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 levels (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 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 less than 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.     

Incorporation of fucose into glycans independent of the GDP-fucose transporter SLC35C1 preferentially utilizes salvaged over de novo GDP-fucose

Mutations in the SLC35C1 gene encoding the Golgi GDP-fucose transporter are known to cause leukocyte adhesion deficiency II. However, improvement of fucosylation in leukocyte adhesion deficiency II patients treated with exogenous fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which remains a mystery. To investigate this phenomenon, we developed and characterized a human cell–based model deficient in SLC35C1 activity. The resulting cells were cultured in the presence/absence of exogenous fucose and mannose, followed by examination of fucosylation potential and nucleotide sugar levels. We found that cells displayed low but detectable levels of fucosylation in the absence of SLC35C1. Strikingly, we show that defects in fucosylation were almost completely reversed upon treatment with millimolar concentrations of fucose. Furthermore, we show that even if fucose was supplemented at nanomolar concentrations, it was still incorporated into glycans by these knockout cells. We also found that the SLC35C1-independent transport preferentially utilized GDP-fucose from the salvage pathway over the de novo biogenesis pathway as a source of this substrate. Taken together, our results imply that the Golgi systems of GDP-fucose transport discriminate between substrate pools obtained from different metabolic pathways, which suggests a functional connection between nucleotide sugar transporters and nucleotide sugar synthases.     

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism

Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.     

Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.     

Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus

Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects. We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP-fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype. Live-born FX(-/-) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet. FX(-/-) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc. Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP-fucose synthesis. Conditional control of fucosylation in FX(-/-) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion.     

Selective loss of sialic acid from rat small intestinal epithelial cells during postnatal development: demonstration with lectin-gold techniques

Post-embedding lectin-gold cytochemistry was employed to investigate the distribution of sialic acid and fucose residues in rat small intestinal epithelial cells during postnatal development. During the suckling phase (postnatal day 1) the apical and basolateral plasma membranes of epithelial cells, as well as the goblet cell mucus was intensely stained with the sialic acid-specific Sambucus nigra L. lectin I-gold complex (SNL I-g). By the weaning period (postnatal day 23), the entire villus contained both SNL I-g-positive and negative cells. In adult small intestine, the plasma membranes of all epithelial cells were unreactive with SNL I-g; however, abundant staining was detectable in goblet cell mucus, cells of the lamina propria, and smooth muscle cells. The distribution of fucose residues as detected with a Ulex europaeus lectin I-gold complex (UEL I-g) was virtually opposite that of sialic acid. At postnatal day 1, staining was restricted to goblet cell mucus, whereas by postnatal day 23, a portion of epithelial cells displayed UEL I-g binding sites along the apical and basolateral plasma membranes. In the adult, the apical and basolateral plasma membranes of all epithelial cells, as well as goblet cell mucus were stained with UEL I-g. These results support biochemical data demonstrating a shift from sialylation to fucosylation of intestinal microvillar glycoconjugates during the weaning phase of postnatal development. Moreover, the results indicate that rather than a general decrease in cellular sialylation, specific individual cells at all positions along the crypt-to-villus axis become devoid of sialic acid.     

Modulation of solubilized brain fucosyltransferase activity by phospholipids

Phospholipids interact on Triton X-100 solubilized GDP-fucose: asialofetuin fucosyltransferase (EC 2.4.1.68) isolated from sheep brain. This enzymatic activity is modulated by charged phospholipids. In particular, phosphatidic acid and analogues markedly inhibit the transfer of fucose from GDP-[14C]fucose. Kinetic studies show that phosphatidic acid interacts as a mixed inhibitor: the velocity and affinity of fucosyltransferase for the GDP-fucose and asialofetuin substrates are strongly decreased. However, this inhibitory effect is not related to stereospecificity, and the different parameters involved in the enzymatic reaction of glycosylation are not modified. The nature of fatty acids and chemical bond (ester or ether) occurring in the carbohydrate chain does not modify the behaviour of phosphatidic acid with respect to fucosyltransferase activity. Further, the physical state of phosphatidic acid (gel phase or liquid crystalline phase) has no influence. However, as the inhibition is closely pH-dependent, these data suggest that phosphatidic acid might directly interact with the active site of the enzyme and induce a conformational change.     

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.     

Spermidine-induced glycoprotein fucosylation in immature rat intestine.

