Evaluation of Fluorescently Labeled Lectins for Noninvasive Localization of Extracellular Polymeric Substances in Sphingomonas Biofilms

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.     

Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: D-glucose or D-mannose for ConA and N-acetyl-D-glucosamine or N-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.     

Lectin binding sites as markers of neonatal porcine uterine development.

To identify lectin binding sites and to determine if lectin binding patterns change with age in developing neonatal porcine uterine tissues, gilts (n = 3/day) were hysterectomized on Day 0 (birth), 7, 14, 28, 42, or 56. Lectin binding was visualized in Bouin's-fixed uterine tissues with seven biotinylated lectins (ConA, DBA, PNA, RCA-I, SBA, UEA-I, and WGA) and avidin-peroxidase staining procedures. Lectin specificities were demonstrated by pre-incubating lectins with appropriate inhibitory sugars (0.2 M). Staining intensity was evaluated visually (absent, weak, moderate, or strong) for three endometrial tissues; luminal epithelium, glandular epithelium, and stroma. Staining intensities for DBA, PNA, SBA, and WGA were not affected by neonatal age. Staining with these lectins was greater in uterine epithelium (moderate or strong) than in stroma (weak). In contrast, binding patterns for ConA, UEA-I, and RCA-I were affected by neonatal age. Strong epithelial staining associated with ConA binding was observed on all days, whereas stromal ConA staining decreased in intensity from moderate to weak after Day 14. Epithelial staining with UEA-I increased from moderate to strong after Day 28, whereas stromal UEA-I staining decreased from moderate to weak after day 28. Staining with RCA-I was homogeneous for luminal epithelium and stroma but variegated for glandular epithelium on and after Day 7. These observations indicate that a variety of lectin binding sites are present in developing neonatal porcine endometrial tissues and that developmentally related alterations in the distribution and/or orientation of glycoconjugates containing alpha-D-mannose, beta-D-galactose, beta-D-acetyl-N-galactosamine, and alpha-L-fucose residues occur between birth and Day 56 as these tissues mature.     

Lectin staining of cultured CNS microglia.

Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.     

Lectin binding to collagen strands in histologic tissue sections

Histologic sections from human skin and uterine ligaments were stained with the following FITC conjugated lectins: Con A, WGA, s-WGA, SBA, DBA, UEA I, PNA, RCA I, BPA, GSA I, GSA II, MPA and LPA. The staining of the connective tissue was similar in the dermis and the uterine ligaments and it was most intense in the extracellular matrix containing collagen strands whereas the fibrocytes remained unstained. The staining was clear with glucose or N-acetylglucosamine binding lectins like Con A, WGA, s-WGA and GSA II, which may be related to the presence of glucose residues in collagenous hydroxylysine. The staining with some of the galactose or N-acetylgalactosamine binding lectins like RCA I, DBA, and BPA was less intense. This may reflect the presence of terminal galactose sugars in the hydroxylysine of collagen. No staining was found with SBA, UEA I, PNA, GSAI, MPA or LPA. The results show that different particularly glucose specific lectins bind to the extracellular matrix and especially to collagenous strands in connective tissue. It is suggested that this might be used in histochemical studies of connective tissue and particularly concerning the changes that may occur in different disease states.     

Specific modifications of hepatoma cell-surface glycoproteins with enzymes : Effects on in vitro growth as investigated by the use of lectins

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.     

138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

Cellular heterogeneity in normal human urothelium: Quantitative studies of lectin binding

Summary A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.     

Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta)

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.     

亲和层析法分离纯化玉米精细胞质膜特异糖蛋白

 亲和层析法分离纯化玉米精细胞质膜特异糖蛋白陈丽邵邻相汪洁杨中汉（北京大学生命科学学院,北京１００８７１）ＩｓｏｌａｔｉｏｎａｎｄＰｕｒｉｆｉｃａｔｉｏｎｏｆＳｐｅｒｍＣｅｌＰｌａｓｍａＭｅｍｂｒａｎｅＧｌｙｃｏｐｒｏｔｅｉｎｓｏｆＺｅａｍａｙｓｂｙＡ...     

