Lectins as Probes to Pneumocystis carinii Surface Glycocomplexes

The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infecteding homogenates were incubated with FTTC-conjugated lectins in a series of concentrations, counlerstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acctylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simpiicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Madura pomifera and Dolichos biflorus agglutinins and Giffonia simpiicifolia Hectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffon'ui simpiicifolia I—β Section had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax ftavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.     

Flow cytometric analyses of lectin binding to Pneumocystis carinii surface carbohydrates.

Pneumocystis carinii obtained from infected rat lung homogenates was incubated with fluorescein isothiocyanate-conjugated lectins, counterstained with the nuclear stain, propidium iodide (PI), and analyzed by dual parameter histograms for lectin-associated green and PI-associated red fluorescence using a fluorescence-activated cell sorter. The presence of glucose/mannose moieties was evidenced by the binding of all organisms to concanavalin A and Wisteria floribunda. From the lectin group specific for N-acetyl-D-glucosamine, P. carinii reacted strongly with wheat germ agglutinin and less intensely with Solanum tuberosum. Reaction with lectins specific for N-acetyl-D-galactosamine/galactose was variable, probably reflecting the secondary binding affinities of the lectins used. Soybean agglutinin, Bauhinia purpurea agglutinin, and Maclura pomifera agglutinin reacted moderately, whereas Dolichos biflorus agglutinin, and Griffonia simplicifolia I reacted less avidly. The organisms reacted partially with Ulex europaeus agglutinin, a lectin specific for fucose, and did not react well with Arachis hypogaea, Viscum album agglutinin, and Griffonia simplicifolia I beta 4, lectins specific for galactose. A very weak fluorescent signal was detected with Limax flavus agglutinin, suggesting little or no sialic acid was present. All lectin-binding reactions were confirmed for specificity by inhibition with the relevant carbohydrates. Flow cytometric analysis of lung-derived Pneumocystis organisms stained with fluorescent surface and nuclear dyes provides a rapid method for characterization of large parasite populations.     

Pneumocystis carinii: surface reactive carbohydrates detected by lectin probes

Pneumocystis carinii obtained from rat lungs (RLH) and in vitro culture (RTC) were reacted with a panel of 14 fluorescein isothiocyanate conjugated lectins. Percentage fluorescence and fluorescent intensity were determined for both trophic and cyst forms. All RLH and RTC derived organisms bound strongly concanavalin A (Con A), and wheat germ agglutinin (WGA). However, differences in soybean agglutinin (SBA) binding between RLH and RTC organisms was observed. Different subsets of the organism bound lectins from Griffonia simplicifolia I, Maclura pomifera, and Bauhinia purpurea, indicating heterogeneity in the surface carbohydrates within each of the RLH and RTC populations. Eight lectins reacted very weakly or not at all: Dolichos biflorus, Arachis hypogaea, Griffonia simplicifolia I-beta 4, Solanum tuberosum, Ulex europeus, Griffonia simplicifolia II, Viscum album, and Limax flavus. The results indicate that P. carinii trophic and cyst forms have surface constituents containing mannose, N-acetylglucosamine and N-acetylgalactosamine as the predominant carbohydrates. Molecules resembling sialic acid and beta-galactose are absent or inaccessible. The surface glycoconjugates identified in these studies may play a role in the adherent properties of P. carinii.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands. An electron microscopic observation using lectins and monoclonal antibodies

Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B4 staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon esculentum to microvascular endothelium: organ-specific localization and partial glycoprotein characterization

The lectins Griffonia simplicifolia I and Lycopersicon esculentum were used to assess the presence of endothelium-specific glycoproteins in the microvasculature of the rat myocardium, diaphragm and superficial cerebral cortex. Organs fixed by intravascular perfusion were processed to obtain semithin (0.5 micron) and thin (less than 0.1 micron) frozen sections that were reacted with biotinylated lectin followed by streptavidin conjugated to Texas Red, for semithin sections, or by streptavidin conjugated to 5-nm colloidal gold particles, for thin sections. Lycopersicon esculentum lectin exclusively labeled the endothelium of all small vessels in all three microvascular beds; it did not bind to components of either the parenchyma or the extracellular matrix. Griffonia simplicifolia I lectin exclusively labeled the endothelium of the entire microvasculature in the myocardium and diaphragm, but marked primarily pericytes in the cerebral microvasculature. It did not label any parenchymal or interstitial organ component. At the electron microscope level, the lectin Griffonia simplicifolia I labeling was associated with the plasmalemma proper and especially with plasmalemmal vesicles and their introits, and Lycopersicon esculentum lectin bound primarily to the luminal plasmalemma in the microvascular beds of the myocardium and diaphragm. In the cerebral cortex, labeling of the microvasculature was clearly different: Griffonia simplicifolia I bound primarily to pericytes and vascular smooth muscle cells whereas Lycopersicon esculentum labeled only the microvascular endothelium. Lysates prepared from the myocardium, diaphragm and cerebral cortex were processed through Griffonia simplicifolia I lectin affinity separation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fraction obtained. A number of putative endothelium-specific glycoproteins was detected and found to differ qualitatively and quantitatively from organ to organ. The most prominent polypeptide, approximately 97 kDa, was present in substantial amounts in the myocardium and diaphragm, but in considerably lower concentration in the cerebral cortex. The reverse applied for a approximately 55 kDa protein. The preferential distribution of the approximately 97 kDa protein parallels differences in Griffonia simplicifolia I lectin binding by fluorescence and electron microscopy on sections of the corresponding organs. The results provide further evidence for the existence of endothelial glycoproteins specific for different microvascular beds and possibly connected with local functional differentiations.     

