Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.     

Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus.     

The case for sequencing the genome of the electric eel Electrophorus electricus

A substantial international community of biologists have proposed the electric eel Electrophorus electricus (Teleostei: Gymnotiformes) as an important candidate for genome sequencing. In this study, the authors outline the unique advantages that a genome sequencing project of this species would offer society for developing new ways of producing and storing electricity. Over tens of millions of years, electric fish have evolved an exceptional capacity to generate a weak (millivolt) electric field in the water near their body from specialized muscle‐derived electric organs, and simultaneously, to sense changes in this field that occur when it interacts with foreign objects. This electric sense is used both to navigate and orient in murky tropical waters and to communicate with other members of the same species. Some species, such as the electric eel, have also evolved a strong voltage organ as a means of stunning prey. This organism, and a handful of others scattered worldwide, convert chemical energy from food directly into workable electric energy and could provide important clues on how this process could be manipulated for human benefit. Electric fishes have been used as models for the study of basic biological and behavioural mechanisms for more than 40 years by a large and growing research community. These fishes represent a rich source of experimental material in the areas of excitable membranes, neurochemistry, cellular differentiation, spinal cord regeneration, animal behaviour and the evolution of novel sensory and motor organs. Studies on electric fishes also have tremendous potential as a model for the study of developmental or disease processes, such as muscular dystrophy and spinal cord regeneration. Access to the genome sequence of E. electricus will provide society with a whole new set of molecular tools for understanding the biophysical control of electromotive molecules, excitable membranes and the cellular production of weak and strong electric fields. Understanding the regulation of ion channel genes will be central for efforts to induce the differentiation of electrogenic cells in other tissues and organisms and to control the intrinsic electric behaviours of these cells. Dense genomic sequence information of E. electricus will also help elucidate the genetic basis for the origin and adaptive diversification of a novel vertebrate tissue. The value of existing resources within the community of electric fish research will be greatly enhanced across a broad range of physiological and environmental sciences by having a draft genome sequence of the electric eel.     

Characterization of desmin in the electric organ of Electrophorus electricus L

The intermediate filament protein of the electric organ from the Electrophorus electricus L. was purified in DEAE-cellulose column after extraction with a Triton X-100 buffer and urea solubilization. The desmin was analysed by SDS-PAGE against desmin purified from chicken gizzard. Characterization of desmin from the electric eel was carried out by peptide mapping and immunoblotting methods.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Synapsin I Is Associated with Cholinergic Nerve Terminals in the Electric Organs of Torpedo, Electrophorus, and Malapterurus and Copurifies with Torpedo Synaptic Vesicles

Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using alpha-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin.     

Purification of acetylcholine receptors from the muscle of Electrophorus electricus

Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.     

Sulfated glycosaminoglycans: their distinct roles in stem cell biology

Sulfated glycosaminoglycan (GAG) chains are a class of long linear polysaccharides that are covalently attached to multiple core proteins to form proteoglycans (PGs). PGs are major pericellular and extracellular matrix components that surround virtually all mammalian cell surfaces, and create conducive microenvironments for a number of essential cellular events, such as cell adhesion, cell proliferation, differentiation, and cell fate decisions. The multifunctional properties of PGs are mostly mediated by their respective GAG moieties, including chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate (KS) chains. Structural divergence of GAG chains is enzymatically generated and strictly regulated by the corresponding biosynthetic machineries, and is the major driving force for PG functions. Recent studies have revealed indispensable roles of GAG chains in stem cell biology and technology. In this review, we summarize the current understanding of GAG chain-mediated stem cell niches, focusing primarily on structural characteristics of GAG chains and their distinct regulatory functions in stem cell maintenance and fate decisions.     

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Eel electric organ: hyperexpressing calmodulin system.

The electroplax of the electric eel Electrophorus electricus is the most abundant source of the calcium-binding protein calmodulin. The electroplax has 250 times the amount of calmodulin and its mRNA than eel skeletal muscle. Our data suggest that there is no major difference in gene copies, the degree of methylation, or genome rearrangement of the calmodulin gene in DNAs from eel electroplax and muscle. Differences in the calmodulin-binding proteins in electroplax and muscle suggest a differential role for the functional expression of calmodulin in cellular regulation.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.     

Alpha-actinin in the electric organ of Electrophorus electricus, L

We have identified Alpha-actinin from the electric organ of the Electrophorus electricus, L. It was analysed by polyacrylamide gel electrophoresis, and identified by immunoblotting. This protein was also found in a membrane fraction of the electric organ enriched with components of the cytoskeleton. Our results suggest that this protein might play a role either in the organization of the microfilaments or its interactions with the membrane to maintain a polarized electrocyte.     

Glycosaminoglycans of Rat Cerebellum: I. Quantitative Analysis of the Main Constituents at Postnatal Day 6

Abstract: Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. ¹⁴C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents—HA, chondroitin sulfate (CS), and heparan sulfate (HS)—were found at concentrations of 1.82, 1.52, and 0.76 μg/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [³H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/μg for HS, 14.2 for HA, and 8.3 for CS.