Marie-Claire Biol

Developmental changes in intestinal glycosylation: nutrition-dependent multi-factor regulation of the fucosylation pathway at weaning time

Developmental changes in the fucoglycoproteins of the intestinal brush-border membranes were determined by lectin affinoblotting after electrophoresis. Whereas only two alpha(1-6)-fucoglycoproteins were detected in brush-border membranes from suckling rats, a large number of N-fucoglycoproteins with alpha(1-2)- and/or alpha(1-6)-linked fucose residues were detected in rat membranes after weaning. Dietary manipulations at weaning time were used to investigate the effect of nutritional factors in the development of fucosylation in the small intestine of prolonged-nursed rats fed with milk (a high-fat, low-carbohydrate diet) compared to rats weaned normally with a standard high-carbohydrate diet. The fucose content of the mucosa glycoproteins was lower in 22-day-old prolonged-nursed rats than in 22-day-old rats weaned normally with the standard diet. The appearance of fucoglycoproteins in the brush-border membranes, which was delayed by prolonged nursing, was accompanied by a concomitant delay in the increase of intestinal fucosyl-transferase activity and in the decrease of GDP-fucose substrate breakdown. The developmental decrease in the activity of the inhibitory protein which regulates the fucosyl-transferase activity was also delayed by prolonged nursing. The intestinal fucosylation of brush-border membrane glycoproteins (which include many digestive enzymes) displayed ontogenic changes on which were superimposed dietary influences at the time of weaning. The complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, is a developmental event which thus seems to be strongly influenced by the manipulation of nutritional factors during the weaning period.

Hormonal regulation of glycosylation process in rat small intestine: Responsiveness of fucosyl-transferase activity to hydrocortisone during the suckling period, unresponsiveness after weaning

The aim of this study was to examine the possible role of certain hormones (especially hydrocortisone) in the developmental variations of intestinal fucosyl-transferase activity in rats. Thyroxine and insulin, injected into suckling rats, did not induce significant modifications of the fucosyl-transferase activity, under the conditions used, whereas this enzyme activity was highly enhanced after administration of glucocorticoids (cortisone and hydrocortisone). Hydrocortisone administration to suckling rats induced a precocious and progressive activation of the fucosyl-transferase activity up to adult level as a function of the duration of treatment. The responsiveness of suckling rats to hydrocortisone, as shown by increased fucosyl-transferase activity, disappeared at the end of the third week (corresponding to the weaning time). These physiological periods of responsiveness and unresponsiveness to hydrocortisone could be related to the binding of the hormones to receptors since the antiglucocorticoid RU 38486 counteracted the effect of hydrocortisone in suckling rats but did not prevent the developmental rise of the fucosyl-transferase activity, when administered in the third week of life. These results suggest that the normal developmental rise of the fucosyl-transferase activity is independent of glucocorticoids.

Nutritional and developmental regulation of glycosylation processes indigestive 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.

Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats.

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an alpha-1, 2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in alpha-1,2-fucosyltransferase level and of the decrease in alpha-l-fucosidase level after treatment with spermine, several alpha-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.

Evidence for the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activities

Soluble endogenous inhibitory activities for glycoprotein: alpha (1-2) and alpha (1-3) fucosyltransferases are demonstrated in rat small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of poly-nucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the glycoprotein substrate. Inhibition is not due to interfering enzymatic activities (glycosyl-nucleotide pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases.

Role of insulin and nutritional factors in intestinal glycoprotein fucosylation during postnatal development

