Application of fluorescein-labelled lectins with different glycan-binding specificities to the studies of cellular glycoconjugates in human full-term placenta

In the present study 13 lectins of plant, fungal and animal origin, characterised by different glycan-binding specificities were used to examine the structure and distribution of specific glycans in the tissues of human placenta. Histochemical analysis was focused on villi (villous syncytiotrophoblast and stroma), cytotrophoblast of the basal plate and amniotic epithelium. It was found that glycoconjugates containing mannose and N-acetyl glucosamine were widely expressed while external fucosyl residues were absent on all studied structures of placenta. Lectins GNA (revealing non-reducing terminal mannosyl residues) and LPA (revealing sialic acid) bound selectively to the villus structures. The presence of sialic acid residues was observed in the superficial plasmalemma of syncytiotrophoblast and on the surface of the foetal capillary endothelium. Lectins LCA and PSA showed specific affinity to the plasmalemma of the cytotrophoblast and to the amniotic epithelium basement membrane. N-acetyl lactosamine-specific fungal lectin PSL selectively labelled amniotic epithelial and cytotrophoblast cell membranes and superficial plasmalemma of the syncytiotrophoblast.     

Trypanosoma cruzi: studies on the interactions of lectins with glycoconjugates of different zymodemes

Detergent extracts were made of eight strains of Trypanosoma cruzi which were representative of the principal zymodemes. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the glycoproteins were reacted with 21 different 125I-labeled lectins and autoradiographed. The staining patterns with particular lectins varied considerably between strains. Concanavalin A stained up to 17 distinct bands in some strains. Other lectins such as peanut lectin only stained two bands in zymodeme 1 strains and none in the other zymodemes. The reaction of N-acetylgalactosamine-specific lectins with some bands indicated the presence of this sugar and this was confirmed by analysis of the extracts. The lectin staining patterns provided an insight into the glycoprotein composition of the bands and should indicate whether combinations of lectins can be used in affinity chromatography systems to purify the glycoproteins.     

Chemical modification studies on the glucose/mannose specific lectins from field and lablab beans.

Seeds of Dolichos lablab var. lignosus (field beans) and variety typicus (lablab beans) contain glucose/mannose specific lectins that have been affinity purified and well characterised (Siva Kumar N., and Rajagopal Rao, D., J.Biosci., 1986, 10, 95-109, (1) Rajasekhar et al., (Biochem.Archives. 1997, 13, 233-240) (2). Purified lectins are glycoproteins with a native molecular mass of 60 kDa and are made of two types of subunits (Gowda et al., 1994, J.Biol.Chem. 269, 18789-18793) (3). Chemical modifications of various groups in purified lectins (using group specific reagents) such as lysine (citraconic anhydride), carboxyl groups (water soluble carbodiimide) tyrosine (N-acetyl imidazole) and tryptophan (2-hydroxy 5-nitro benzylbromide) revealed that 14 out of 21 residues of lysines 7 out of 92 residues of carboxyl groups, 16 out of 24 tyrosine residues and 2 out of 10 tryptophan residues were modified. Lysine and carboxyl group modification led to 95% loss in haemaglutinating activity compared to control while tyrosine and tryptophan modifications led to complete loss of lectin activity. Arginine and histidine modifications led to only 50% loss in activity. The extent of modification and loss in activity was same when the lysine and carboxyl groups were modified in the presence and absence of the inhibitory sugar methyl alpha-D-glucopyranoside at 0.1 M concentration. However protection of modification and lectin activity was observed when the tyrosine and tryptophan residues were modified in the presence of the inhibitory sugar. Earlier CD studies carried out (1) and extensive chemical modification studies reported here substantiate the involvement of tyrosine and tryptophan residues in the sugar binding site of these lectins.     

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.     

