Fucose Content Research Articles

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin.

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120.

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.

Content of fucose, neutral hexoses and sialic acids in duodenal juice in duodenal ulcer and chronic acalculous cholecystitis

The aim of our work was to estimate the state of fucoglycoprotein and sialoglycoprotein secretion in duodenal ulcer disease. We examined 125 patients aged from 16 to 60 years divided into five groups. The 1st group included 29 patients (17 men and 12 women) with acute exacerbation of peptic ulcer disease; the 2nd group included 20 patients (11 men and 9 women) with moderate exacerbation of the disease; the 3rd group included 21 patients (11 men and 10 women) with clinical remission; the 4th group included 38 patients (18 men and 20 women) with exacerbation of chronic acute cholelithiasis. The 5th control group included 17 (10 men and 7 women) healthy people. The duration of the disease varied from one year to 20 years.

Alterations in carbohydrate composition of serum IgG from patients with rheumatoid arthritis and from pregnant women.

The carbohydrate composition of IgG purified from serum of patients with rheumatoid arthritis (RA), pregnant women, and blood donors has been determined by gas-liquid chromatography. Comparison of the results indicates that IgG from patients with RA contains significantly less galactose but more N-acetylglucosamine than normal IgG, whereas the fucose and sialic acid contents are not changed. The carbohydrate content of IgG in RA is reduced. IgG in pregnancy contains more galactose and more sialic acid than normal IgG, whereas fucose, N-acetylglucosamine, and the total carbohydrate content are not changed. These data suggest a temporal compensation of the RA associated undergalactosylation of IgG in female patients with RA during pregnancy, a period during which remission of the disease is often observed.

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly ( P < 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.

A study on assay method for fucose and sialic acid contents in sputum and serum of patients with lung diseases

A study on assay method for fucose and sialic acid contents in sputum and serum of patients with lung diseases

The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin.

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.

Effect of banana powder ( Musa sapientum var. paradisiaca) on gastric mucosal shedding

Banana pulp powder ( Musa sapientum Linn. var. paradisiaca) was studied for its effects on gastric mucosal resistance. Banana-treated (0.5 g/kg orally, twice daily for 3 days) rats of either sex showed: (i) a significant increase in the [ 3H]thymidine incorporation into mucosal cell DNA; (ii) a significant increase in the total carbohydrate (sum of total hexoses, hexosamine, fucose and sialic acid) content of gastric mucosa; (iii) a significant decrease in gastric juice DNA and protein; (iv) a significant increase in the total carbohydrates and carbohydrate/protein ratio of gastric juice. Aspirin treatment to rats caused similar effects as banana on the [ 3H]thymidine incorporation into mucosal cell DNA but showed opposite effects on the other parameters. These results suggest that banana treatment increased and aspirin decreased the gastric mucosal resistance as evidenced by a respective decrease and increase in gastric juice DNA, the latter serving as an index of the rate of mucosal shedding. Increased cellular mucus may be the factor for increased mucosal resistance. The results of the present study tend to confirm that plantain banana powder strengthens mucosal resistance and promotes the healing of ulcers.

褐藻中の硫酸多糖に関する研究 ＩＶ 日本産褐藻類中のフコース含有多糖量について

The yields of hot-water extracts (HWE), water-soluble alginates (WSA) and fucose-containing polysaccharides (FCP) from brown algae of various sources in Japan were investigated. In addition, the contents of fucose, uronic acid and suifate in the FCP were determinated. The following results were obtained. 1) The yields of HWE varied from 2.44% for Sargassum thumbergii to 19.44% for Laminariareligiosa. The HWE contents in Laminariaceae are generally higher than those in Sargassaceae. 2) The yields of WSA varied widely from nondetectable amounts for Cladosiphon okamuranus to 9% for Laminaria religiosa, which was the highest among the Laminariaceae. 3) The yields of FCP varied widely from 0.5% for Sargassum thumbergii to 10% for Eisenia bicyclis. In general, the FCP contents tended to be the highest in the algae growry at the upper intertidal zone where the alga is exposed to the sunlight for a longer more space time. 4) The FCPs from Cladosiphon okamuranus, Ishige okamurae, Ishige sinicola, and Sargassum horneri seemed to be a simple fucan sulphate constructed mostly from fucose. On theother hand, it seemed that the PCPs from most of the other samples have more complicated constitution.