In rat small intestine, during postnatal development, the glycoprotein fucosylation is markedly increased at weaning. At the same time, a rise in the intestinal spermidine level was observed, partly due to the increase in the spermidine content of solid food given to animals at this period as compared to the spermidine content of milk. In order to mimic the spermidine increase observed in weanling rat intestines, we had treated suckling rats with spermidine by oral ingestion to study its role as maturation factor of the small intestine. In spermidine-treated suckling rats, the spermidine and N-acetyl-spermidine contents were highly increased. Spermidine treatment induced the rise in alpha-1,2-fucosyltransferase activity and the precocious appearance in the brush-border membrane of some alpha-1,2-fucoproteins in weaned rats. Such results indicate that spermidine could be a maturation factor implicated in the appearance of alpha-1,2-fucoproteins naturally observed at weaning time.     

Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine: I. Evidence of a developmental shift from terminal sialylation to fucosylation

We present evidence of a change from sialylation to fucosylation of intestinal microvillus membrane oligosaccharides during postnatal development in the rat. The initial high sialic acid to fucose molar ratio in native and delipidated membranes was completely reversed after weaning. The specific binding of 125I-labeled wheat germ agglutinin to neuraminidase-sensitive sites in the native and delipidated membranes decreased markedly from early suckling to weaning ages. The binding of 125I-labeled Ulex europeus agglutinin I showed an age-related pattern opposite to that of wheat germ agglutinin. The changes in membrane reactivities to these lectins were entirely consistent with the existence of a developmentally-controlled shift from terminal sialyl to fucosyl substitutions among various glycoconjugate classes. This could play a key role on the functional transformation experienced by the intestinal epithelium of suckling rats.     

Biological function of fucosylation in cancer biology

Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins or glycolipids. Fucosylation comprises the attachment of a fucose residue to N-glycans, O-glycans and glycolipids. O-Fucosylation, which is a special type of fucosylation, is very important for Notch signalling. The regulatory mechanisms for fucosylation are complicated. Many kinds of fucosyltransferases, the GDP-fucose synthesis pathway and GDP-fucose transporter are involved in the regulation of fucosylation. Increased levels of fucosylation have been reported in a number of pathological conditions, including inflammation and cancer. Therefore, certain types of fucosylated glycoproteins such as AFP-L3 or several kinds of antibodies, which recognize fucosylated oligosaccharides such as sialyl Lewis a/x, have been used as tumour markers. Furthermore, fucosylation of glycoproteins regulates the biological functions of adhesion molecules and growth factor receptors. Changes in fucosylation could provide a novel strategy for cancer therapy. In this review, the biological significance of and regulatory pathway for fucosylation have been described.     

Gastrointestinal mucins of Fut2-null mice lack terminal fucosylation without affecting colonization by Candida albicans

Posttranslational modification of apomucins by the sequential action of glycosyltransferases is required to produce mature mucins. The Secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (EC 2.4.1.69) that catalyzes addition of terminal alpha(1,2)fucose residues on mucins and other molecules in mucosal epithelium. Mutant mice containing targeted replacement of Fut2 with the bacterial reporter gene lacZ were studied to determine the affect of the loss of Fut2 on glycosylation of mucins in the gastrointestinal tract. By whole organ X-gal staining, lacZ activity is prominently expressed in the foveolar pit and chief cells of the glandular stomach, Brunner's glands of the duodenum, and goblet cells in the large intestine of Fut2-LacZ-null mice. Staining with Aleuria aurantia agglutinin demonstrates loss of L-fucosylated epithelial glycans throughout the gastrointestinal tract of Fut2-LacZ-null mice, however, histologic appearance of the tissues appears normal. Analysis of oligosaccharides released from insoluble colonic mucins, largely Muc2, by mass spectrometry shows complete lack of terminal fucosylation of O-linked oligosaccharides in Fut2-LacZ-null mice. Precursor glycans accumulate with no evidence of compensation by other fucosyltransferases or sialyltransferases on mucin glycosylation. Because Candida albicans has been reported to adhere to intestinal mucins creating a potential reservoir associated with vaginitis, Fut2-LacZ-null and wild-type mice were inoculated by gastric lavage with C. albicans. We observe no difference in colonization between genotypes suggesting mucin terminal fucosylation does not significantly influence C. albicans-host interaction in the intestine, highlighting that infections caused by the same organism at different mucosal surfaces are not equal.     

Acceptor requirements for GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase activity solubilized from pea epicotyl membranes.