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence,spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. Examination of the percentage enhancement and association constants (1.51×10⁶, 6.56×10⁶ and 4.17×10⁵ M^–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×10⁵, 1.3×10⁴, and 11.7×10⁵ M^–1 sec^–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10^–2, 83.3 sec^–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Glycosidic residues in the mouse submandibular gland: biochemical determination and visualization with plant lectins

Biochemical and histochemical studies were carried out on the mouse submandibular gland to define the presence and the localization of glycosidic residues. The results obtained by applying peanut, winged pea, wheat germ, soybean and Con A lectins to unfixed and fixed sections tallied completely with biochemical determinations of the various glycosidic residues. The data from this study have been compared with those obtained by other authors who had performed biochemical and histochemical studies on the gland; similarities and discrepancies as to the presence and the localization of the sugars are discussed.     

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Summary Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Vitamine-like substances L-carnitine and acetyl-L-carnitine: from biochemical studies to medicine

Recently reported data clarify our understanding of the molecular aspects of carnitine in medicine. Carnitine is a compound necessary for the transport of acyl-CoA across the inner mitochondrial membrane for their beta-oxidation. Only L-isomer of carnitine is biologically active. The D-isomer may actually compete with L-carnitine for absorption and transport, increasing the risk of carnitine deficiency. By interaction with CoA, carnitine is involved in the intermediary metabolism by modulating free CoA pools in the cell. Detoxification properties and anabolic, antiapoptotic and neuroprotective roles of carnitine is presented. Carnitine deficiency occurs as a primary genetic defect of carnitine transport and secondary to a variety of genetic and acquired disorders. The pathophysiological states associated with carnitine deficiency have been summarized. L-Carnitine is effective for the treatment of primary and secondary carnitine deficiencies. Acetyl-L-carnitine improves cognition in the brain, significantly reversed age-associated decline in mitochondrial membrane potential and improved ambulatory activity. The therapeutic effects of carnitine and acetylcarnitine are discussed.     

Activation of rhodopsin: new insights from structural and biochemical studies

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.     

Oxalate oxidase from Ceriporiopsis subvermispora: biochemical and cytochemical studies.

The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s-1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.     

Two different families of hydroxyproline-rich glycoproteins in melon callus: biochemical and immunochemical studies

Two different families of hydroxyproline-rich glycoproteins, HRGP₁ and HRGP₂, have been isolated from melon callus and separated by ion exchange chromatography on CM-sepharose. HRGP₁ corresponds to an arabinogalactan protein. The sugar portion of HRGP₁ accounts for 94% of the molecule and contains galactose (66%) and arabinose (34%); these residues are present as polysaccharide side chains attached to hydroxyproline. Hydroxyproline is the main amino acid residue (46%) of the protein moiety. The arabinogalactan protein nature of HRGP₁ has been checked by its ability to positively react with the β-glucosyl Yariv antigen; the ³H-labeled deglycosylated HRGP₁ also called HRP₁ migrates upon electrophoresis as a single band of molecular weight 76,000. HRGP₂ was fractionated by affinity chromatography on heparin-Ultrogel into three different glycoproteins, HRGP_2a,2b and _2c. Two of these glycoproteins behave as polycations (HRGP_2b and _2c) and are chemically distinct from HRGP_2a. HRGP_2b is the most abundant component and contains 41% protein and 50% sugar. Hydroxyproline, lysine, tyrosine, and arabinose are the most prominent residues of their respective moiety. The glycosylation pattern of hydroxyproline indicates that HRGP_2b is related to and possibly a precursor of the wall HRGP; as in melon cell wall HRGP, Hyp-Ara₃ predominates, and small amounts of a putative Hyp-Ara₅ a hitherto unreported hyp-arabinoside, are recorded. The molecular weight of HRP_2b, the protein portion of HRGP_2b is 55,000 ± 5,000, as estimated after deglycosylation of the molecule with trifluoromethane sulfonic acid. Antibodies have been raised against HRGP_2b and HRP_2b. Immunodiffusion shows that each antigen (HRGP_2b or HRP_2b) reacts with its own IgG, and cross-reacts with the heterologous IgG, thereby indicating the presence of common (unglycosylated) and specific (glycosylated and deglycosylated) epitopes. The arabinogalactan protein HRGP₁ is not recognized by either antibody and HRGP_2b does not react with the Yariv antigen. Immunoprecipitation of ³H-labeled HRP₁ and HRP_2b in the presence of goat antirabbit IgG, followed by gel electrophoresis, allows to recover HRP_2b only. Again, HRP_2b is immunoprecipitated by the two antisera.     