Characterization of carbohydrates of adult Echinococcus granulosus by lectin-binding analysis

The identification of lectin-binding structures in adult worms of Echinococcus granulosus was carried out by lectin fluorescence; the distribution of carbohydrates in parasite glycoconjugates was also studied by lectin blotting. The lectins with the most ample recognition pattern were ConA, WGA, and PNA. ConA showed widespread reactivity in tegument and parenchyma components, including the reproductive system, suggesting that mannose is a highly expressed component of the adult glycans. Although reproductive structures appeared to be rich in N-acetyl-D-glucosamine (GlcNAc)-N-acetyl neuraminic acid (NeuAc) and galactose (Gal) as demonstrated by their strong reactivity with WGA and PNA, respectively, some differences were observed in their labeling patterns. This was very clear in the case of the vagina, which only reacted with WGA. Furthermore, WGA and ConA both had reactivity with the excretory canals. RCA, the other Gal binding lectin used, only reacted with the tegument, suggesting that widespread PNA reactivity with the reproductive system is related to the presence of the D-Gal-beta-(1,3)D-GalNAc terminal structure. UEA I failed to bind to any parasite tissues as determined by lectin fluorescence, whereas DBA and SBA showed a very faint staining of the tegument. However, in transferred glycans, N-acetyl-D-galactosamine (GalNAc) and fucose (Fuc) containing glycoproteins were distinctly detected.     

Lectin receptors on the peri- and early post-implantation mouse embryo

Summary Mouse embryos at the blastocyst, blastocyst outgrowth, and primitive streak (day 7.5) stages of development were analysed for expression of lectin receptors using a panel of six FITC-conjugated lectins with affinities for five distinct saccharides (BSL, ConA, DBA, LTL, UEA and WGA). Blastocyst trophoblast expressed receptors for all the lectins but later tissues of the trophectoderm lineage lost receptors for distinct but overlapping subsets of the lectin panel. The inner cell mass (ICM) of the early blastocyst lacked receptors only for UEA. Differentiation of primary endoderm was accompanied by the aquisition of UEA receptors but subsequent differentiation into visceral and parietal endoderm involved the loss of receptors for both fucose binding lectins (UEA and LTL). Embryonic ectoderm in the day 7.5 egg cylinder retained receptors only for ConA and WGA. Thus, in general, differentiation during the peri- and early post-implantation period was associated with a differential loss of lectin receptors in all cell lineages of the mouse conceptus.     

Lectin binding sites in Paramecium tetraurelia cells. I. Labeling analysis predominantly of secretory components

Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis. When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in approximately 6-10 vacuoles, which are several microns in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (otherwise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts). Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of "tips"), while ConA (fluorescence concentrated over "bodies") and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (approximately 1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable "matrix". Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.     

Binding of wheat germ agglutinin to extracellular network produced by cultured human fibroblasts

The occurrence of diverse carbohydrate moieties on the cell surface and in the extracellular matrix, makes lectins the suitable probes to study the distribution of appropriate determinants produced in cell culture. Biotin-labelled wheat germ agglutinin (WGA) was used in microscopic and photometric detection of lectin binding to monolayer of human skin fibroblasts. The incubation of confluent fibroblast monolayer with labelled WGA reveals two principal patterns of binding of this lectin: to cell surface structures and, predominantly, to extracellular fibres; the alignment and density of extracellular network are not uniform. After binding of WGA to confluent culture, light microscopic analysis revealed the ubiquitous fibrillar network between and over cells, with some regions of increased compactness and altered orientation of fibrils. Binding to cell surfaces (manifested as specks) was predominant for the fibroblasts at the logarithmic phase of growth. N-acetylglucosamine (0.2 M) and native lectin (100 microg/ml) had a partial inhibitory effect on WGA binding to the extracellular network. Treatment with neuraminidase (0.1 unit/ml) of untreated or prefixed monolayers resulted in a significant decrease in WGA binding to fibrils (and increase in PNA binding), indicating that terminal sialic acid residues are mainly involved in the network-WGA interaction. Mild trypsinization (10 microg/ml) removed the target sites, which retained the ability to bind WGA, being spotted on hydrophobic Immobilon P paper; biotinylated lectin, bound to adsorbed glycopeptides, could be eluted and quantified in solid-phase inhibition assay.     

