Cellular Glycoconjugates Research Articles

Comparative Histochemical and Biochemical Analysis of Endogenous Receptors for Glycoproteins in Human and Pig Peripheral Nerve

Endogenous sugar-binding proteins were localized in sections of human and pig peripheral nerves by the application of two types of labelled ligands: neoglycoproteins (chemically glycosylated carrier proteins that had proven to be histochemically inert) and desialylated, naturally occurring glycoproteins. These proteins allowed evaluation of the presence and distribution of endogenous receptors for carbohydrates, commonly present in cellular glycoconjugates. (Neo)glycoprotein binding was similar, but not identical, for the two types of mammalian peripheral nerves. The pig nerve differed from the human nerve in more pronounced staining when using different types of beta-galactoside-terminated (neo)glycoproteins and charge-carrying neoglycoproteins, such as bovine serum albumin, bearing galactose-6-phosphate residues, glucuronic acid residues, and sialic acid residues. Comparative biochemical analysis of certain classes of sugar receptors by affinity chromatography and gel electrophoresis revealed the presence of sugar receptors that can contribute to the histochemical staining in a pattern with certain significant differences among rather similar expression for the two species. The assessment of sugar receptor distribution by application of (neo)glycoprotein binding among morphologically defined regions in nerves may hold promise in detecting developmental regulation and changes during nerve degeneration and subsequent regeneration after trauma or pathological states. Correlation of these results to changes in the structure and abundance of glycoconjugates, which are the potential physiological ligands of endogenous sugar receptors commonly detected by plant lectins, may help to infer functional relationships.

Elderberry bark lectin-gold techniques for the detection of Neu5Ac (?2,6) Gal/GalNAc sequences: applications and limitations

The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites of Sambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine.

Characterization of mono-, di-, and tri-O-acetylated sialic acids on human cells.

The presence of mono-, di-, and tri-O-acetylated sialic acids on human cells was demonstrated by using radiochromatographic and chemical techniques. Human melanoma cells and fresh colon tissue were biosynthetically labeled with 6- (3H) glucosamine. Radiolabeled sialic acids were hydrolytically removed from cellular glycoconjugates, purified by ion-exchange chromatography, and separated by paper chromatography on the basis of the number of O-substitutions on each sialic molecule. This analytical technique characterized radiolabeled sialic acids that migrated with the same Rf as synthetic mono-, di-, and tri-O-acetylated 14C-labeled sialic acids. The mono-O-acetylated sialic acids were characterized by their sensitivity to sodium periodate oxidation and a crude mouse liver esterase preparation. The di- and tri-O-acetylated sialic acids were characterized by their resistance to sodium periodate oxidation and sensitivity to the action of crude mouse liver esterase. Chromatographically separated di- and tri-O-acetylated sialic acids from normal human colon tissue were characterized by their respective ion molecular weights by using fast-atom bombardment-mass spectrometry. Using these methods, we chemically characterized mono, di-, and tri-O-acetylated sialic acids expressed on human cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.

Expression of cellular glycoconjugates in transfilter-induced metanephric mesenchyme.

Expression of glycoconjugates during transfilter-induced differentiation of metanephric mesenchyme was studied by using fluorochrome- and peroxidase-coupled lectins. All cells in the uninduced metanephric mesenchyme expressed mannose, beta-D-galactose (beta-Gal), N-acetylglucosamine (GlucNAc), and terminal sialic acids. Additionally, solitary cells showed terminal alpha-D-galactose alpha-D-galactose (alpha-Gal) typical of mouse endothelial cells. During culture, undifferentiated mesenchymal cells seemed to disappear from induced explants, and many of the stromal cells between the evolving tubules presented terminal alpha-Gal residues. Similar positivity could be revealed in monolayer cultures of induced mesenchymes. A number of tubules in induced explants displayed alpha-L-fucosyl (Fuc) residues, characteristic of mature proximal tubules. Some terminal Ga1NAc residues, recognized only by Dolichos biflorus agglutinin, emerged in a few tubular cells after prolonged culture. The early tubules and glomerular bodies displayed a basement membrane presenting both terminal Ga1-(beta 1-3)-Ga1NAc and Ga1NAc residues. These positivities disappeared later from many tubular structures and glomerular bodies but persisted in tubules expressing proximal tubular differentiation. The glomerular bodies displayed only one cell type, reminiscent of maturing podocytes, presenting terminal Ga1-(beta 1-3)-Ga1NAc and Ga1NAc residues. Later these saccharide residues became covered by sialylation, as they could then be revealed only after treatment with neuraminidase. The results indicate that the segment-specific expression of saccharide residues during differentiation of nephron in vitro resembles the sequence seen in vivo. This study also suggests that the basement membranes surrounding the nephron show a stepwise, segment-specific maturation. Despite the presence of endothelial cells in the metanephric explants, only avascular glomeruli evolved in this differentiation model.

