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PubMed1987

Peanut lectin binding to the alveolar lining layer in hyaline membrane disease.

The authors have studied the histochemical pattern of Peanut lectin (PNA) binding sites in lungs of seven newborns with hyaline membrane disease (HMD) and ten controls. The alveolar lining layer was positive in HMD and no changes in the PNA pattern was noted after neuraminidase digestion. Hyaline membranes were generally unstained but occasional reactivity was encountered in some parts. No reaction with PNA was observed in control lungs, but positivity was seen after neuraminidase pretreatment. Our histochemical data document the presence of accessible galactosyl residues with absence of terminal sialic acid in the alveolar lining layer of newborns with HMD. The authors suggest that PNA reactivity in HMD reflects an histochemical feature described in fetal lungs at the pseudoglandular and canalicular stages.

Fluorescein-5-isothiocyanate↗
PubMed1988

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens.

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determined in vitro by a classical rosette assay in 12 postmenopausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, alpha-mannosyl [IgG(Con A)] or beta-galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P less than 0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Influence of sulphate groups in the binding of peanut agglutinin. Histochemical demonstration with light- and electron-microscopy.

The influence of sulphation of mucus glycoproteins in the binding of peanut agglutinin (PNA) to tissue sections has been investigated by means of histochemical techniques at the light- and electron-microscopic level. A sequential methylation-saponification procedure was applied for the desulphation of tissue samples. Labelling by peroxidase- and colloidal gold-conjugated PNA was compared in control and desulphated samples of rat intestinal mucosa. The high-iron-diamine (HID) technique was used as a control for the effectiveness of the desulphation technique, and the Alcian Blue, pH 2.5 (AB 2.5), PAS and phosphotungstic acid-HCl (acid-PTA) techniques served as controls for the integrity of the oligosaccharide chains, respectively. In general, a marked increase of PNA reactivity was observed in desulphated samples when compared with control sections. These findings indicate that sulphation of galactose inhibits the binding of PNA to carbohydrate moieties in tissue sections. Staining patterns obtained with HID, PNA and the desulphation-PNA sequence in the goblet cells of the large intestine suggest a modification of the secretory product stored in these cells as the cell matures and moves from the lower crypt region toward the luminal surface. These modifications were not detected in the small intestine. Ultrastructural detection of PNA-binding sites suggests that galactose residues are incorporated into the oligosaccharide chains of O-linked glycoproteins at the medial cisternae of the Golgi apparatus. However, sulphation occurs at the trans side of the Golgi complex and the trans Golgi network. In conclusion, desulphation procedures are useful for revealing PNA-binding sites.

Animals↗
PubMed1996

The lectin-binding sites for peanut agglutinin in invasive breast ductal carcinomas and their role as a prognostic factor.

The present study was designed to analyze the expression of lectin-binding sites for peanut agglutinin (PNA) in paraffin sections of primary invasive ductal carcinoma not otherwise specified and to consider PNA lectin histochemistry as a further aid in the prognostic evaluation of breast cancer. The expression of lectin-binding sites was studied using the avidin-biotin complex/ immunoperoxidase technique, and analyzed in relation to the different clinical, pathological, and biological parameters of the primary disease, i.e. the presence or absence of nodal metastases, pre- or post-menopausal age, size of the tumor, mitotic activity index, morphometric prognostic index, DNA content, S-phase fraction, and steroid receptor status. The results show significant differences in PNA binding patterns among malignant epithelial breast cells. There was no expression of PNA-binding sites in 14 out of 157 tumors, while 64 showed mostly apical (membrane) staining and 124 non-apical (membrane and/or cytoplasmic) staining. Apical staining was mostly observed in patients without lymph node metastasis, with positive steroid receptor status, and those who were postmenopausal diagnosis; non-apical staining was mostly observed in lymph-node-positive premenopausal patients negative for steroid receptors and with aneuploid tumor cells. Our results indicate that, in malignant breast cells, there is an alteration of cell-surface glycoconjugates, shown by heterogeneity within a histopathologically defined group, which is related to different properties of tumor cells. The apical PNA binding pattern indicates a better differentiation of tumor cells while non-apical PNA binding suggests a higher metastatic potential. Specific PNA lectin binding patterns should be considered as a further reliable prognostic factor in breast cancer.

Breast Neoplasms↗
PubMed1991

Peanut lectin: a histochemical marker for phaeochromocytomas.

