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The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three major glycoproteins that show considerable heterogeneity due to extensive post-translational modifications. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the ZP reveals three to four glycoproteins which have been nominated ZPI. ZP2, ZP3 and ZP4. As cloning and characterization of the ZP genes of a variety of mammalian species including domestic animals show a high homology, three classes of ZP genes, ZPA, ZPB and ZPC can be discerned. The corresponding proteins were named ZPA, ZPB and ZPC. Whereas in the mouse ZPB is the primary sperm receptor. the situation is more complicated in other species. For instance, in the pig ZPA has been shown to possess receptor activity. Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the ZP and carbohydrate-binding proteins of the sperm surface. In domestic animals zona proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play a role in granulosa cell differentiation. The role of ZP glycoproteins in immunocontraception is briefly discussed.

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three glycoproteins (ZPA, ZPB, ZPC), products of the gene families ZPA, ZPB and ZPC that have been found to be highly homologous within mammalian species. Most data on the structure and function of the ZP are obtained from studies in mouse. New data from pig and other domestic animals, however, indicate that the mouse model does not hold for all other species. Whereas in the mouse ZPB is the primary sperm receptor, in the pig ZPA has been shown to possess receptor activity. Contrary to the mouse, where the growing oocyte is the only source of zona glycoproteins, in domestic animals these proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play also a role in granulosa cell differentiation. In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins (oviductins) that become closely associated with the ZP of the ovulated oocyte. Once bound to the ZP, oviductin molecules could act as a protective layer around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains., (Copyright 2001 S. Karger AG, Basel)

The duplication of genes for recognition molecules and the ensuing diversification of the members of such families generate complex groups of homologous proteins. One example are galactoside-specific lectins whose sequences display constant features related to sugar binding, the galectins. Based on the inverse abundance of the chicken galectins CG-14 and CG-16 in adult intestine and liver, these two lectins represent a model to comparatively study expression of the related proteins and the galectin-reactive sites (glycoproteins and glycolipids) biochemically and histochemically. Functional overlap and/or acquisition of distinct functions would be reflected in qualitative and/or quantitative aspects of ligand display. Using five different stages of embryogenesis, differential regulation of the two galectins was detected in liver and intestine. The clear preference for one galectin (CG-14) was observed in intestine already at rather early stages, whereas equivalence for both proteins was noted in liver from day 12 to day 18 prior to hatching, as seen by ELISA assays and Western blot analysis. Presentation of galectin-reactive glycoproteins showed a tendency for gradual increase in both organs. Galectin-blotting analysis revealed primarily very similar patterns of positive bands at the different stages of development and only few quantitative and qualitative changes. The reactivity of glycolipids in a solid-phase assay was more variable, even surpassing the response of extracts of the adult organ at several embryonic stages. While the localization patterns of the galectins and galectin-reactive sites were nearly indistinguishable in the liver, intestinal tissue differed with respect to the placement and accessibility of binding sites. Thus, the results suggest a differential regulation of galectin activities in the two organs. As a sum they resemble the course of development of availability of glycoprotein ligands in vitro. These findings support the notion for a partial functional redundancy in this family. The described approach to employ galectin-specific antibodies and the labeled galectins as tools to assess presentation of ligands is suggested to be of general relevance to address the question of distinct vs. overlapping functions of related recognition molecules.

Carrier-immobilized carbohydrates are valuable tools for assessing the glycoligand-binding capacity of cell surfaces. A panel of 10 types of fluorescent neoglycoproteins has been synthesized to determine the extent of their specific binding to endothelial cells in vitro that have been obtained from porcine brain, ovary, and testis. Different sugar moieties revealed a nonuniform capacity to bind to the endothelial cells as determined by flow cytometric analysis, the histogenetic origin of the preparations being an important factor. Binding of mannose, xylose, and glucuronic acid was especially pronounced, but the extent of cell-associated fluorescence was significantly different dependent on the source of the endothelial cells. These results clearly reveal that endothelial cells in vitro display the capacity to specifically recognize defined carbohydrate moieties. Moreover, endothelial cells of different tissue origin can exhibit variability in this property, potentially endowing this cell type with site-dependent molecular properties.

We used several biotinylated neoglycoproteins as tumor markers to detect and localize endogenous carbohydrate-binding proteins in cultured hepatoblastoma, melanoma, and bladder carcinoma tumor cells. The neoglycoproteins used consisted of cellobiose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, lactose, maltose, mannose, melibiose, and xylose. In addition, naturally occurring asialofetuin that was chemically disialylated was also used. Binding to the cultured tumor cells was made visible with the avidin-peroxidase technique. Depending on the type of neoglycoprotein used, markedly different expression of cytoplasmic and nuclear receptors for sugars (endogenous lectins) was obtained from rat hepatoblastoma, human melanoma, and bladder carcinoma tumor cells. The most pronounced staining differences were documented for asialofetuin and the neoglycoproteins containing fucose, N-acetyl-galactosamine, and lactose.

Modifications in bull sperm plasmamembrane during epididymal passage were investigated by the use of four different lectins: Concanavalin A (Con A); Ricinus communis I (RCA1); Wheat germ agglutinin (WGA); Ulex europaeus agglutinin I (UEA1). During sperm passage from caput to cauda epididymidis agglutination by RCA1 and WGA distinctly increased. Similar but somewhat less pronounced difference in the agglutinability was found for Con A. No agglutination was observed with UEA1. Ultrastructural examination of Con A binding sites on sperm plasma membrane with a Con A-horseradish peroxidase-gold technique (Con A-HRP-G) revealed a significant increase in the number of gold granules on the sperm tails during the epididymal passage of spermatozoa. No change in WGA-binding sites was observed between caput and cauda spermatozoa using a WGA-peroxidase method.

In the present study, the topographical distribution of carbohydrate binding sites on the plasma membrane of bovine epididymal spermatozoa was investigated using 15 different fluorescent neoglycoproteins and asialoglycoproteins. With mannose-bovine serum albumin (BSA)-fluoresceinthiocarbamyl (FTC), mannose-6-phosphate-BSA-FTC, lactose-BSA-FTC, maltose-BSA-FTC, asialolactoferrin-FTC and asialotransferrin-FTC a marked fluorescence was observed in the postacrosomal area. These results further substantiate the concept that carbohydrate binding sites of the spermatozoan plasma membrane and corresponding carbohydrates of the zona pellucida play a significant role in gamete interactions.

The goal of this study was to demonstrate the distribution of glycoproteins in the various segments of the Japanese quail nephron, using lectins labeled with HRP or FITC. Each one of the six labeled lectins had a characteristic distribution pattern along the nephron. The study shows that lectins are useful markers for certain nephron segments or for cell types in certain segments of the renal tubules. PNA marks the thin portion of the medullary loop, DBA marks the thick portion; it is thus possible to differentiate the nephron segments in the medullary cone of the kidney. Con A binds selectively with the epithelioid, granular cells of the tunica media of the vasa efferentia. The histochemical technic using labeled lectins makes it possible to identify certain renal structures that could not, or only with difficulty, be differentiated using conventional histology. Therefore, lectins as specific markers are gaining in importance for further studies of the morphology and physiology of the kidney.

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish ( Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues. [ABSTRACT FROM AUTHOR]