Digoxigenin-labelled Lectins Research Articles

Glycohistochemistry of the lateral tympanic membrane in the syrinx of the Denizli cock

The Denizli cock is a special race that has harmonious long crowing. In this study, the syrinx of the Denizli cock was studied by using glycohistochemical analyses. Paraffin sections were stained with Alcian blue (AB) at pH 1 and pH 2.5 for demonstration of sulfated and carboxylated carbohydrates, respectively. At AB-pH 1, the lateral tympanic membrane (LTM) and also the medial tympanic membrane (MTM) exhibited a weak reactivity, whereas they were stained intensely at AB-pH 2.5. The frozen sections were incubated with 5 digoxigenin-labeled lectins for lectin histochemistry: peanut agglutinin, Maackia amurensis leucoagglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. In LTM, positive stainings were observed for the 5 lectins. These results suggest that LTM exhibits an intense reactivity with AB-pH 2.5, indicating carboxylated carbohydrates, and it also has terminal saccharide moieties consisting of b(1->3) linked galactose, a(2->3) linked sialic acid, mannose, b (1->4) linked galactose, and a (2->6) linked sialic acid.

Identification of the glycosylation pattern of meat proteins in tissues from bovine myelin

A variant form of Creutzfeld-Jacob disease in humans is associated with the consumption of food contaminated with the bovine central neural system. This has focused attention on the need for procedures to detect tissues of the neural system in meat and meat products. A method was developed for the identification of myelin glycoproteins in bovine neural tissue. The glycosylated structures of glycoproteins in different protein mixtures from central nervous system (CNS) and peripheral nervous system (PNS) isolated from bovine myelin were identified by the specific lectin-glycoprotein reactions. Digoxigenin-labeled lectins bonded to the terminal glycoconjugate sequences of nine CNS glycoproteins ranging from 15 to 200 kDa and four PNS glycoproteins ranging from 22 to 105 kDa. Datura stramonium (DSA) recognized the epitope Galbeta1-4GlcNAc by two CNS and three PNS glycoproteins. Maackia amurensis (MAA) recognized the epitope NeuAcalpha2-3Gal by four CNS and two PNS glycoproteins. The peanut Arachis hypogaea (PNA) reacted with the high molecular CNS glycoprotein (200 kDa) with the sequence Galbeta1-3GalNAc. Galanthus nivalis (GNA) bonded to mannose subunits linked alpha1-3 and alpha1-6 by six CNS glycoproteins with molecular weights between 17 and 200 kDa. Four of these glycoproteins were recognized from monoclonal antibodies against the "HNK-1 epitope".

The Effects of Hypoand Hyperthyroidism on Nuclear, Cytosolic, Endoplasmic and Mitochondrial Fractions of Sialoglycoproteins in Rabbit Hepatocytes

Enhanced sialylation of glycoproteins occurs during hypoand hyperthyroidism. The role of sialic acid (SA) in cell membranes is well-standing, but its role in intracellular structures is still under analysis. We wanted to evaluate the influence of thyroid hormones on the sialylation ratio of intracellular proteins presented in cytosolic, mitochondrial, endoplasmic and nuclear fractions of rabbit hepatocytes. Twenty-one New Zealand male rabbits were divided into three groups. Hypothyroidism (N = 7) or hyperthyroidism (N = 7) was induced by adding propylthiouracyl (2 mg/l) or L-thyroxine (12 mg/l), respectively, to the drinking water for four weeks. Results were compared with healthy (euthyroid) control animals (N = 7). After isolation of intracellular fractions, standard SDS-PAGE electrophoresis and transfer onto nitrocellulose membrane were performed. Identification of SA residue was carried out with digoxigenin-labelled lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). We noticed significantly higher level of SNA than MAA linkage sialoglycoproteins in all evaluated fractions. The sialylation ratio was significantly lower (P < 0.05) in the nuclear fraction in case of hyperthyroidism (detected with both agglutinins). In contrast to the nuclear fraction the content of SNA-detected sialoglycoproteins was significantly reduced in mitochondrial fraction of hyperthyroid hepatocytes (P < 0.05). Non-significant augmentation of MAA-detected sialoglycoproteins was observed in the mitochondrial fractions in both hypoand hyperthyroidism. The fluctuations of sialoglycoproteins in endoplasmic fraction were not significant. Our work showed that the subcellular structures are rich in SA residues. Differing effects of thyroid hormones on sialylation ratio suggest an important role for hypo- and hyperthyroidism in sialoglycoprotein metabolism.