Differential interaction of reovirus type 3 with sialylated receptor components on animal cells

In this report we study the interaction of reovirus type 3 Dearing (RV3) with vertebrate erythrocytes whose membrane glycoconjugates differ in the degree and position of O-acetylation of their sialic acid (NeuAc) residues. Binding to erythrocytes required the presence of NeuAc on cellular glycoconjugates, since pretreatment with sialidase (neuraminidase) abolished hemagglutination by RV3. Furthermore, we found that RV3 binds efficiently to and hemagglutinates all erythrocyte preparations possessing exclusively NeuAc, or a mixture of NeuAc and 4- O-acetyl-NeuAc (4- O-Ac-NeuAc), but poorly to erythrocytes bearing a mixture of 9- O-Ac-NeuAc and NeuAc, suggesting that RV3 binds preferentially to NeuAc-containing glycoconjugates. To gain further evidence for this hypothesis we treated chicken erythrocytes with influenza C virus neuraminate, 9- O-acetylesterase, to convert their 9- O-Ac-NeuAc residues to NeuAc. When hemagglutination assays were carried out on these cells, we observed a 16-fold increase in the hemagglutination titer for RV3 compared to untreated cells. When we treated bovine submaxillary mucin (BSM) with influenza C virus, we observed a dramatic increase in its potency as an inhibitor of RV3 hemagglutination. Concomitant with this, the 9- O-Ac-NeuAc residues on BSM were converted to NeuAc. Taken together and in conjunction with a previous report (A. F. Pacitti and J. R. Gentsch, 1987, J. Virol. 61, 1407–1415) , these results suggest that the virion attachment protein exhibits a strong preference for NeuAc over 9- O-Ac-NeuAc as a receptor component on erythrocytes.

Expression of cellular glycoconjugates in transfilter-induced metanephric mesenchyme: [formula omitted]. Department of Pathology, University of Helsinki, SF-00290 Helsinki, Finland

Expression of cellular glycoconjugates in transfilter-induced metanephric mesenchyme: [formula omitted]. Department of Pathology, University of Helsinki, SF-00290 Helsinki, Finland

Glycosylation mutants and the functions of mammalian carbohydrates

Glycosylation mutants of mammalian cells synthesize novel carbohydrate structures that are expressed on cellular glycoconjugates. Such mutants provide many avenues for investigating the functions of carbohydrates, both in their effects on the behavior of individual macromolecules (endogenous or introduced into the cell) and on the array of structures expressed at the surface of cells.

Glycosylation of developing human glomeruli: lectin binding sites during cell induction and maturation.

Expression of cellular glycoconjugates during differentiation of human fetal kidney was studied using fluorochrome-labeled lectins. Each lectin revealed a characteristic binding pattern during the phenotypic change of the nephrogenic mesenchyme and during distinct stages of nephron development. The uninduced mesenchymal cells were positive for Pisum sativum (PSA), Concanavalin A (ConA), Wistaria floribunda (WGA), and Ricinus communis (RCA-I) lectins. However, these lectins failed to react with the uninduced cells of the S-shaped bodies, whereas Maclura pomifera (MPA), Triticum vulgaris (WGA) and, after neuraminidase treatment, Arachis hypogaea (PNA) agglutinins bound intensely to the presumptive podocytes. During later stages of nephrogenesis, MPA positively on the podocytes weakened and could not be observed in adult kidney glomeruli. Binding sites for Helix pomatia (HPA) agglutinin in glomeruli were also expressed only transiently during nephrogenesis. During further development PSA, ConA, WFA, and RCA-I reacted with mesangial cells in addition to the glomerular basement membranes. The segment-specific lectin binding patterns of the tubuli emerged in parallel with the appearance of brush border and Tamm-Horsfall antigens of the proximal and distal tubuli. The results show that nephron site-specific saccharides appear in a developmentally regulated manner and in parallel with morphologic maturation of the nephron. Lectins therefore appear to be useful tools for study of induction and maturation of various nephron cell types.