Role of carbohydrates in insect resistance in Phaseolus vulgaris

The bruchid beetles Acanthoscelides obtectus (Say) and Zabrotes subfasciatus Boheman, are major storage pests of the economically important legume Phaseolus vulgaris (haricot bean). In the present study, resistance of a wild line of P. vulgaris G12953, towards these two insects is confirmed. Mature seeds from both resistant and susceptible lines were fractionated and tested in feeding trials in an attempt to elucidate the basis of resistance. The heteropolysaccharide fraction isolated from the resistant line and a susceptible line were each incorporated into artificial beans over a concentration range up to 10% dry wt. At a concentration of 4%, the approximate physiological concentration within the seed, the heteropolysaccharide fraction from the resistant line was very toxic resulting in 80–85% larval mortality of A. obtectus with an LC 50 of 2.5%. Furthermore, surviving larvae showed a marked increase in their development period. The corresponding fraction from a susceptible line, on the other hand, even at twice the physiological concentration, had a negligible effect upon larval development both in terms of adult emergence and development period. The effects of albumin proteins and globulin proteins on larval development of A. obtectus were also tested by incorporation into artificial beans at a range of concentrations up to 10%. Whilst those from the susceptible line had little effect upon development both the albumins and globulins from the resistant line were found to cause a slight decrease in adult emergence and a small increase in the median development period. These effects, however, are not sufficient to account for the observed seed resistance of G12953. Mature seeds from the resistant line and nine susceptible lines were also screened for the following secondary plant compounds: proteinase inhibitors, α-amylase inhibitors, lectins (phytohaemagglutinins), saponins, tannins and alkaloids. Although some of these antimetabolites were found to be present none, except for α-amylase inhibitors, were thought to play any significant role in conferring seed resistance. It is concluded that this example of insect resistance in seeds of P. vulgaris, line G12953, is due, at least in part, to the presence of a heteropolysaccharide which has an unusually high arabinose and fucose content. In the absence of other antimetabolic compounds the levels of this heteropolysaccharide present in G12953 are sufficiently high to account for the observed resistance. However, it is possible regarding seed resistance towards Z. subfasciatus, that α-amylase inhibitors may be having a synergistic effect.

Extracellular Polysaccharides From Shaken Liquid Cultures of Zea mays

Callus initiated from root and embryo of Zea mays proliferated largely through aberrant root growth. In shaken liquid cultures (liquid cultures), the callus remained as clumps of roots but some cells were released into the medium. The extracellular polysaccharides (ECPS) from these culture supernatants are similar with respect to monosaccharide and glycosyl linkage composition to the slime secreted by roots of young Zea mays seedlings but are distinguished from ECPS produced by suspension cultured cell lines of other species by their high fucose content. Callus initiated from root and embryo therefore provides a model for studies of fucosyl-containing polysaccharides secreted by seedling roots.

Studies of Double-Labeled Mouse Thyrotropin and Free -Subunits to Estimate Relative Fucose Content

The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with [35S]methionine and [3H]fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of 3H/35S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The 3H/35S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated [3H]fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones.

Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine

To study an enzymatic basis for the postnatal changes in intestinal glycosylation, the activities of sialyl- and fucosyltransferases were determined in the particulate fraction of mucosal cells prepared from rat small intestine of various ages. The results show that sialyltransferase activity was present in increased levels compared to adults during the preweaning period (1–2 weeks) and subsequently declined 5-fold to adult levels after weaning, while fucosyltransferase activity was decreased compared to adults in the first 3 weeks of life, rapidly increased at 4 weeks, and reached adult level (10-fold) by 5 weeks. The changes in both sialyl- and fucosyltransferase activities were reflected by the membranous content of glycosidic-bound sialic acid and fucose, respectively. Cortisone injection precociously induced a decreased sialyltransferase activity and an increased fucosyltransferase activity in 2-week-old suckling rats. This study indicates that the activities of sialyl- and fucosyltransferases were reciprocally related and modulated by cortisone action in the developing intestine. These enzyme changes may be responsible for the previously noted shift from sialylation to fucosylation of the intestinal mucosa during maturation.