Sialic acid in human testis and changes with aging

The aim of the present study was to investigate the distribution of the glycoconjugates sialoderivatives in the human testis. Orchidectomy specimens from men aged 18-30 years (Group 1) and from men aged 70-93 years (Group 2) were obtained at autopsy. The study was performed using digoxigenin-labelled lectins, namely Maackia amurensis II lectin (MAA), Sambucus nigra agglutinin (SNA) and Arachis hypogaea lectin (PNA), in addition to enzymatic and chemical treatments (neuraminidase, KOH-neuraminidase, mild oxidation-neuraminidase, mild oxidation-KOH-neuraminidase, strong oxidation-neuraminidase, strong oxidation-KOH-neuraminidase), to characterise the different glycosidic linkages of the sialoderivatives and to obtain information regarding their structure. In all Group 2 samples, sialic acids linked alpha-2,3 to galactose and alpha-2,6 to galactose/N-acetyl-D-galactosamine (Gal/GalNAc), revealed by MAA and SNA, respectively, were observed in testicular interstitial tissue and in the lamina propria. Sialic acid linked alpha-2,6 to Gal/GalNAc was detected in only some samples from Group 1. After treatment, PNA showed structural changes and/or the gradual disappearance of sialic acid linked to D-galactose-beta(1-3)-N-acetyl-D-galactosamine in testicular components with aging. These findings indicate that changes in the metabolism of sialoderivatives in the testis could be related to morphofunctional changes in various testicular components typical of this organ during aging. This suggests that sialoderivatives are important in the functionality of the mature testis in men, as well as its involution.

The sialoglycoconjugates in the oviducts of fertile and postmenopausal women

ObjectivesThe aim of the study was to investigate the distribution of the sialoderivatives of the glycoconjugates in the oviduct of normally menstruating and postmenopausal women. MethodsBiopsies of ampullary and isthmic portions of the oviduct were obtained from fertile women, in proliferative and secretive phases, and from postmenopausal subjects. The study was carried out using digoxigenin-labelled lectins (MAA, SNA and PNA) in addition to the use of enzymatic and chemical treatments to characterize the different glycosidic linkages of the sialoderivatives and to obtain information on their structure. ResultsNo appreciable difference in sialoderivatives distribution was observed among the oviducts, particularly at the epithelium luminal surface, of the fertile women in the two menstrual cycle phases or among those of the fertile and some postmenopausal women, independently from age. Moreover, no appreciable difference of distribution was observed between the ampullary and the isthmic portions in both the study groups. ConclusionsIn the fertile women sialoderivatives present at the luminal surface of the epithelial cells could play a role in sperm capacitation and mobility, and facilitate the transit of the egg and of the early embryo along the oviducts. The similar distribution of sialoderivatives in the oviduct epithelium of some postmenopausal women of different age, compared to the fertile ones, suggests a maintaining of some functions of the organ, independently from the age of the woman. This could be due, in some cases, to the influence of remaining still sufficient sex hormonal levels.

Excreted/secreted glycoproteins of G. intestinalis play an essential role in the antibody response

In the present work, glycoproteins in the excretory/secretory products of G. intestinalis were identified and the reactivity in serum of immunized mice with these molecules was evaluated by western blotting before and after chemical treatment or enzymatic deglycosylation. Glycoproteins of 58 and 63 kDa were revealed in E/S products after periodic acid-Schiff (PAS) stain. Studies of carbohydrate specificity using digoxigenin-labeled lectins, revealed the presence of O-glycans and N-glycans. Chemical treatment of excretory/secretory products with sodium meta-periodate or enzymatic deglycosylation with N-glycosidase F reduced the reactivity in serum for proteins of 36, 58 and 63 kDa, respectively. These results show the presence of glycoproteins in E/S products of G. intestinalis and suggest that the antibody response is directed against glycoepitopes. The expression of carbohydrate moieties in the E/S-G. intestinalis may play an essential role in the antibody response and may be a target for serodiagnosis or immune intervention in human giardiasis.

Lectin histochemistry of the boar bulbourethral glands.

The present study describes, for the first time, the glycosidic content of boar bulbourethral glands using lectin histochemistry. Fourteen horseradish peroxidase- or digoxigenin-labelled lectins with different carbohydrate specificities were used in samples obtained from 3 healthy Landrace boars. The results obtained indicate that endpiece and duct cells synthesize and secrete mainly O-glycoproteins with alpha- and beta-D-N-acetylgalactosamine, beta-D-galactose-beta(1-->3)-D-N-acetylgalactosamine, D-N-acetylglucosamine and neuraminic acid residues. Glycoproteins secreted by bulbourethral glands have a role in the protection and lubrication of the urethra. In addition, they may be also involved in the regulation of the sperm metabolic activity and in the maintenance of the structural integrity of acrosomal and plasma membranes.

