1 Shanghai Institute for Pediatric Research, Xinhua Hospital and Shanghai Second Medical University, Shanghai, People's Republic of China 200092; 2 Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, Charlestown 02129; and 3 Program in Glycobiology, Shriver Center for Mental Retardation, Waltham, Massachusetts 02452
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ABSTRACT |
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Regional differences in the
ontogeny of mouse intestinal -2,6-sialyltransferase activities
(
-2,6-ST) and the influence of cortisone acetate (CA) on this
expression were determined. High ST activity and
-2,6-ST
mRNA levels were detected in immature small and large intestine, with
activity increasing distally from the duodenum. As the mice matured, ST
activity (predominantly
-2,6-ST) in the small intestine decreased
rapidly to adult levels by the fourth postnatal week. CA precociously
accelerated this region-specific ontogenic decline. A similar decline
of ST mRNA levels reflected ST activity in the small, but not the
large, intestine. Small intestinal sialyl
-2,6-linked
glycoconjugates displayed similar developmental and CA
induced-precocious declines when probed using Sambucus nigra
agglutinin (SNA) lectin. SNA labeling demonstrated age-dependent
diminished sialyl
2,6 glycoconjugate expression in goblet cells in
the small (but not large) intestine, but no such regional specificity
was apparent in microvillus membrane. This suggests differential
regulation of sialyl
-2,6 glycoconjugates in absorptive vs. globlet
cells. These age-dependent and region-specific differences in sialyl
-2,6 glycoconjugates may be mediated in part by altered
-2,6-ST
gene expression regulated by trophic factors such as glucocorticoids.
sialyl -2,6 glycoconjugates; hormonal regulation; ontogeny of
the gut; lectin
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INTRODUCTION |
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SIALIC ACIDS ARE
A FAMILY of negatively charged sugars whose most common form in
mammals is N-acetyl neuraminic acid (NANA). Sialic acid can
be attached to penultimate galactose (Gal),
N-acetylglucosamine (GlcNAc), or
N-acetylgalactosamine (GalNAc) sugars of glycoconjugates (glycoproteins, mucins, and glycolipids). In addition, sialic acid
can form polysialic acid side chains of up to 50 residues in length via
the 8-hydroxyl group (39). Sialic acid-containing glycoproteins represent a major class of cell surface receptors important for both physiological transcellular communication (20, 53, 54) and pathogenesis of major pathogens (63).
During postnatal development, rodent intestinal membrane
glycoconjugates undergo a striking alteration from an initially high
sialic acid-to-fucose ratio to a low ratio during maturation (11,
35, 52). This inversion has been attributed to diminished
sialylation and increased fucosylation of glycoconjugates during the
transition from the suckling to the adult phenotype (50).
Sialylation involves transfer of sialic acid from its
activated form [cytidine monophosphate (CMP)-NANA] onto the
acceptor sugar and is catalyzed by a sialyltransferase (ST) family of
enzymes. The glycan sequences most commonly found in intestine are
NANA2,6Gal and NANA
2,3Gal in membrane glycoproteins (43), NANA
2,6GalNAc, NANA
2,6Gal, and NANA
2,3Gal
in mucins (32), and NANA
2,3Gal and NANA
2,8NANA in
gangliosides (62). Because there is a specific enzyme for
each transfer reaction, the existence of up to 19 individual ST enzymes
has been proposed (20, 53). Despite the similarity in the
reaction catalyzed, all ST cloned to date exhibit very little homology,
except for a short consensus sequence (the sialyl motif) to which the
activated sugar donor is thought to bind (28, 53). Among
these ST,
-galactoside
-2,6-ST (EC 2.4.99.1), which forms the
terminal NANA
2,6Gal
1-4GlcNAc sequence on the carbohydrate
side chain of glycoproteins, was the first to be purified from rat
liver (60). The corresponding cDNA has since been cloned
(61), allowing molecular analysis of tissue-specific
expression of ST mRNA (34, 36) and its regulation by
glucocorticoids and cytokines (57).