Changes in connective tissue composition in the skin lesion in psoriasis

We studied the content of chondroitinseric acid, glucuronic acid, hexosamines, fucose, and N-acetylneuraminic acid, as well as the activity of trypsin-like proteases in the papule tissues (biopsied skin from the lesion focus, including papillary layer of derma, basal and thorny layers of epidermis) and in parakeratotic scales of 104 psoriasis patients (53 of them were diagnosed with progressing stage, 37 with stabilizing stage, 14 with regressing stage).

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man.

Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose-(beta 1----4)-N-acetylglucosamine (Gal(beta 1----)-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal beta-Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(beta 1----3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal beta-Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose. The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize Po protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin.

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.

Saliva Secretion Rate and Saliva Composition as a Model to Determine the Effect of Antidepressant Drugs on Cholinergic and Noradrenergic Transmission

Three principally different antidepressant drugs--amitriptyline (with effects on both noradrenaline and serotonin reuptake), maprotiline (a selective inhibitor of reuptake of noradrenaline) and zimelidine (a selective inhibitor of reuptake of serotonin) were tested after a single dose and, as concerns maprotiline and zimelidine, after long-term use in healthy volunteers as regards the effects on saliva secretion rate and saliva composition. Amitriptyline gave a strong decrease in both unstimulated and stimulated saliva secretion rate, indicating a strong anticholinergic effect, and a strong increase in the activity of amylase and the content of protein, fucose and hexose in saliva, indicating a strong agonistic effect on noradrenaline transmission. Maprotiline gave an intermediate decrease in saliva secretion rate, and the effect remained after 14 days of treatment. Single doses gave increases in saliva amylase activity and protein content. After 14 days this effect was increased, and there was also a pronounced increase in fucose and hexose content indicating a strong agonistic effect on noradrenaline transmission. Zimelidine gave a low decrease in saliva secretion rate, indicating a mild anticholinergic effect. No certain effect on the saliva composition could be settled after a single dose, but after 14 days there were consistent increases in amylase activity and protein, fucose and hexose content, indicating a facilitation of noradrenaline transmission. Zimelidine as well as its active metabolite, norzimelidine, are selective inhibitors of serotonin reuptake. The action of zimelidine is discussed in view of recent hypotheses about a modulating serotoninergic influence on the noradrenergic system.

Analysis of Fucose and Sialic Acid Content of Sputum from Patients with Chronic Airway Diseases

Analysis of Fucose and Sialic Acid Content of Sputum from Patients with Chronic Airway Diseases

Effect of hormone administration on the sialylation and fucosylation of intestinal microvillus membranes of suckling rats.

Cortisone, thyroxine, epidermal growth factor, or insulin were administered to 8-day-old rats for 4 days. In comparison to saline-injected controls, cortisone treatment: 1) lowered the sialic acid and raised the fucose content of the intestinal microvillus membranes, 2) increased [3H]fucose incorporation into these membranes, and 3) decreased the membrane binding of 125I-wheat germ agglutinin, while increasing the binding of 125I-ulex europeus agglutinin I and 125I-peanut agglutinin. Thyroxine treatment had similar effects on fucose content and 125I-ulex europeus agglutinin I binding, but did not alter [3H] fucose incorporation or sialic acid content. At the doses used, epidermal growth factor and insulin had no significant effects. The effect of cortisone treatment on sialic acid and fucose was commensurate with a 5- to 6-day acceleration of postnatal intestinal maturation. The changes in lectin binding, however, suggested qualitative differences between developmental and cortisone-induced membrane glycosylation. In addition, this study demonstrates significant quantitative and qualitative differences in the response of intestinal glycosylation to pharmacologic doses of the four hormones.