Fifty-five neuroendocrine tumours and 6 adrenocortical tumours were examined immunohistochemically for the expression of neuron-specific enolase (NSE), chromogranin and synaptophysin. The results were compared with the staining patterns obtained with peanut lectin (PNA) using a streptavidin-biotin staining technique. In separate experiments, sections were preincubated with neuraminidase for the demonstration of masked PNA binding sites. Two of the 24 phaeochromocytomas, 1 of the 6 medullary carcinomas of the thyroid gland, 5 out of the 7 islet cell tumours of the pancreas and all 4 extra-adrenal paragangliomas were negative with PNA. When the sections were first incubated with neuraminidase all these tumours were positive with PNA. Six adrenocortical tumours and 7 neuroblastomas were examined and found to be negative with PNA with or without neuraminidase pre-treatment. Seven carcinoid tumours were examined and found to be positive with PNA only in tubular areas and negative in solid areas; pre-treatment with neuraminidase did not alter the staining pattern. Immunoreactivity for NSE was absent in only 1 of the neuroendocrine tumours. A higher proportion of neuroendocrine tumours was positive with anti-chromogranin than with anti-synaptophysin.

Adrenal Gland Neoplasms↗
PubMed1988

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: histochemical, ultrastructural cytochemical, and biochemical characterization.

The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained the trans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgi trans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.

Animals↗
PubMed1988

Lectin binding patterns in normal and neoplastic colonic mucosa. A study of Dolichos biflorus agglutinin, peanut agglutinin, and wheat germ agglutinin.

The cellular distribution of the carbohydrates labeled by Dolichos bifluorus agglutinin (DBA), peanut agglutinin (PNA), and wheat germ agglutinin (WGA) were studied in 21 normal colonic mucosae, 17 transitional mucosae, 9 nonneoplastic polyps (NNP), 27 adenomas, and 25 colorectal carcinomas. In normal mucosa DBA bound selectively to mucin of the goblet cells in the upper colonic crypt and to apical cytoplasm of the superficial columnar cells with a strong linear pattern. PNA binding was present only in the supranuclear portion (Golgi area) of the cells. WGA showed a strong reactivity in the goblet-cell mucin and in the supranuclear portion and apical cytoplasm of columnar cells. Transitional mucosa (TM) showed a decrease in DBA binding to goblet-cell mucin, which was replaced by an increase in PNA reactivity. The DBA linear pattern in the apical cytoplasm of columnar cells was unmodified, however. Changes similar to those of TM were observed in juvenile and Peutz-Jeghers polyps. Adenomas showed a progressive loss of DBA reactivity and an increase in PNA positivity related to the degree of dysplasia. This change was more evident in the linear pattern of apical cytoplasm. Only 32% of the carcinomas reacted with DBA and those were mucinous and well-differentiated adenocarcinomas. WGA was positive in all carcinomas with a different pattern than in normal mucosa. These findings suggest that the different lectin-binding patterns in normal and neoplastic colonic mucosa are related to the degree of cellular differentiation. In the process of malignant transformation the carbohydrate distribution undergoes progressive changes through the adenoma-carcinoma sequence. These changes are related to the degree of dysplasia in adenomas and to the degree of differentiation in carcinomas.

Adenoma↗
PubMed1986

Identification of osteoclasts by rhodamine-conjugated peanut agglutinin.

Rhodamine-conjugated Arachis hypogaea (peanut agglutinin, PNA) lectin, which is specific for beta-D-galactosyl and beta-D-gal-[1,3]-D-galNAC residues, was used to identify osteoclasts in paraffin-embedded bone sections of fetal rat calvaria and a human bone-derived tumor, osteoclastoma. All multinucleated osteoclasts were positive for PNA-lectin. This was also confirmed by studying smears of isolated osteoclasts from rat and chicken long bones. In addition to multinuclear cells, some mononuclear cells on the endosteal surface of the rat calvaria and some bone marrow cells also revealed specific binding of PNA lectin. Isolated rat peripheral monocytes were also studied, and these showed specific binding of PNA lectin which was maintained unchanged for at least 3 days in culture. Different lectins could be useful as membrane markers for studying bone cell origin and maturation.

Animals↗
PubMed1999

Anti-T antibodies and peanut-agglutinin-binding glycoproteins in sera of patients with gastric cancer.