Characterization of the Alzheimer's Disease-associated CLAC Protein and Identification of an Amyloid β-Peptide-binding Site

Amyloid beta-peptide (Abeta) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with Abeta in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Abeta plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Abeta. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Abeta-(1-40) and Abeta-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Abeta. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Abeta deposits.

Histochemical study of glycoconjugates in active and photoperiodically-regressed testis of hamster (Mesocricetus auratus)

The objective of the present study was to characterize glycoconjugates of hamster testis in gonadally-active and -inactive states by lectin histochemical methods. Thirteen HRP- or digoxigenin-labeled lectins were used in samples obtained from fertile and photoinhibited hamsters. In gonadally-active hamsters, spermatozoa tails were stained with Con-A, HPA, PNA, UEA-I, LTA, AAA, WGA and LFA and weakly with GNA and RCA-I. Spermatozoa acrosomes were labeled with HPA, SBA, WGA and PNA. Spermatid acrosomes were labeled with SBA, RCA-I, PNA, and WGA. Staining with GNA and Con-A was found in the Golgi phase and HPA staining was found in the Golgi phase and maturated spermatids. Cytoplasm of spermatocytes was labeled with Con-A, GNA, LTA, AAA, RCA-I, HPA, WGA and LFA, whereas spermatocyte membranes were stained with Con-A, LTA and AAA. Spermatogonia were strongly labeled with Con-A and moderately labeled with AAA, WGA and LFA. Sertoli cells were positive after staining with Con-A, AAA, WGA, and LFA. The lamina propria was positive after staining with UEA-I, LTA, AAA and LFA. Leydig cells showed strong labeling with SBA, Con-A, GNA, SNA and MAA, moderate labeling with WGA, weak labeling with RCA-I, AAA and LFA. In gonadally-inactive hamsters, spermatocytes showed increased staining with HPA, PNA and AAA, whereas staining with Con-A, GNA and LTA had disappeared. Spermatogonia showed an increased labeling with AAA and WGA, but labeling with Con-A and LFA had disappeared. Sertoli cells were strongly labeled with GNA. Con-A and GNA staining was decreased in Leydig cells of gonadally-inactive hamsters but PNA and HPA staining was increased. The lamina propria in regressed testes showed intense labeling with PNA. These results suggest that histological, morphological and hormonal changes occurring in hamster testis during exposure to a short photoperiod are reflected in altered patterns of expression and distribution of N- and O-linked glycans.

Characterization of the glycoconjugates of boar testis and epididymis

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

Differences of alpha3beta1 integrin glycans from different human bladder cell lines.

Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.

Age-related profile of beta-N-acetylhexosaminidase glycosylation in rat liver.

Beta-N-acetylhexosaminidase was prepared from a liver lysosomal fraction obtained from rats between 18 days of gestation (group I) and 72 weeks of age (groups II-VI). A glycan chain analysis was performed after an electrophoresis and blotting, followed by a very sensitive detection system with highly specific digoxigenin-labelled lectins. The presence of high-mannose/hybrid type glycans, as well as their fucosylated forms was shown in all the experimental groups. Complex-type glycans with terminal sialic acid or galactose were present in all the groups except for 1-week-old rats in which only a positive reaction with lectins from Galanthus nivalis and Aleuria aurantia was observed. Thus it may be assumed that age-related changes in the glycosylation pattern occur on the first days after birth.

Does glycosylation of lysosomal proteins show age-related changes in rat liver?

We studied the pattern of lectins binding by liver lysosomal proteins from rats between 18 days of gestation and 72 weeks of age. An analysis of the carbohydrate structure was carried out after an electrophoresis and blotting, followed by a very sensitive detection system with highly specific digoxigenin-labelled lectins. The only age-related differences were observed in the reaction with sialic acid—(MAA; Macckia amurensis, SNA; Sambucus nigra) and fucose—(AAA; Aleuria aurantia) specific lectins. Sialylation increased and fucosylation decreased with age. We also observed a specific reaction with Galanthus nivalis (GNA), Phaseolus vulgaris (PHA-L) and peanut agglutinin (PNA), without any significant changes with age.

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose β-(1-4)- N-acetylglucosamine β-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid α-(2-6) terminally linked.

Qualitative analysis of the carbohydrate composition of apolipoprotein H.