In the colon of the rat, mucus glycoprotein contains carbohydrate
chains that terminate in sialic acid attached by -2,3 and
-2,6
linkages (12, 48). However, the adult rat colon contains less
-2,6-ST activity than
-2,3-ST activity, and the
-2,6-ST activity is more predominant in the small intestine. Furthermore, the
-2,6-transferase activity in the small intestine decreases from
birth to weaning in the developing rat (12). The amount of
mRNA and protein of this
-2,6-ST likewise decrease in the small
intestine during rat development, with the most pronounced decrease
occurring first in the distal small intestine (55). Administration of hydrocortisone in vivo results in a reduction of ST
mRNA in immature rat small intestine (18) but causes
upregulation in vitro in organ culture (19), indicating
that regulation of ST can be mediated by corticosteroids
(18). This decrease in ST is accompanied by a reciprocal
increase in fucosyltransferase in rats, indicating an inverse
relationship in the activities of the two principal enzymes involved in
glycosylating the nonreducing termini of the glycans of rat intestinal
glycoprotein (9, 52). This inversion of enzyme activities
corresponds to a shift from sialylation to fucosylation of intestinal
membrane glycoconjugates (50, 52). These changes in
glycosylation may explain the differential expression of isoforms of
membrane glycoproteins during postnatal gut development. For example,
alkaline phosphatase (56) and
-glutamyltransferase
(25) both have sialic acid-rich immature isoforms and
sialic acid-poor adult isoforms. Similarly, lactase has a sialic-rich
neonatal isoform and a fucose-rich adult isoform (6).
These observations support the hypothesis that intestinal maturation
involves developmental control of membrane glycoconjugates. Their
glycosylation is regulated primarily by the altered expression of
specific glycosyltransferases, possibly mediated by modified enzyme
gene transcription, activity, and stability. The shift in glycosylation
from terminal sialic acid to terminal fucose may also relate to
differential susceptibility to enteric pathogens in immature mammals.
The expression of sialic acid in cell surface glycoconjugates of mouse
intestine is somewhat different from that of the rat, and its
developmental expression and control of expression have not been well
defined. Genetic deletion of specific glycosyltransferases in mice has
been reported (14, 27, 29). Mice homozygous for the
1,4-galactosyltransferase deletion die during the third postnatal
week. In wild-type mice, this period coincides with dramatic increases
of the enzyme in the small intestine and changes in the brush-border
membrane glycoproteins (9, 11); these changes coincide
with a shift in microflora. The inability of the galactosyltransferase
knockout mouse to effect these changes in gut physiology could
contribute toward lethality in
1,4-galactosyltransferase mutant
mice. Inactivation of fucosyltransferase (Fuc-TVII) is not lethal in
mice, but the mice do respond inappropriately to experimental colitis
of the gut (3, 14).
Thus intestinal glycosyltransferases may be central to the normal
development of the gut and can participate in enteric pathophysiology. In addition, the documentation of normal development of ST provides an
important basis for understanding the microbial-epithelial interaction
in the developing mouse intestine. To better understand the regulation
of regional specificity of intestinal sialylation during intestinal
development, we quantitatively studied the expression of -2,6-ST
enzyme activity, its mRNA accumulation, and microvillus membrane (MVM)
sialylation in the mouse intestine from duodenum to colon during
postnatal development. The effect of glucocorticoids, known trophic
factors that promote precocious gut maturation, on
2,6-ST expression
in the suckling mouse was also investigated using cortisone acetate (CA).
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MATERIALS AND METHODS |
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Reagents.
Fetuin, submaxillary mucin, 1-acid glycoprotein,
neuraminidase (Clostridium perfringens), BSA,
2-mercaptoethanol, OCT, phenylmethylsulfonyl fluoride (PMSF), and
ultra-pure sucrose were purchased from Sigma (St. Louis, MO).