Vitamin A modifies the glycopeptide composition of cultured Sertoli cells.

Sertoli cells obtained from prepubertal rat testes were cultured in the presence or absence of retinol. Incorporation of monosaccharides and glycopeptide composition of the cells were studied under two experimental conditions. The results indicate that retinol increases the amount of mannose and glucosamine incorporated into cellular glycoconjugates. The labeled glycopeptides obtained from control and retinol-treated cells were separated by size and lectin affinity. Gel filtration analysis showed no size differences between the glycopeptides obtained from control and vitamin A-treated cells. Affinity chromatography on Concanavalin A and Wheat Germ Agglutinin of 3H-mannose-labeled glycopeptides showed that Sertoli cells cultured in the presence of retinol contain a higher percentage of high mannose-type glycopeptides compared with control cells. The effect of retinol on Sertoli cell glycopeptide composition is partially reversed by the administration of FSH.

Lectin-binding sites in normal, hyperplastic, adenomatous and carcinomatous human colorectal mucosa.

The carbohydrate structures of cellular glycoconjugates in normal, hyperplastic, adenomatous and carcinomatous human colorectal mucosa were analysed with six fluorescein isothiocyanate-conjugated lectins. In normal, hyperplastic and adenomatous colorectal mucosa showing mild or moderate dysplasia Concanavalin A (Con A), Lens culinaris (LCA), and wheat germ (WGA) agglutinins stained goblet cell glycoconjugates (actual mucin goblet itself) while peanut (PNA), Vicia villosa (VVA), and Griffonia simplicifolia-II (GSA-II) agglutinins showed a supranuclear staining of goblet cell glycoconjugates. After neuraminidase treatment of tissue sections PNA and VVA stained mucin goblets of mature cells in normal mucosa, while less differentiated cells in the lower crypt displayed a supranuclear staining with VVA. The mucin goblets in adenomatous mucosa with mild or moderate dysplasia did not stain with PNA and VVA, neither before nor after neuraminidase treatment. Areas of in situ cancer in adenomas and carcinomas displayed a strong and direct binding of Con A, LCA, WGA and PNA in an apical linear distribution, while the binding of VVA and GSA-II was heterogeneous. We conclude that there are alterations in the carbohydrate structures of cellular glycoconjugates, which can be related to goblet cell differentiation in normal colorectal mucosa and to the degree of dysplasia in adenomas. Heterogeneous and incompletely glycosylated glycoconjugates appear to be synthesized by the majority of colorectal carcinomas.

Histochemical investigation of cellular and tissue glyco-conjugates with lectins in semithin sections

Histochemical investigation of cellular and tissue glyco-conjugates with lectins in semithin sections

Cellular differentiation of basal cell carcinoma studied with fluorescent lectins and cytokeratin antibodies.

The expression of cellular glycoconjugates and cytokeratin polypeptides in 8 basal cell carcinomas (BCC) was studied using fluorochrome-coupled lectins and different keratin-antibodies. Peanut agglutinin and Wistaria floribunda agglutinin, binding to all layers of normal human epidermis, also stained all cells in the basal cell carcinomas. Dolichos biflorus agglutinin, which in normal epidermis binds only to the basal cells, gave a mottled staining pattern in most of the tumors. Instead, Ulex europaeus I agglutinin and soybean agglutinin, which in normal epidermis only bind to the spinous and granular cell layers, did not stain tumor cells in basal cell carcinomas. Rabbit antibodies to human 43-50kD epidermal keratin polypeptides and 2 monoclonal cytokeratin antibodies, PKK1 reacting only with follicular epithelium, and PKK2 reacting also with the basal epidermal cells, brightly stained all cells of the basal cell carcinomas studied, whereas antibodies to human 60-67kD epidermal keratin polypeptides did not bind to the carcinoma cells. The results suggest that the cells in basal cell carcinomas resemble epidermal basal cells both by their glycoconjugate pattern and keratin expression. However, the tumor cells also express cytokeratins, which can be found only in the follicular epithelium, but not in normal interfollicular epidermis.

