Glycoproteins from Teratocarcinomas and Organs of Adult Mice: Analysis by Affinity Chromatography on Lectin-conjugated Agarose1

Glycoproteins were isolated from particulate fraction of four teratccarcinomas and several organs of adult mice by affinity chromatography on lectins conjugated with agarose [concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA)] and were compared by SDS gel electrophoresis. Glycoprotein components were found to be very similar in three lines of solid teratocarcinoma, namely F9, STT-2 and OTT-10A. Teratocarcinoma OTT6050, which is an ascitic form called embryoid bodies, also gave glycoprotein profiles somewhat similar to those of other teratocarcinomas. However, glycoprotein profiles of most adult organs were significantly different from those of teratocarcinomas. The following points were of special interest. 1) RCA receptors from the four teratocarcinomas gave a strong band with an apparent molecular weight 145,000 daltons, which was either weak or absent in the receptors from adult organs. 2) The WGA receptors of all adult organs except muscle and small intestine gave more intense bands than those of the teratocarcinomas. 3) Glycoproteins with molecular weights of more than 240,000 daltons were present in WGA receptors, RCA receptors and PNA receptors of teratocarcinoma OTT 6050, but not in the receptors of other teratocarcinomas.     

Reactivity of five N-acetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse

The expression of receptors for N-acetylgalactosamine-recognizing lectins, namely Helix pomatia agglutinin (HPA), Sophora japonica agglutinin (SJA), Bauhinia purpurea agglutinin (BPA), Vicia villosa agglutinin (VVA), and Wistaria floribunda agglutinin (WFA) was studied in early mouse embryos and teratocarcinoma cells. Each of these lectins as well as Dolichos biflorus agglutinin (DBA) bound differently to early embryonic cells, with the exception of VVA and WFA which showed indistinguishable reactivities. SJA reacted intensely with visceral endoderm, but hardly at all with parietal and primitive endoderm. Therefore, SJA will be useful for analyzing the mechanism of visceral-endoderm formation. Furthermore, the inner cell mass (ICM) of early blastocysts reacted intensely with DBA, while the ICM of late blastocysts reacted only faintly with this lectin. Primary endoderm derived from the ICM reacted faintly with SJA, HPA, and DBA, and these reactivities increased again during the differentiation of the endoderm. Therefore, these three lectins could be used in the analysis of early stages during the differentiation of endoderm from the ICM. The results illustrate the highly complex nature of developmentally regulated alterations of cell-surface carbohydrates during the early stages of embryogenesis.     

Characteristics of the distribution of lectin receptors in intrahepatic cholangiocellular carcinoma

Summary The receptors of peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andUlex europaeus agglutinin I (UEA-I) were localized in intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, intrahepatic bile ducts and normal, cirrhotic and pericarcinomatous liver using the avidin—biotin—peroxidase complex method. It was found that epithelial cells of normal bile ducts had many UEA-I receptors, fewer DBA receptors and no PNA receptors. The positive rates of PNA, UEA-I and DBA receptors in 18 cases of intrahepatic cholangiocellular carcinoma were 88.9%, 61.1% and 33.3% respectively, which were significantly higher than those in hepatocellular carcinoma (16.0%, 4.0% and 4.0% respectively). Hepatocytes in normal, cirrhotic and pericarcinomatous liver had no receptors for these three lectins. It is suggested that lectin receptor distribution in intrahepatic cholangiocellular carcinoma is obviously different from that in normal bile duct cells and in hepatocellular carcinoma, and might be used as an auxiliary index in its clinical diagnosis.     

The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

Lectins influence chondrogenesis and osteogenesis in limb bud mesenchymal cells

The role of cell surface glycoproteins in cell behavior can be characterized by their interactions with plant lectins. This study was designed to identify the effects of lectins on chondrogenesis and osteogenesis in limb bud mesenchymal cells in vitro. Limb bud mesenchymal cells from mouse embryos were cultured in high-density micromass culture. Wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin (RCA) were added separately to the culture media. Cells were cultured for 5 or 9 days, and cell viability was assayed by neutral red on day 5. The micromasses were stained with alcian blue, alizarin red S and Von Kossa stains, and alkaline phosphatase assays were also done. Dolichos biflorus agglutinin induced an increase in chondrogenesis, calcium precipitation and proteoglycan production. ConA and PNA did not affect chondrocyte differentiation but induced chondrocytes to produce more proteoglycan. Wheat germ agglutinin reduced chondrification and ossification but induced mesenchymal cells to store lipid droplets. Ricinus communis agglutinin 1 was toxic and significantly reduced cell survival. In conclusion, DBA was the most effective inducer of ossification and chondrification. Wheat germ agglutinin induced adipogenesis instead. These assays showed that lectins play important roles in limb bud development.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of glycosylated sites in embryonic rabbit kidney by lectin chemistry