Agglutinating antibodies to neuraminidase-treated red blood cells (anti-T agglutinins) are known to be reduced in patients with gastric cancer. The antigenic determinant of anti-T agglutinin is known to have a disaccharide structure [Gal(beta1-3)GalNAc], the same specificity as peanut agglutinin (PNA). We examined sera of 27 patients with gastric cancer and 30 controls for anti-T agglutinins, anti-T antibodies and PNA-binding glycoproteins. Anti-T agglutinins were titrated by a microtiter hemagglutination method. Levels of anti-T antibodies were determined by enzyme immunoassay using synthetic glycoconjugate [Gal(beta1-3)GalNAc O-alpha-linked to human serum albumin] as an antigen. Levels of PNA-binding glycoproteins in sera were measured by sandwich enzyme-linked lectin assay using wheat germ agglutinin and peroxidase-conjugated PNA. Titers of anti-T agglutinins were significantly lower in patients with gastric cancer than in controls (P = 0.041). Levels of anti-T antibodies were not significantly different in patients with gastric cancer and controls; however, decreased levels of anti-T antibodies were more frequent in patients with gastric cancer than in controls (P = 0. 001). Levels of PNA-binding glycoproteins were significantly higher in sera of patients with gastric cancer than in controls (P = 0.001). The levels of anti-T antibodies inversely correlated with the levels of PNA-binding glycoproteins in sera of patients with gastric cancer (r = -0.44, P = 0.021). These results suggest that the decrease in anti-T antibodies in sera of patients with gastric cancer might be due to immune complex formation between circulating PNA-binding glycoproteins and anti-T antibodies.

Adult↗
PubMed1996

Reactivity of Trypanosoma cruzi strains with peanut agglutinin (PNA) correlates with number of in vitro infected host cells.

Reactivities of 4 lectins with intact trypomastigote forms derived from 8 different Trypanosoma cruzi strains were compared with their capacity to infect in vitro cultured LLC-MK(2)cells. A sensitive and reproducible titration method for lectin binding sites (ELLA: Enzyme Linked Lectin Assay) was employed, in which reactivities were scored through optical densities in an ELISA reader. Tissue culture trypomastigotes from the strains Y, CL, SC4, SC24, SC25, SC28, SC32 and SC33 were investigated for expression of different cell surface carbohydrate residues using Concanavalin A (ConA), Peanut agglutinin (PNA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA) conjugated to peroxidase. The reactivity of the strains to PNA lectin was SC28 > SC32 > SC33 > SC25> SC24 > Y> CL> SC4. The optical density values obtained were highly correlated (r2=0.986, p< 10(-4)) with the number of parasitized LLC-MK(2) cells 24 hours after infection by trypomastigotes from each corresponding strain. We concluded that galactose and N-acetyl-D-galactosamine residues that are present on the surface of trypomastigotes are important in host-cell recognition.

Animals↗
PubMed1985

Effect of pH on oligomeric equilibrium and saccharide-binding properties of peanut agglutinin.

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.

Carbohydrate Metabolism↗
PubMed1987

Properties of the lectin from the hog peanut (Amphicarpaea bracteata).

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.

Adenine↗
PubMed1987

Light-microscopic studies on spatial and temporal binding of the lectins concanavalin A, wheat-germ agglutinin and peanut agglutinin in early rat odontogenesis.

The spatial distribution and temporal expression of alpha-D-mannosyl(glucosyl)-, N-acetyl-D-glucosaminyl- and beta-D-galactosyl residues as detected by peroxidase-conjugated lectins correlated with early odontogenic events in six principal developmental stages (fetal days 13.5, 14, 15, 17, 18.5 and 19.5). The odontogenic epithelium of 13.5- and 14-day-old fetuses was characterized by strong concanavalin A (Con A) binding and between days 17 and 19.5, the stellate reticulum displayed strong peanut agglutinin (PNA) binding. Between 15 and 19.5, differentiation of dental ectomesenchyme was characterized by a rhythmic expression of terminal galactosyl residues shown by PNA-binding. At the developing dental basement membrane, there were various carbohydrate-specific regions. At days 13.5 and 14, the odontogenic basement membrane was specific for N-acetyl-D-glucosamines detected by wheat-germ agglutinin (WGA). The results suggest that the carbohydrates present at the inner dental basement membrane at days 17 to 19.5 may be involved in cell-matrix interactions during cytodifferentiation.

Animals↗
PubMed1996

Identification of cryptic peanut agglutinin-reactive sites in human lipoprotein(a).

After treatment of human lipoprotein(a) (Lp(a)) with neuraminidase, formerly cryptic sites became available for binding to peanut agglutinin (PNA) lectin and a Thomsen-Friedenreich antigen (T-antigen)-specific monoclonal antibody. The PNA-reactive sites were localized to the apo(a) moiety of Lp(a) and O-specific carbohydrate side chains. Lp(a) with larger isoforms of apo(a) contained more potential PNA reactivity per molecule of Lp(a) apoB than did smaller isoforms. Very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) did not contain comparable amounts of the cryptic PNA-reactive sites.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1983

Characterization of chicken lymphocyte subsets separated by peanut agglutinin.