Synthesis of neoglycopeptides and analyses of their biodistributionin vivo to identify tissue specific uptake and novel putative membrane lectins

Complex typeN-linked oligosaccharides derived from fetuin, fibrinogen and thyroglobulin were coupled to acetyltyrosine affording a series of neoglycopeptides with retention of terminal structures and the -anomeric configuration of their reducing endN-acetylglycosamine residue. The neoglycopeptides thus synthesized could be labelled to high specific activities with¹²⁵I in the aromatic side chain of tyrosine. Analysis of the fate of these neoglycopeptides in conjunction with inhibition with asialofetuin and oligosaccharides of defined structure in micein vivo revealed the uptake of galactosylated biantennary compound by kidneys, in addition to the known itinerary of triantennary galactosylated complex oligosaccharide from fetuin to liver and the galactosylated biantennary chain with fucosylation in the core to bone marrows. On the other hand, the agalacto, aglucosamino biantennary chains with and without fucosylation in the core region are taken up by submaxillary glands while the conserved trimannosyl core with fucose is primarily concentrated in stomach tissue. These studies thus define new routes for the uptake of complexN-linked glycans and also subserve to identify lectins presumably involved in their recognition.     

Muscarinic receptor-detergent complexes with different biochemical properties: selective solubilization, lectin affinity chromatography and ligand binding studies

Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.     

Porphyrin-retinamides: synthesis and cellular studies

A series of four porphyrin-retinamides containing either all-trans- or 13-cis-retinoid acid residues, directly linked to the para-phenyl position of meso-tetraphenylporphyrin or via a low-molecular-weight PEG spacer, have been synthesized. The biological properties of these conjugates were evaluated in a model cell line, human HEp2, and in neuroblastoma SK-N-DZ cells, which exhibit moderate expression of retinoic acid receptors and retinoic acid-induced differentiation. The directly linked porphyrin-retinamides were taken up by a greater extent (20-50% more) in SK-N-DZ than in HEp2 cells. However, the PEG-containing conjugates accumulated maximally within both cell lines and approximately by the same amount, probably due to their increased amphiphilicity. Among all conjugates, the porphyrin-PEG-13-cis-retinamide accumulated the most in both cell lines (about 5 times more than the non-pegylated conjugates). None of the porphyrin-retinamide conjugates were toxic toward HEp2 cells at concentrations up to 100 microM, and only the hydrophobic non-pegylated conjugates were moderately toxic to SK-N-DZ cells [IC50 (dark) = 56-92 microM, and IC50 (at 1 J/cm2) = 6-8 microM]. All conjugates preferentially localized within cellular vesicles that correlated well to the lysosomes and, in addition, the PEG-containing porphyrin-retinamides were also found in the ER.     

Toshiaki Osawa: biochemistry of lectins and their applications in immunochemistry and cellular biology

Lectins are proteins that agglutinate cells and exhibit an antibody like, sugar-binding specificity. Professor Toshiaki Osawa has discovered, purified and characterized many plant lectins that display diverse biological activities. Using lectins as biochemical tools, he developed methods to determine the biochemical structures of glycoprotein glycans that react with lectins; separated and characterized glycoproteins and cell populations; analysed the mechanisms by which lectins activate cells; and characterized several cytokines produced by immune cells stimulated by lectins. The studies on lectins, the field he took strong leadership, developed into an essential hub of the biology of multicellular organisms.     

Genetic and biochemical studies with ataxia telangiectasia

Summary This article summarizes the genetics and clinical features of ataxia telangiectasia (AT) and then reviews recent cytogenetic, cellular, and biochemical studies which support the hypothesis that a defect in DNA repair is responsible for the various manifestations of the disease. The biochemical evidence further indicates that the defect specifically reduces the cellular capacity to remove bases and nucleotides damaged by ionizing radiation, without affecting the cells' ability to scavenge free radicals or to rejoin breaks in the sugar-phosphate backbone of DNA. Suggestions for additional research to more precisely identify the repair defect will also be presented.