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates.

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP- N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [ 3H]glucosamine-HCl or N-[ 3 H]acetylmannosamine showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, 2- acetamido-1,3,4,6- tetra-O- acetyl-2- deoxy- d-mannopyranose (Ac 4-NAcMan) and the 2-trifluoroacetamido derivative (Ac 4F 3-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of N-[ 3 H]acetylmannosamine into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac 4F 3-NAcMan and Ac 4-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac 4-NAcMan was relatively reversible, whereas that produced by Ac 4F 3-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of N-[ 3 H]acetylmannosamine into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac 4F 3-NAcMan caused accumulation of radioactivity from N-[ 3 H]acetylmannosamine in CMP- N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound 3H caused by this agent. In contrast, similar exposure to Ac 4-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from N-[ 3 H]acetylmannosamine in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with 3H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac 4-NAcMan labeled with 14C in the 4- O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro- d-galactose, on the L1210 leukemic cell ectosialyltransferase system

In L1210 leukemia cells, 6-deoxy-6-fluoro- d-galactose specifically inhibited the incorporation of [ 3H]- d-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6- 3H]-6-deoxy-6-fluoro- d-galactose to a nucleotide sugar was similar to that found for [ 3H]- d-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro- d-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5′-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro- d-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.

The biosynthesis of a mannolipid containing a metabolite of retinoic acid by 3T12 mouse fibroblasts.

Retinol and retinoic acid (RA) increase the adhesive properties of spontaneously transformed mouse fibroblasts (BALB/c 3T12-3 cells) and the incorporation of [2-3H]mannose into cellular glycoconjugates. Therefore we searched for a mannolipid of retinoic acid similar to mannosylretinylphosphate (MRP) in these cells. Radioactively labeled RA was incorporated into a compound of chromatographic characteristics similar to those of standard MRP. This metabolite contained the same 3H:14C ratio as the precursor [11, 12-3H, 15-14C]retinoic acid, demonstrating that no decarboxylation had occurred. A doubly labeled mannolipid was obtained from cells incubated with [2-3H]mannose and [15-14C]retinoic acid. This mannolipid was readily cleaved by mild acid, yielding [3H]mannosephosphate and a compound that migrated as standard anhydroretinol on thin layer of silica gel in toluene:chloroform:methanol (4:1:1) at Rf 0.93. Standard all trans-MRP yields all-trans-anhydroretinol under these conditions. An ion pair reverse phase HPLC system was developed to further characterize the mannolipids obtained from retinol and retinoic acid in 3T12 cells. [15-3H]Retinol and [15-14C]retinoic acid were incorporated into mannolipids that cochromatographed upon HPLC with standard MRP. The mixture of the [15-3H]retinol and [15-14C]retinoic acid derived mannolipids was subjected to mild acid hydrolysis, after purification by HPLC. Nearly 100% of the compounds was hydrolyzed, yielding all-trans-[3H]anhydroretinol and a 14C]labeled product which was eluted from HPLC as a slightly more polar compound than all-trans-anhydroretinol. The retinoic acid-derived mannolipid (MXP) represented approximately 4% of the total radioactivity in the methanolic extract of 3T12 cells incubated for 20 hours in the presence of labeled retinoic acid. However, if the cells were incubated for an additional 20 hours in the absence of the radioactive precursor, MXP represented 40% of the total extracted radioactivity. These results demonstrate that 3T12 cells synthesize mannosylretinylphosphate from retinol and a mannosylretinoidphosphate (MXP) from retinoic acid. These results exclude the possibility of a reduction of retinoic acid to retinol, but suggest that a closely related compound is formed from RA and that this retinol-like compound (X) is incorporated into MXP.

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B. An in vitro study of human airway.

The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial explants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline 14C-labeled glycoconjugate release but caused a dose-dependent (10(-7)-10(-4) M) inhibition of 14C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 X 10(-5) M methacholine. Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited 3H-threonine or 3H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10(-5) M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10(-5) M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine. Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.