The reciprocal cell biological interaction between mesenchymal and epithelial tissue plays a critical role during nephrogenesis. It is unknown to date whether the tissues interact during nephron induction by pure diffusion of substances or whether cellular contacts via gap junctions or focal adhesion molecules are involved. In neonatal rabbit kidney the interface between both tissues shows unique features. It consists of a distinct space, which is filled with specific extracellular matrix consisting of glycosylated proteins such as fibronectin, laminin, collagen, and proteoglycans. In the present experiments we tested by histochemistry whether it is possible to detect additional glycosylated proteins using Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus I agglutinin (UEA I), and Peanut agglutinin (PNA) as molecular markers. All tested lectins showed distinct labeling patterns in embryonic renal tissue. Within the collecting duct ampulla, DBA and UEA I revealed intensive cellular reaction. In contrast, PNA and SBA reacted at the basal aspect of the collecting duct ampulla tip in addition to a cellular reaction. To identify the individual molecules labeled by the lectins, embryonic tissue was fractionated and separated by electrophoretic methods. For the first time, we were able to show by two-dimensional electrophoresis and subsequent western blot experiments that lectins bind to a series of individual protein spots, which have not been identified to date.     

Isolation and characterization of peanut agglutinin-resistant embryonal carcinoma cell-surface variants

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell-surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 × 10^–5 following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild-type cells and two different PNA-resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA-resistant cells also showed reduced binding of monoclonal antibody against stage-specific embryonic antigen 1 (SSEA–1) in indirect cytotoxicity tests. All eight PNA-resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well-differentiated teratocarcinomas. The PNA-resistant cells behaved like their wild-type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo-specific cell surface structures (s) that bind PNA and anti-SSEA-1 antibody.     

Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development

Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI-and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI- and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

Lectin binding to rat spermatogenic cells: Effects of different fixation methods and proteolytic enzyme treatment

Summary The binding of a panel of eight different fluorescein-conjugated lectins to rat spermatogenic cells was investigated. Particular attention was paid to the effects of different fixation methods and proteolytic enzyme digestion on the staining pattern.Concanavalin A (Con A), wheatgerm agglutinin (WGA), succinylated WGA (s-WGA) and agglutinin from gorse (UEA I) stained the cytoplasm of most germ cells as well as the spermatid acrosome. In contrast, peanut agglutinin (PNA), castor bean agglutinin (RCAI) and soy bean agglutinin (SBA) mainly stained the acrosome. The staining pattern varied depending on the fixation method used. PNA was particularly sensitive to formalin fixation, while SBA, DBA and UEA I showed decreased binding and Con A, WGA, s-WGA and RCA I were insensitive to this type of fixation. Pepsin treatment of the sections before lectin staining caused marked changes in the staining pattern; staining with PNA in formalin-fixed tissue sections was particularly improved but there was also enhanced staining with SBA and horse gram agglutinin (DBA). On the other hand, in Bouin- and particularly in acetone-fixed tissue sections, pepsin treatment decreased the staining with several of the lectins, for example WGA and UEA I.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro

Summary The binding of several plant lectins, Concanavalin A (ConA), Lens culinarisA (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope.In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro-and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.     

Glycoprotein profiling of stem cells using lectin microarray based on surface plasmon resonance imaging

Lectin microarrays have emerged as a novel platform for glycan analysis during recent years. Here, we have combined surface plasmon resonance imaging (SPRi) with the lectin microarray for rapid and label-free profiling of stem cells. In this direction, 40 lectins from seven different glyco-binding motifs and three different cell lines—mouse embryonic stem cells (mESCs), mouse-induced pluripotent stem cells (miPSCs), and mouse embryonic fibroblast stem cells (MEFs)—were used. Pluripotent mouse stem cells were clearly distinguished from non-pluripotent stem cells. Eight lectins—DBA, MAL, PHA_E, PHA_L, EEL, AAL, PNA, and SNA—generated maximal value to define pluripotency of mouse stem cells in our experiments. The discriminant function based on lectin reactivities was highly accurate for the determination of stem cell pluripotency. These results suggested that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.