A histochemical comparison of human corneal stromal glycoconjugates with eight other species. Distinct species-dictated differences in binding sites of Griffonia simplicifolia I

Frozen sections of human, calf, rabbit, rat, cat, dog, goat, lamb, and hog corneas were stained with various lectins using an avidin-biotin-peroxidase complex to study glycoconjugates of stromal matrix. Staining of the stromal matrix and keratocytes with an alpha-galactose-specific lectin, Griffonia simplicifolia I (GSA-I) was species-dependent. The stromal matrices of cat, dog, and hog corneas invariably reacted intensely with this lectin, whereas those of the human, calf, rabbit, rat, and lamb did not react. A positive reaction with GSA-I could be abolished in each instance by preincubation of the sections with alpha-galactosidase. The stromal matrices and keratocytes of all nine species reacted positively with wheat germ agglutinin, concanavalin A, and Ricinus communis agglutinin but did not react with soybean agglutinin. Results of this study may help select an appropriate animal model for further investigate human corneal stromal glycoconjugates.     

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent.     

Reduction of spontaneous metastases through induction of carbohydrate cross-reactive apoptotic antibodies

The selective targeting of tumor-associated carbohydrate Ags by the induction of serum Abs that trigger apoptosis of tumor cells as a means to reduce circulating tumor cells and micrometastases would be an advantage in cancer vaccine development. Some plant lectins like Griffonia simplicifolia lectin I and wheat germ agglutinin mediate the apoptosis of tumor cells. We investigated the possibility of using these lectins as templates to select peptide mimotopes of tumor-associated carbohydrate Ags as immunogens to generate cross-reactive Abs capable of mediating apoptosis of tumor cells. In this study, we show that immunization with a mimotope selected based on its reactivity with Griffonia simplicifolia lectin I and wheat germ agglutinin induced serum IgM Abs in mice that mediated the apoptosis of murine 4T1 and human MCF7 cell lines in vitro, paralleling the apoptotic activity of the lectins. Vaccine-induced anti-carbohydrate Abs reduced the outgrowth of micrometastases in the 4T1 spontaneous tumor model, significantly increasing survival time of tumor-bearing animals. This finding parallels suggestions that carbohydrate-reactive IgM with apoptotic activity may have merit in the adjuvant setting if the right carbohydrate-associated targets are identified.     

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 sperm-oocyte interaction.     

A survey of lectin binding in Paramecium

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.     

Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limax flavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies.     

Glycolipid-lectin interactions: detection by direct binding of 125I-lectins to thin layer chromatograms

Glycolipids that bind 125I-labeled lectins are detected by autoradiography after thin layer chromatography of glycolipid standards or crude lipid extracts. Soybean agglutinin, Bandeiraea simplicifolia I isolectins A4 and B4, and Helix pomatia lectin are used to detect corresponding cell surface, glycolipid receptors in human and bovine erythrocytes. When lipid extracts from A and AB erythrocyte stroma are analyzed with Helix pomatia lectin, a polymorphic expression of blood group A glycolipid determinants is detected. The Bandeiraea simplicifolia isolectins react weakly with human erythrocyte glycolipids but bind at least 4 glycolipids in bovine stroma extracts. Soybean agglutinin reacts with glycolipids in all erythrocytes analyzed. This technique extends lectin specificity studies from inhibition analyses in aqueous systems using available, known structures to identification of specific, lectin-binding glycolipids in crude lipid extracts of cell membranes.     

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands

Summary Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B₄ staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins. However, difference was also observed between monoclonal antibody and lectin staining, that is, monoclonal anti-A antibody reacted weakly but consistently with granules from blood group A nonsecretors but DBA (HPA) did not; staining with UEA-I was observed in granules from the secretor individuals of any blood groups whereas monoclonal anti-H antibody reacted with granules from blood group O and some A secretor individuals but not from B and AB secretor individuals; GSAI-B₄ reacted uniformly with granules throughout the cells whereas monoclonal anti-B antibody bound to limited number of granules in the same cells. This was confirmed by the double labeling experiments with the lectin and the antibody. These results suggest that the different types of antigens as to the binding ability for monoclonal antibodies and lectins are expressed on different granules in the same cell.     

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human sperm surface mapping with lectins.