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[14C]fucose as donor. Native seed XG (Mr greater than 10(6) Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P4. Specific activities (Bq/microgram carbohydrate) of fragments with Mr ranging from 10(6) to 10(4) Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltransferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB3H4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.     

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of d-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports d-mannose by both Na⁺-dependent and Na⁺-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na⁺-independent mechanism was higher than that of the Na⁺/mannose transport system. At birth, the Na⁺-independent d-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal d-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.     

Plasma cholesterol-lowering effect on rats of dietary fiber extracted from immature plants

Crude dietary fiber samples were prepared from beet, cabbage, Japanese radish, onion and mung bean sprouts (BF, CF, RF, OF and MF, respectively). These samples contained total dietary fiber at the levels of 814, 699, 760, 693 and 666 g/kg, respectively. To examine the effect of these dietary fiber sources on the plasma cholesterol concentration, male Sprague-Dawley rats were fed on a fiber-free (FF) diet or on an FF diet supplemented with 5% or 10% dietary fiber. Dietary fiber extracted from vegetables, wood cellulose (CL), pectin (PE) and guar gum (GG) were used as the fiber sources. Compared with the rats fed on the FF diet, a significant reduction in the plasma cholesterol concentration was observed in the rats fed on BF, CF, RF, MF, PE or GG after a 21-d feeding period. Cecal acetate, n-butyrate and total short-chain fatty acids were significantly higher in the rats fed on these dietary fibers, except for CF, than in those fed on the FF diet. A negative correlation was apparent between the total dietary fiber content, hemicellulose content and pectin content of each dietary fiber source and the plasma cholesterol concentration. These results suggest that some vegetable fibers exert a plasma cholesterol-lowering effect through cecal fermentation of these fibers.     

Surface Glycosyltransferase Activities During Development of Neuronal Cell Cultures

Neurons in culture obtained from dissociated cerebral hemispheres of 8-day-old chick embryos showed measurable activities of galactosyl-, fucosyl-, and sialyl-transferases at the external surface of their plasma membrane. Important changes in these activities were observed during cell proliferation and maturation, in particular the surface fucosyltransferase activity, and/or the amount of intracellular fucosylated acceptors increased during synaptogenesis, between 3 and 5 days in culture (d.i.c.). A sodium dodecyl sulfate radioelectrophoretic analysis of the fucosylated neuronal acceptors labelled with [14C]fucose showed, during synaptogenesis, the high labelling of two protein bands of 116 and 50 X 10(3) daltons. The fucosylation of glycoconjugates occurred preferentially, in neurons, upon glycoproteins whereas in glial cell cultures glycolipids were more fucosylated. The reasons for such a difference are not yet understood but the results suggest that the surface fucosyltransferase activity and fucosylated proteins in particular may play a role during the synaptogenesis of neurons in culture.     

Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants

Protein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glyco-engineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (α1,4 fucosylation and β1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of α1,4 fucose and β1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.     

Nutritional and developmental regulation of glycosylation processes in digestive organs.

We review the nutritional and developmental variations of the glycosylation processes in digestive organs, since glycoproteins play a prominent part as mucins or digestive enzymes in these tissues. The biosynthesis of the glycannic chains is demonstrated to be largely sensitive to various exogenous (such as nutritional) or endogenous (such as developmental) factors. Although the metabolic regulation by dietary variations appears as rather complex, according to the variety of experimental conditions and the diversity of the organs studied, available data demonstrate that this regulation does exist, depending on the quantity or sometimes the quality of the major or minor components of the diet, which induce significant variations in the glycosylation processes. The synthesis of the internal core of N-glycans is essentially regulated by diet-induced variations of the phosphoryl-dolichol level, whereas the modulation of the biosynthesis of the external part of N-glycans or the biosynthesis of O-glycans is controlled by diet-induced variations in the systems transferring fucose, galactose, sialic acid or hexosamines. Modifications in intestinal glycosylation during post-natal development in the rat control the quality of the glycannic chains of mucins and brush-border enzymes. The post-natal maturation of the intestinal rat tissue is characterized by a shift from sialylation to fucosylation, depending on coordinate changes in glycosyltransferase activities, in sugar-nucleotide breakdown or synthesis or in the activity of regulatory proteins. These activities are largely sensitive to dietary manipulations at weaning and to hormonal stimulations before weaning. However, glucocorticoid hormones do not appear as the triggering signal for the induction of these changes.