The reactivity of chicken lymphocytes isolated from bursa, thymus, spleen, and peripheral blood (PBL) with peanut agglutinin (PNA) has been investigated. High numbers of cells binding PNA (PNA+ cells) were found in bursa (74.5%) and thymus (85.2%), as well as in the peripheral organs (spleen and PBL, 43.8 and 70%, respectively). In the latter organs, the levels of PNA+ cells exceeded by far the values reported for mammals. Separation of PNA+ and PNA- cells from different organs was achieved by selective agglutination with the lectin or by affinity chromatography on immobilized PNA. Experiments combining the use of limiting concentrations of neuraminidase to cleave off sialic acid from the surface membrane of splenocytes that do not bind PNA (PNA- cells), followed by agglutination with the lectin, suggested that the high binding of PNA to mature lymphocytes is most likely due to a low degree of sialylation of chicken cell surface glycoconjugates. The functional properties in vitro of PNA+ and PNA- spleen cell fractions were assayed in mitogen responses (PHA, Con A, PWM), the mixed-lymphocyte reaction (MLR), and the in vitro anti-SRBC antibody response. In all three systems the responses of the PNA+ fraction were found to be significantly higher than those of the PNA- cells. Mixing experiments revealed that irradiated (1500 R) PNA- cells preferably suppress pure T-cell responses of unfractionated spleen cells, whereas PNA- cells exerted a stronger suppressive effect on responses involving B cells. Thus, in the chicken, PNA may be a valuable tool to distinguish subsets of suppressor cells with different target specificity.

Animals↗
PubMed1984

Post-natal ontogenesis of graft-versus-host reactivity of peanut agglutinin lectin-negative thymocytes in the chicken.

Chicken thymocyte fractionation by peanut agglutinin lectin yields two cell subpopulations which differ in their GvH competence when injected into major histocompatibility complex (MHC)-(B locus) incompatible embryos. The nonagglutinated PNA- fraction displayed a degree of alloreactivity similar to that of peripheral blood lymphocytes while the PNA+ cells were nonreactive. Analysis of chickens at the age of 4 to 24 weeks showed that the development of GvH-reactive PBL preceded by 12 weeks the maturation of GvH-reactive PNA- thymocytes. Possible interpretations for this difference between the time of appearance of GvH reactivity of PNA- thymocytes and PBL are discussed.

Animals↗
PubMed1986

Identification of peanut agglutinin-binding glycoproteins on immature human thymocytes.

Previous studies in our laboratory have shown that peanut agglutinin (PNA), a lectin specific for the disaccharide Gal beta 3GalNAc, binds to immature (cortical) thymocytes of mouse and man and not to the mature (medullary) cells. Using lectin overlay of protein blots and lectin-affinity chromatography, we have found that the major PNA-binding glycoproteins on total as well as on immature (PNA+) human thymocytes correspond to two bands of Mr 170,000 and 180,000. Another glycoprotein, of Mr 110,000, also binds PNA but to a lesser extent. All three glycoproteins contain sialic acid as demonstrated by cell surface labeling with NaIO4-NaB3H4, binding of wheat germ agglutinin, and reaction with alkaline phosphatase-hydrazide. After treatment with sialidase, binding of PNA to these glycoproteins is significantly enhanced.

Chromatography, Affinity↗
PubMed1989

Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes. I. Distribution of cryptic and exposed TF antigens on murine lymphocytes from different lymphoid organs: detection with an anti-TF monoclonal antibody and peanut agglutinin.

Cryptic Thomsen-Friedenreich (TF) antigens were detected by the lectin peanut agglutinin (PNA) on the surface of murine lymphocytes after treatment of cells with neuraminidase. Thereby, a particular TF antigen could be distinguished using a monoclonal anti-TF antibody 49H8. In contrast to the known general galactoside specificity of PNA, the mAb was restricted to Gal beta(1-3)GalNAc/GlcNAc. Preincubation of cells with PNA abolished mAb 49H8 binding completely. However, only the intensity of staining with PNA was reduced by prior incubation of cells with the mAb. Cryptic TF antigens detected by the mAb were expressed on 39% of murine bone marrow cells, 88% of thymocytes, 62% of lymph node cells, and 65% of spleen cells. On the other hand, over 80% of the lymphatic cells carried cryptic PNA binding sites independent of the lymphoid organ they derived. In the thymus, a subpopulation of cells (76%) could be detected by PNA without neuraminidase treatment. Twenty-eight percent of thymocytes carried exposed mAb binding sites, too. All of them were shown to express further binding sites for PNA constantly. Therefore, a subpopulation of PNA-reactive, immature thymocytes can be distinguished by the mAb 49H8. During activation of splenic lymphocytes with PHA, the lymphoblasts completely lost their cryptic mAb binding sites while PNA reactivity was not affected. We conclude that the anti-TF mAb recognizes a particular TF antigen exposed on thymocytes and present in a cryptic form on other lymphocytes. The number of cells carrying mAb 49H8 binding sites varied, dependent on the organ from which the lymphocytes derived. PNA-reactive lymphocytes are distributed homogeneously in the lymphoid organs.

Animals↗