Lectin-binding properties of hamster egg zona pellucida and plasma membrane during maturation and preimplantation development

The structure and organization of the zona pellucida and plasma membrane of the hamster egg at various stages of maturation and development were examined using lectin-mediated agglutination and the binding of fluorescent-labeled lectins. Ricinus communis I and Dolichos biflorus lectins specifically agglutinated the zona pellucida of both unfertilized and fertilized eggs, while wheat germ agglutinin (WGA) only agglutinated eggs which had been pretreated with protease. Six other lectins failed to agglutinate even eggs pretreated with protease. A comparison of the lectin-binding sites on the zona pellucida of eggs in various stages of maturation and development revealed that the intensity of binding and distribution of fluorescent-labeled lectins remain unchanged. Zona-free eggs were agglutinated by every lectin tested except those recognizing -fucose-like residues. Fertilized zona-free eggs were slightly more agglutinable by concanavalin A (ConA), Lens culinaris and WGA than unfertilized eggs. When the surfaces of zona-free eggs were examined with fluorescent ConA, Ricinus communis I and WGA, maximal binding was seen when eggs reached full maturity and binding decreased during the later stages of preimplantation development.     

Morphological study of lectin binding in the rabbit temporomandibular joint disc

Summary Glycoconjugates of the extracellular matrix are important for the normal mechanical functions of connective tissue structures such as the temporomandibular joint disc. Since lectins are known to bind to sugar residues with high affinity, a variety of lectins were used to study the presence and distribution of glycoconjugates in the temporomandibular joint disc. Discs were removed from 6 to 8-month-old rabbits and either sectioned in a cryostat and processed for light microscopy or fixed in 2% glutaraldehyde and processed for electron microscopy. The frozen sections were incubated with fluorescein- or peroxidaseconjugated lectin solutions. Ultrathin sections mounted on grids were incubated with lectins combined with a colloidal gold marker system for electron microscopical study. Our results indicate thatCanavalia ensiformis agglutinin (ConA) showed little or no binding to the discal tissue.Triticum vulgaris agglutinin (WGA) andMacluras pomifera (MPA) were bound strongly to both the synovium and the extracellular matrix and WGA also bound to the territorial matrix of chondrocyte-like cells.Glycine max andArachis hypogoea agglutinins (SBA and PNA), were localized in the synovium and extracellular matrix but to a lesser degree than WGA and MPA. WGA, MPA,Griffonia simplicifolia II andUlex europaeus were bound by discal fibroblasts. WGA was also localized in lysosomes of synovial A-cells (macrophages). The electron microscopical studies with lectins and colloidal gold marker systems indicated that some areas of the disc may be fibrocartilagenous as had been suggested by earlier immunohistochemical studies using monoclonal antibodies to characteristic glycosaminoglycans (GAGs) in cartilage.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Protein microarrays to study carbohydrate-recognition events

In order to expand areas in which protein microarrays can be used to solve important biological problems, we have investigated ways in which the technique can be employed for functional glycomics. Initially, our protein microarrays were used for the rapid identification of carbohydrate-binding proteins using trifunctional carbohydrate probes and fluorescent dye-labeled polysaccharides. Glycan probes were selectively bound to the corresponding lectins immobilized on the solid surface. In addition, these microarrays were also employed for profiling of carbohydrates on Jurkat T-cell surfaces. These cells adhered to ConA, RCA(120), SNA and WGA, indicating expression of alpha-Man, Gal, NeuNAcalpha2,6Gal and GlcNAc residues on their surfaces. Furthermore, we determined binding affinities between WGA and carbohydrates by measuring IC(50) values of GlcNAc that inhibited 50% of trivalent GlcNAc binding to WGA immobilized on the solid surface. All the experiments show that protein microarrays can be used to study carbohydrate-recognition events in the field of glycomics.