Ten fluorescein isothiocyanate (FITC)-linked lectins [Bauhimia purpurea, Concanavalin A, Dolichos biflorus (DBA), Griffonia simplicifolia I, Griffonia simplicifolia II, Maclura pomifera, Arachis hypogea (PNA), Glycine max, Ulex europaeus (UEA) and Triticum vulgaris agglutinin] have been used to study their binding features on the human ejaculate spermatozoa. Qualitative changes in the labeling pattern have been observed in unfixed and acetone-treated spermatozoa. Furthermore, ultrastructural localization of some of the colloidal gold-linked lectins, namely PNA, UEA and DBA, has been attempted to delineate the binding domains of the specific sugars on the sperm surface. It needs to be emphasized that flow-cytometric methods employed in our study, which provide quantitative slant to qualitative data, should be utilized to evaluate the functional status of the spermatozoa.     

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.     

A new method of lectin histochemistry for the study of brain angiogenesis. Lectin angiography

In an attempt to analyse the kinetics of angiogenesis in the brain, we developed a new lectin-histochemical staining technique for identifying the vasculature. Three horseradish-peroxidase-conjugated lectins, i.e., Griffonia simplicifolia agglutinin 1 (GS1), Ricinus communis agglutinin 1 (RCA1) and soybean agglutinin (SBA), selectively stained vascular walls in brain-tissue sections. When these lectins were injected into the circulation of ether-anesthetized animals via the pulsating left ventricle, they bound specifically to the inner surface of endothelial cells and revealed the three-dimensional architecture of the vascular network within thick tissue preparations. When this technique, referred to a lectin angiography, was combined with 5-bromo-2-deoxyuridine (BudR) immunohistochemistry, proliferating capillary cells could be easily identified in three-dimensional structures of the developing vasculature. Because of its simplicity and wide applicability, lectin angiography should be useful for analysing the kinetics of angiogenesis in developmental, regenerative, and pathological conditions in various tissues and organs.     

Localization of prostatic glycoconjugates by the lectin-gold method.

The glycoconjugates of the lateral prostate were examined ultrastructurally by lectin-gold histochemistry in combination with a low-temperature embedding technique using Lowicryl K4M. The binding patterns of concanavalin A, wheat germ agglutinin, Griffonia simplicifolia, soybean agglutinin, peanut agglutinin, Ricinus communis agglutinin isolectin I, Griffonia simplicifolia isolectin B4, Ulex europaeus isolectin I and Phaseolus vulgaris agglutinin P have been documented in the subcellular compartments of the lateral prostate. The results show that the granular endoplasmic reticulum (GER) is rich in glycoproteins with mannosyl residues while the Golgi cisternae, secretory granules and microvilli are less so. The mannose (Man) and N-acetylglucosamine (GlcNAc) residues present in the GER of the epithelial cells may be associated with the initial assembly of the N-linked oligosaccharides of glycoproteins. The secretory granules exhibited different reactivities to lectins. Most of the lectin-binding sites confined to the limiting membranes may play a role in the transport of plasmalemma glycoconjugates to the apical plasma membrane. The epithelial Golgi stack is rich in GlcNAc, galactose (Gal), N-acetylgalactosamine (GalNAc) and sialic acid residues, and a compartmental organization of the Golgi stack is apparent which might be associated with the sequential addition of sugar residues to the oligosaccharides. The plasma membrane contains abundant Man, GlcNAc, Gal, GalNAc and complex carbohydrates, especially in the microvilli, and a differential lectin labelling was noted between the apical and basolateral plasma membrane. The present study showed that fucose-containing glycoconjugates were detected in the apical plasma membrane of the lateral prostate. The stromal extracellular matrices as well as the epithelial basement membranes demonstrated weak lectin reaction. Man, GlcNAc, Gal residues and complex sugars were also noted in the stromal tissues of the lateral prostate including the extracellular matrix, capillaries and smooth muscle.     

Lectin binding to parietal cells of human gastric mucosa

A light microscopic and ultrastructural analysis of lectin receptors on parietal cells from human gastric mucosa was performed utilizing 12 biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex. Peanut agglutinin conjugated directly to peroxidase was also used. Several fixatives and fixation regimens were evaluated for optimal preservation of parietal cell saccharide moieties. Formalin proved to be the most practical fixative for light microscopic studies. A periodate-lysine-paraformaldehyde (PLP) combination provided good preservation of lectin binding capacity but yielded relatively poor ultrastructure. Conversely, glutaraldehyde provided excellent preservation of ultrastructure but a somewhat diminished lectin binding activity, which was overcome by using long incubation times and high concentrations of reagents. Parietal cells reacted strongly with Bandieraea simplicifolia, Dolichos biflorus, peanut agglutinin, and soybean agglutinin (all specific for galactosyl/galactosaminyl groups) and weakly with Ulex europaeus (specific for fucose). At the light microscopic level a beaded, perinuclear staining pattern was observed which, ultrastructurally, corresponded to an intense staining of intracytoplasmic canaliculi. The membranes of the intracytoplasmic canaliculi were characterized by an abundance of galactosyl residues, a paucity of fucosyl groups, and a lack of mannosyl and glucosyl residues. The biochemical and physiological significance of these findings is discussed.