This study deals with the role of insulin in the regulation of the intestinal glycoprotein fucosylation process during postnatal development in the rat. Circulating insulin level was found to increase at weaning time in parallel with α-1,2-fucosyltransferase activity and with the appearance of α-1,2-fucoproteins in brush-border membranes. Insulin treatment of young suckling rats induced a precocious increase in fucosyltransferase activity and in the biosynthesis of its substrate (GDP-fucose), but the sensitivity to insulin disappeared after weaning. The insulin level was lower in 22-day-old rats that received prolonged nursing (on a high-fat diet) compared with age-matched normally weaned rats (on a high-carbohydrate diet), whereas the appearance of α-1,2-fucoproteins and the increase in activity of α-1,2-fucosyltransferase were delayed, as was the decrease in the degradation of GDP-fucose. In 22-day-old animals that received prolonged nursing and insulin treatment, the α-1,2-fucosyltransferase activity reached a level close to that observed in age-matched weaned rats, and several α-1,2-fucoproteins appeared in brush-border membranes with a molecular mass similar to that found in weaned rats. These results suggest that changes in insulin levels at weaning time (as caused, in the present case, by dietary modifications) may be responsible for the regulation of the glycoprotein fucosylation process, essentially by increasing fucosyltransferase activity.This study deals with the role of insulin in the regulation of the intestinal glycoprotein fucosylation process during postnatal development in the rat. Circulating insulin level was found to increase at weaning time in parallel with alpha-1, 2-fucosyltransferase activity and with the appearance of alpha-1, 2-fucoproteins in brush-border membranes. Insulin treatment of young suckling rats induced a precocious increase in fucosyltransferase activity and in the biosynthesis of its substrate (GDP-fucose), but the sensitivity to insulin disappeared after weaning. The insulin level was lower in 22-day-old rats that received prolonged nursing (on a high-fat diet) compared with age-matched normally weaned rats (on a high-carbohydrate diet), whereas the appearance of alpha-1, 2-fucoproteins and the increase in activity of alpha-1, 2-fucosyltransferase were delayed, as was the decrease in the degradation of GDP-fucose. In 22-day-old animals that received prolonged nursing and insulin treatment, the alpha-1, 2-fucosyltransferase activity reached a level close to that observed in age-matched weaned rats, and several alpha-1,2-fucoproteins appeared in brush-border membranes with a molecular mass similar to that found in weaned rats. These results suggest that changes in insulin levels at weaning time (as caused, in the present case, by dietary modifications) may be responsible for the regulation of the glycoprotein fucosylation process, essentially by increasing fucosyltransferase activity.

Implication of insulin and nutritional factors in the regulation of intestinal galactosyltransferase activity during postnatal development

In the rat small intestine, galactosyltransferases are the enzymes implicated in the biosynthesis of glycoproteins of the brush-border membranes and mucins. During postnatal development, the circulating insulin level increased at weaning in parallel with the activities of intestinal galactosyltransferases on O-glycans and N-glycans. This study deals with the role of insulin in the regulation of galactosyltransferase activities during postnatal development. The treatment of immature suckling rats with insulin induced a precocious increase in the activities of the O-glycan and N-glycan galactosyltransferases, partly reproducing the increase in galactosyltransferase activity normally found at weaning, since the O-glycan galactosyltransferase activity increased more quickly than the N-glycan galactosyltransferase activity. The sensitivity of the two galactosyltransferase activities to insulin disappeared after weaning, a period when drastic diet changes occur. In 22-day-old rats submitted to prolonged nursing (high-fat diet), the activities of the O-glycan and N-glycan galactosyltransferases were lower than those found in age-matched normally weaned rats (high-carbohydrate diet), indicating a delay in the maturation of the intestine of prolonged-nursing rats. The circulating insulin level of these animals stayed lower than that of the age-matched weaned rats. When the prolonged-nursing animals were treated with insulin, the O-glycan and N-glycan galactosyltransferase activities reached levels similar to those of the weaned rats. These observations suggest that insulin is one of the maturation factors for intestinal glycoprotein galactosylation and may be partly responsible for the natural enhancement of intestinal galactosyltransferase activities observed during postnatal development in relation to the dietary changes at weaning.

Glycosyltransferase Activities in the Rat Intestinal Mucosa: Comparison between Standard Commercial and Semi-Synthetic Diets

Two groups of rats were fed either a commercial (C group) or a semi-synthetic (S group) diet of very similar quantitative composition, which induce similar growth in the animals. Glycosyltransferase activities are very different in the two groups: fucosyltransferase is lower in S group than in C group, regardless of the expression of the results (specific activity versus protein or DNA). The other activities (galactosyl-, N-acetylglucosaminyl-, N-acetylgalactosaminyltransferases) are modified when expressed versus DNA content; N-acetylneuraminyltransferase specific activity is not altered. Since the weight of mucosa is significantly lower in the S group than in the C group, all the total activities are decreased in the S group. The fucosylation process is further characterized by partial purification of the enzyme and study of the synthesis of GDP-fucose. In each case, interfering reactions (glycosyl-nucleotide pyrophosphatases and proteinases) are controlled. The results give evidence that glycosyltransferases are very sensitive to the qualitative composition of the diet.

Regulation of a soluble intestinal glycoprotein: Fucosyl-transferase by fatty acids in vitro

Abstract 1. 1. A soluble intestinal fucosyl-transferase (EC 2.4.1.68), partially purified by DEAE-cellulose chromatography, is inhibited by fatty acids in vitro . By testing the action of 25 compounds, the structural features needed are defined. 2. 2. The inhibition is due neither to pH alterations, nor to differential solubility of the fatty acids, nor to modifications of glycosyl-nucleotide pyrophosphatases. 3. 3. The inhibition requires at least a double bond and is stronger when the double bond is in a cis configuration. 4. 4. The mechanism of the inhibition is different for the two substrates: competitive for the glycoproteic exogenous acceptor (desialyzed fetuin) and noncompetitive for the other substrate GDP-fucose.

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.