CMP-N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid
(CMP-[14C]NeuAc; 0.1 µCi, sp act, 1.8 mCi/mmol) was
purchased from New England Nuclear Life Sciences (Boston, MA). TaqMan
reverse transcription reagents and TaqMan Gold RT-PCR kits were
purchased from Perkin-Elmer (San Ramone, CA). Streptavidin-horseradish
peroxidase conjugate was purchased from Amersham Life Sciences
(Piscataway, NJ). Biotinylated Sambucus nigra agglutinin
(SNA), fluorescein-conjugated SNA, and Maackia amurensis II
were purchased from Vector Laboratories (Burlington, CA). Control
glycoprotein transferrin was purchased from Roche Diagnostics
(Indianapolis, IN). CA was from Merck, Sharp and Dohme (West Point,
PA). All other reagents were of analytic or molecular biology grade
from Fisher Biotech (Pittsburgh, PA) or Sigma.
Animals. Timed-pregnant dams of Black Swiss mice were purchased from Taconic Farms (Germantown, NY) at 16-18 days of gestation. All dams were housed individually in opaque polystyrene cages. The dams gave birth in our animal facility with a 12:12-h light-dark cycle and were fed mouse chow and water ad libitum. On the due date, cages were checked every 4 h for the presence of pups. The date of birth of the pups was designated as day 0. The following day (day 1), litters were reduced to 9 pups per dam. To avoid circadian influences, all pups were killed between 1200 and 1400.
Cortisone treatment. Each litter of suckling mice was divided into two groups. At 10 days of age, one group was injected subcutaneously with a single dose (5 mg/100 g body wt) of CA in a saline suspension. The other control group was injected with the same volume of normal saline (0.9% NaCl). The animals were maintained with their dams until they were killed at 14 days of age.
Preparation of the microsomal fraction.
Animals were killed by cervical dislocation, and the entire small
intestine and whole colon were removed and thoroughly flushed with
ice-cold 0.9% NaCl. The small intestine was divided into the duodenum,
jejunum, and ileum. The small intestine from the stomach to the
ligament of Treitz was defined as the duodenum. The proximal and distal
halves of the remaining small intestine were defined as the jejunum and
ileum, respectively. Intestine samples were placed on a glass plate
maintained at 4°C and cut open; mucosa was harvested by scraping with
a microscope glass slide. All subsequent procedures were performed at
4°C. A 10% mucosal homogenate in 0.1 M Tris · HCl buffer (pH
7.4) was centrifuged at 1,000 g for 15 min to remove nuclei
and cellular debris. The supernatant was then centrifuged at 105,000 g for 1 h in a Beckman L6-65 ultracentrifuge,
producing a microsomal pellet (9, 52). Enzyme activities
were determined from this fraction (reconstituted in the same buffer),
which was either stored at 80°C or used immediately for the enzyme assay.
Protein determination. Protein was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL) modified for use in 96-well microtiter plates as specified by the supplier. Absorbance was measured at 560 nm (BT 2000 microkinetics reader spectrophotometer, Fisher). The protein concentration of each sample was calculated using a BSA standard curve.
ST assay.
Asialofetuin, asialo bovine submaxillary mucin, and asialo
1-acid glycoprotein were prepared from their sialylated
parent compounds by incubation with neuraminidase (1 U/10 mg
glycoprotein) at 37°C, pH 5.0, in 100 mM acetate buffer in 150 mM
NaCl for 8 h followed by boiling for 15 min. Combined
-2,3-ST and
-2,6-ST activity on both N- and O-linked
glycans was assayed using asialofetuin as an exogenous acceptor.
Because of the ability of this acceptor to measure all of these
activities, this acceptor was used to monitor changes in ST activity in
specific regions of intestine as a function of time. To determine the
relative contribution of each of the ST activities to the change in
general activity, individual ST activities were measured in selected
tissues through the use of specific substrates. Asialo submaxillary
mucin was the receptor used to measure the addition of sialic acid by
-2,3 and
-2,6 linkages to O-linked glycans
(mucin-type). Asialo
1-acid glycoprotein was used to
measure the addition of sialic acid by
-2,3 and
-2,6 linkages to
N-linked glycans, with a preference for
-2,6 linkages
(37,61). Lacto-N-tetraose was used to measure the activity that catalyzes the addition of sialic acid by
-2,3 linkages to N-linked glycans, whereas
N-acetyllactosamine was used to measure the activity in
which addition of sialic acid by
-2,6 linkages to
N-linked glycans occurs (37, 61).
