Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Lectin-binding spectra in the hyperplastic human tonsil

Summary In order to determine the effect of routine fixation on the lectin affinity of tissue structures, we used unconjugated lectins and an indirect immunoalkaline-phosphatase method for frozen sections, and the peroxidase-anti-peroxidase method for paraffin-embedded, formalinfixed tissue sections. Fourteen hyperplastic human tonsils were used, and the results of the binding spectra of each lectin were compared. In general, the binding spectrum defected in the paraffin sections was part of the broader range of affinity obtained in the frozen sections. The lectin receptors on the cell surface were especially affected by formalin fixation. On the other hand, the paraffin sections, because of their superior morphology, showed a better localization of the cytoplasmic reaction product and discriminated the cell types more accurately. Thus, the two tissue preparations are rather complementary. In the tonsil peanut agglutinin (PNA) and periodic acid/Concanavalin A (PA/Con A) proved to be suitable tools for distinguishing exactly between the crypt and the surface epithelium. Ulex europaeus agglutinin I (UEA) is a reliable endothelial marker with a strong affinity to the crypt epithelium. In the frozen sections, PNA regularly stained follicular-centre cells on their cell surface. PNA, Helix pomatia agglutinin (HPA), soybean agglutinin (SBA) and Con A stained the histiocytic population, especially PNA which additionally stained an asteroid histiocyte. This cell probably corresponds to the antigen-presenting histiocyte of the T-system.Abbreviations PNA Peanut agglutinin - UEA Ulex europaeus agglutinin I - HPA Helix pomatia agglutinin - SBA Soybean agglutinin - Con A Concanavalin A - PHA Phaseolus vulgaris agglutinin - SaR swine-anti-rabbit immunoglobulins - PaP peroxidase-anti-peroxidase-complexes - HRP horseradish peroxidase - PA periodic acid - DAB diaminobenzidine - AP alkaline phosphatase - PBS phosphate buffered saline solution - pls paraffin section - fzs frozen section - s surface - c cytoplasmic     

Transmural exocytosis in maize root cap

Summary The maize root cap is a tissue known for its high production of a fucose-rich slime. At the cellular periphery, two kinds of components exist which are indistinguishable: the cell wall barrier and the slime which passes through. Two complementary probes were used, both at the light and the electron microscope level, in order to distinguish the different components. The lectinUlex europaeus agglutinin I was used as a probe targetting the slime and the enzyme cellulose 1,4--D-glucan cellobiohydrolase I was used to probe the cellulose framework. Both probes were used either alone or sequentially for double labeling. The cytochemical PATAg test was optionally used with the enzyme-gold complex labeling. After several technical improvements (multistep method, increase in accessibility), UeA I was used to follow the exocytic pathway of the slime from the Golgi apparatus to the exterior of the cell. The results indicate the occurrence of at least two populations of Golgi apparatus vesicles, and one is directly engaged in the transport of the fucoserich slime. The slime accumulated in pockets between the plasmamembrane and the outer tangential cell wall. The CBH I-gold complex showed the existence and the maintenance of a thin but continuous cellulosic layer, even when the cells slough. The double labeling showed the fucose-rich compounds within the cell wall. Data emphasize the role of the cell wall as a filtering barrier and a mechanical protection in the course of differentiation.Abbreviations CBH I EC 3.2.1.91, cellulose 1,4--D-glucan cellobiohydrolase I - CMC carboxymethyl cellulose - CPB citrate phosphate buffer - FITC fluorescein isothiocyanate - IC internal cell - PA-TAg periodic acid-thiocarbohydrazide-silver proteinate - PBS phosphate buffered saline - PC cell with accumulation pockets - PEG polyethyleneglycol - SC sloughed cell - UeA I Ulex europaeus agglutinin I - VI UeA I-labelled Golgi-derived vesicles - V2 UeA I-unlabelled Golgi-derived vesicles     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Summary Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin-or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (ConA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especiàlly those of N-acetylgalactosamine.     

A Survey of Lectin Binding in Paramecium1

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA₆₀) and agglutinin (RCA₁₂₀), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit ofBranchiostoma belcheri

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins (Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA),Helix pomatia agglutinin (HPA), Concanavalin A (Con A),Ulex europaeus agglutinin I (UEA I) andRicinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.     

Enterocytes in the follicle-associated epithelia of rabbit small intestine display distinctive lectin-binding properties

Glycoconjugate expression in follicle-associated epithelia has been examined by application of a panal of lectins to fixed preparations of rabbit small intestine, including Peyer's patches. Each of the lectins examined (wheat germ agglutinin, peanut agglutinin,Ulex europaeus agglutinin I andBandeiraea simplicifolia agglutinin II) exhibited a lower affinity for the apical surface of the specialised M cells than to columnar enterocytes within the Peyer's patch follicle-associated epithelium. Peanut agglutinin differed from the other lectins examined in that it displayed a markedly higher affinity for enterocytes within the follicle-associated epithelium than the neighbouring villi. This observation reveals that the specialised development of the follicle-associated epithelium involves expression of distinctive surface properties within the enterocyte population in addition to the more widely documented heterogeneous development of enterocytes and the specialised M cells.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

The localization of galactosyl residues and lectin receptors in the mucilage and the cell walls ofCosmocladium saxonicum (Desmidiaceae) by means of fluorescent probes

