showed different labelling patterns. Data obtained at the light microscopical level indicated that WGA was specific to the dense vesicles and the outer portion of the envelope, while LCA exhibited specificity for the ring vesicles and the inner portion of the envelope. At the ultrastructural level, gold-LCA labelling was seen associated with the cisternal elements (containing ring-shaped structures), ring vesicles, and the inner layer of the fully formed envelope. These data demonstrated that 1) the ring vesicles are formed by fusion of cisternal elements containing ring-shaped structures; 2) the two species of cortical vesicles are chemically heterogeneous; and 3) the components of each type of vesicle contribute to different integral parts (the outer and inner layers) of the hatching envelope.     

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin

Summary In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin — probes of sialic acid and N-acetylglucosamine on the cell surface — was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration — rabbits with sodium iodate induced retinopathy — was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pgiment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.     

Localization, morphology and function of the mitochondria-rich cells in relation to transepithelial Na(+)-transport in chicken lower intestine (coprodeum)

The correlation between morphology of the mitochondria-rich cells (MR cells) in chicken lower intestine, coprodeum, and dietary sodium levels, has been investigated, using hens with differing dietary intake of NaCl and plasma aldosterone levels. Additionally, the function of the MR cells was evaluated in relation to proton secretion/exchange. Epithelium from the coprodeum was examined by optical, transmission and scanning electron microscopy, and Na(+)-transport across the coprodeal epithelium was measured electrophysiologically in Ussing-chambers. To investigate the function of MR cells, lectin-, enzyme- and immunohistochemistry methods were used. The MR cells were generally located in the epithelium on the upper parts of the sides of mucosal folds. Long microvilli, high but variable toluidine blue affinity/electrondensity and numerous mitochondria were the main features distinguishing them from the surrounding epithelial cells. Two main MR cell types were observed, differing in microvillous morphology, diameter and toluidine blue affinity/electrondensity. This probably reflected differences in maturity and activity. The MR cells expressed a positive carbonic anhydrase reaction and a proton exchange similar to the absorptive intestinal epithelial cells, but exhibited no specific demonstrable proton secretion. A close correlation between the ultrastructure of the MR-cells, dietary sodium levels, plasma aldosterone and transepithelial Na-transport was observed.     

Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition

Microglia, the tissue macrophages of the brain, play a crucial role in recognition and phagocytic removal of apoptotic neurons. The microglial receptors for recognition of apoptotic neurons are not yet characterized. Here we established a co-culture model of primary microglia and cerebellar granule neurons to examine the receptor systems involved in recognition/uptake of apoptotic neurons. Treatment with 100 microM S-nitrosocysteine induced apoptosis of cerebellar neurons as indicated by nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet of the plasma membrane. Microglial cells were added to neurons 2 h after apoptosis induction and co-cultured for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4', 6-diamidino-2-phenylindole/propidium iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N-acetylglucosamine or galactose, suggesting that recognition involves asialoglycoprotein-like lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid vesicles enriched in phosphatidylserine and O-phospho-L-serine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Expression pattern of glycoconjugates in the integument of Bufo ictericus (Anuran, Bufonidae): biochemical and histochemical (lectin) profiles

Mucous consists of glycoproteins and proteoglycans produced by specific secretory cells (mucocytes). In anurans the cutaneous mucous is produced by intradermal glands and displays both mechanical and chemical protection functions. Indeed, mucous maintains the integument moist and facilitates gas exchange (cutaneous respiration). In this work, the carbohydrate moiety distribution was investigated in the integument of Bufo ictericus using conventional and lectin histochemistry to describe the pattern of cutaneous glycoconjugate expression, including both secretory and structural proteoglycans. As a preliminary step, the descendent chromatography in Whatmann 1MM paper was undertaken to prepare the histochemical trials involving the lectins. In B. ictericus, the integument exhibits the basic morphological structure found in lower terrestrial vertebrates: the epidermis is a keratinized squamous stratified epithelium supported by spongious and compact layers. The spongy dermis contain secretory portion of both mucous and serous (or poison) glands. The paper chromatography identified galactose, fucose and mannose as characteristic sugar residues. The secretory cells of the mucous gland in the dermis, as well as the interstice between the stratum corneum and the subjacent stratum spinosum in the epidermis exhibit alpha-l-fucose and alpha-galactose residues. The serous glands give no reaction. The alpha-mannose residue was detected in the extracellular matrix of spongious dermis, but not in the dermal glands. The different glycoconjugate location reflects in two glycoconjugates categories: the secretory which participate in the water flow regulation, and the structural which is involved in the dermal maintenance.