The specific binding of digoxigenin-labeled lectins to carbohydrate moieties is used to characterize the carbohydrate chains bound to apolipoprotein H. Our results show that apolipoprotein H is rich in sialic acid linked alpha(2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha(2-3)-linked to galactose. Galactose is beta(1-4)-linked to N-acetylglucosamine and beta(1-3)-linked to N-acetylgalactosamine. High-mannose N-glycan chains are barely detectable. After N-glycosidase F treatment the molecular weight is substantially reduced. The main band is 32,500 daltons. Carbohydrate O-linked chains, which are mainly represented by sialic acid, are alpha(2-6)-linked to galactose or N-acetylgalactosamine. Galactose is also organized in O-linked chains and it is beta(1-4)-linked to N-acetylglucosamine and beta(1-3)-linked to acetylgalactosamine. Biochemical analysis of carbohydrate structures reveals that no specific carbohydrate complex is bound to a single isoform.

Age-related patterns of lectin-binding nuclear glycoproteins of hamster liver

We studied the age-related patterns of lectin-binding liver nuclear glycoproteins from hamsters between 5 and 35 weeks of age. The examinations of the carbohydrate structures of liver nuclear glycoproteins in relation to the age of hamsters were carried out after electrophoresis and blotting by a very sensitive immunological detection system with highly specific digoxigenin-labelled lectins. The results reported in the present study do not show any significant changes in the patterns of nuclear glycoproteins with regards to the age of hamsters except the decrease in affinity of wheat germ agglutinin (WGA) to 63 kDa glycoprotein bearing single O-linked N-acetylglucosamine residues.

Changes of Glycoprotein Patterns in Sera of Humans under Stress

Stress exhibits adverse effects on many vital processes in which glycoproteins play a significant role(e.g. cell-cell/matrix interactions, immune response, neoplastic growth, implantation, prenatal development), yet only scarce attention has been directed towards studying stress induced changes in glycoprotein patterns. Using SDS-electrophoresis, blotting and digoxigenin-labelled lectins (Sambucus nigra agglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Maackia amurensis agglutinin and peanut (Arachis hypogaea) agglutinin),sera were analysed from 30 individuals chosen randomly from a severely stressed population of 309 male volunteers with no specific medical symptoms. Significant changes were found in glycoprotein pattern and content, compared with healthy controls of matching age and sex. Occasionally minor non-specific deviations from the reference values for several analytes (haemoglobin, glucose, bilirubin and alanine aminotransferase) were detected in the tested group, but glycoprotein GP4S (Mr = 45 000), detected by Datura stramonium agglutinin and Sambucus nigra agglutinin, appeared in 96.7% of samples of the stressed population. The same population also revealed an approximately 500-fold increase of GP37 in comparison with the control sera. These results suggest that stress, as a non-specific syndrome, induces specific biochemical changes, which could be of diagnostic relevance as risk makers before any more serious symptoms of stress-related consequences have developed.

An improved multiwell immunoassay using digoxigeninlabelled lectins to study the glycosylation of purified glycoproteins

An improved multiwell immunoassay using digoxigeninlabelled lectins to study the glycosylation of purified glycoproteins

Lectins and also bacteria modify the glycosylation of gut surface receptors in the rat

Oral exposure to lectins or the presence or absence of bacteria in the rat small intestine were shown by histological methods using anti-lectin antibodies or digoxigenin-labelled lectins to have major effects on the state of glycosylation of lumenal membranes and cytoplasmic glycoconjugates of epithelial cells. Taken together with the dramatic effects of exposure to lectins on gut function, metabolism and bacterial ecology, this can be used as a basis for new perspectives of biomedical manipulations to improve health.

Identification of organ-specific glycosylation of a membrane protein in two tissues using lectins.

Since glycosylation of proteins is performed by the host cell, and variable sugar groupings can confer heterogeneity on the same polypeptide, we wished to see whether membrane proteins, in particular the ubiquitous transmembrane Na,K-ATPase, could be glycosylated differently in different organs. Using a highly sensitive enzyme-linked antibody detection system of bound digoxigenin-labelled lectins on nitrocellulose sheets containing electroblotted alpha and beta subunits of kidney and brain Na,K-ATPase, isolated from various rat strains, in combination with isoform-specific immunoblots, we discovered that brain Na,K-ATPase was highly mannosylated in contrast to renal Na,K-ATPase. Thus, we describe the existence of organ-related glycoforms of an integral ubiquitous membrane protein, i.e. diversification of the same polypeptide by organ-typical sugars. At the same time, the presence of the same glycosylation pattern can make distinct protein isoforms occurring in a same organ more homogeneous. Such organ-related glycoforms may serve for tissue identification and as tissue-specific receptors.