Summary The localization of galactosyl residues and lectin binding sites in mucilage and cell walls of the colony forming green algaCosmocladium saxonicum (Desmidiaceae) has been studied using fluorescent probes. In mucilaginous filaments, which are secreted through pores of the cell wall, and in the primary cell wall galactosyl residues in -bound configuration are exposed, as indicated by indirect immunofluorescence using antiserum to monogalactosyl diglyceride residues. Concanavalin A receptors are present mainly at the surface of the secondary cell wall, whereasRicinus communis agglutinin, type I, receptors are predominantly associated with mucilaginous connecting strands, which join adjacent cells within a colony. NoUlex europaeus agglutinin receptors were found. Application of the fluorochrome calcofluor white ST resulted in labeling both, the primary and the secondary cell wall. The data, obtained with the fluorescent probes were compared with those obtained by thin layer chromatography of hydrolysed mucilage.This work includes parts of a doctoral thesis of B. S. carried out under the supervision of Prof. Dr. M.Mix.     

Differences of microvascular endothelium in the bovine corpus luterum of pregnancy and the corpus luteum of the estrous cycle

Summary— The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells (‘cobblestone growth pattern’ and ‘arcuate growth pattern’) were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to be status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.     

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.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

A putative carbohydrate-binding domain of the lactosebindingCytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of thel-fucose-bindingUlex europaeus anti-H(O) lectin

The complete amino acid sequence of a lactose-bindingCytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of thel-fucose-bindingUlex europaeus lectin I (UEA-I).Abbreviations BPA Bauhinia purpurea lectin - Con A concanavalin A - CMA-I Cytisus multiflorus lectin I - CMA-II Cytisus multiflorus lectin II - CSA-I Cytisus sessilifolius lectin I - CSA-II Cytisus sessilifolius lectin II - CSII Cytisus scoparius lectin II - ECorL Erythrina corallodendron lectin - GSIV Griffonia simplicifolia lectin IV - HPLC high performance liquid chromatography - LAA-I Laburnum alpinum lectin I - LAA-II Laburnum alpinum lectin II - LOL Lathyrus ochrus lectin - LTA Lotus tetragonolobus lectin - MAH Maackia amurensis haemagglutinin - PSA Pisum sativum lectin - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid - UEA-I Ulex europaeus lectin I - UEA-II Ulex europaeus lectin II - VFA Vicia faba lectin     

Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

Summary Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and subunits of amphibian epidermal Na⁺,K⁺-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.Abbreviations BSA bovine serum albumin - CHAPS (3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate) - Con A Canavalia ensiformis agglutinin - DTT dithiothreitol - Gal galactose - GalNAc N-acetyl-D-galactosamine - GlcNAc N-acetyl-D-glucosamine - GS I Griffonia simplicifolia agglutinin I - GS II Griffonia simplicifolia agglutinin II - HRP horseradish peroxidase - LFA Limax flavus agglutinin - LCA Lens culinaris agglutinin - NDPAGIF non-denaturing polyacrylamide gel isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase - PBS phosphate buffered saline - PMSF phenyl methyl sulphonyl fluoride - RCL replacement cell layer - SBA soybean agglutinin (Glycine max) - SB stratum basal - SDS sodium dodecyl sulphate - SG stratum granulosum - SS stratum spinosum - UEA I Ulex europaeus agglutinin I - WGA wheat germ (Triticum vulgaris) agglutinin     

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments. Thus, carbohydrate content varied with the development and maturation of the oocytes, and the PAS results were in agreement with the lectin-binding results. Charged carbohydrate residues were observed in the egg envelope and Golgi bodies.These results suggest that the appearence of Con A-, HPA-, RCA-I- and UEA-I-binding glycoconjugates in the nuclei of developing oocytes show a varying pattern indicating different phases of nuclear activity which correlate with different carbohydrate synthetic activities of the oocyte.     

Affinity chromatography of branched oligosaccharides in rat liver β-glucuronidase

Rat liver microsomal and lysosomal β-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[¹⁴C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal β-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal β-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively).     

Lectin histochemistry of mammalian Brunner's glands

Summary Lectin histochemical study was performed on twenty-eight specimens of formalin-fixed paraffin embedded tissues of proximal duodenum from human, cat, dog and Rhesus (macaque) monkey to demonstrate the pattern of carbohydrate residues in submucosal glands of Brunner as compared to that of the duodenal absorptive and goblet cells. Ten different biotinylated lectins were used as probes, and avidin-biotin-peroxidase (ABC) or avidin-gold-silver (AGS) complexes were used as visualants. Brunner's gland cells of the four species studied exhibited a similar lectin-binding pattern which differ from other duodenal cells. The epithelium of Brunner's gland stained intensely with Ricinus communis agglutinin-I (RCA-I), succinylated-WGA (S-WGA) and wheat-germ agglutinin (WGA), moderately with Bandeirea simplicifolia agglutinin-I (BS-I), Concanavalia ensiformis agglutinin (Con A) peanut agglutinin (PNA) and Ulex europaeus agglutinin-I (UEA-I) and occasionally with Dolichos biflorus agglutinin (DBA), Lens culinaris agglutinin (LCA) and soybean agglutinin (SBA). Desialylation with neuraminidase resulted in only a slight elevation in binding intensities of PNA, DBA and SBA, indicating that glycoconjugates of the Brunner's gland cells are rich in asialo-oligosaccharides, which differs from duodenal epithelial cells. In addition, these histochemical reagents were useful in localizing Brunner's gland elements in the duodenal mucosa.