RNA isolation.
Tissue samples were dissolved in GIT buffer (20 vol; 4 M
guanidine-isothiocyanate, 50 mM Tris, pH 7.6, 2% sarkosyl, and 100 mM
2-mercaptoethanol). RNA was deproteinized by extraction with phenol,
chloroform, and isoamyl alcohol and stored at 20°C
(8). Purity of the RNA was determined by the ratio of
absorbances at 260 to 280 nm, and the concentration was determined from
optical density at 260 nm.
Quantitative real-time RT-PCR assays.
PCR primers and TaqMan probes for -2,6-ST were designed using
application-based primer design software (Primer, Perkin-Elmer) according to the published mouse
-2,6-ST cDNA sequence (GenBank accession no. D16106) (17). Primer and probe sequences
were as follows: forward, 5'-CGGGACCAGGAGTCAGGTT-3'; reverse,
5'-ACATTCACGTGGTCTCGAAGG-3'; and probe,
5'-AGCGTAGAAGGCCTGCGCTGCC-3'. The TaqMan probe was labeled with a
reporter fluorescent dye, 6-carboxyfluorescein, at the 5' end and a
fluorescent dye quencher, 6-carboxytetramethyl-rhodamine, at the 3'
end. PCR primers and a TaqMan probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Perkin-Elmer.
Preparation of MVM.
Mucosal scrapings were homogenized in 500 mM mannitol, 10 mM HEPES, 5 mM EGTA, and 1 mM PMSF (pH 7.4), for 5 min, diluted with ice-cold
distilled water (1:5), and filtered under suction through fine nylon
mesh (mesh size, 40 µm; Tetko, Elmsford, NY). MgCl2 (1 M)
was added to the crude homogenate (with stirring) to a final
concentration of 10 mM, and the suspension was allowed to stand for an
additional 15 min. The homogenate was then centrifuged at 5,000 g for 15 min. The pellet was discarded, and the supernatant was centrifuged at 28,000 g for 30 min. The resulting pellet
was suspended and homogenized in 100 mM mannitol, 10 mM HEPES, 5 mM EGTA, and 1 mM PMSF (pH 7.4) and centrifuged again at 28,000 g for 30 min. This purified MVM pellet was suspended and
homogenized in 10 mM Tris · HCl and 1 mM PMSF (pH 7.4) and
stored at 80°C (6, 9, 31). The above procedures were
all performed at 4°C.
Slot-blot analysis with SNA lectin.
MVM protein (25 µg) was loaded onto a 0.45-µm nitrocellulose
membrane with the use of the Bio-Dot SF microfiltration apparatus (Bio-Rad, Hercules, CA). Sialylglycoconjugates were detected on the
blot using biotin-conjugated SNA lectin (Vector Laboratories, Burlingame, CA). SNA recognizes both sialic acid -2,6-galactose moieties and sialic acid
-2,6-galactosamine moieties
(46). The slot blot was blocked with solution containing
1% BSA in TTBS (0.05 mM Tris · HCl, 0.15 M NaCl, 1 mM
MgCl2, and 1 mM CaCl2, pH 7.5) at room
temperature for 1 h and washed twice for 10 min in TTBS. The
membrane was subsequently probed with biotin-SNA (0.1 µg/ml) for
1 h, washed three times for 10 min each with TTBS, then incubated
with streptavidin-horseradish peroxidase conjugate in the blocking
solution (1:5,000 dilution) for 1 h. After washing the blot three
times for 10 min each in TTBS, the blot was processed with SuperSignal
West Pico chemiluminescent substrate (Pierce, Rockford, IL) and exposed
to enhanced chemiluminescence Hyperfilm (Amersham, Piscataway, NJ).
Densitometric analysis of the sample bands was performed with National
Institutes of Health image analysis software. The amount of sialyl
-2,6 glycoconjugate in each sample was expressed in densitometry
units. Transferrin was loaded and used as a positive control.
Lectin-fluorescent staining with SNA.
Analysis of -2,6 sialyl glycoconjugates was performed on frozen
tissue sections using FITC-conjugated SNA (Vector Laboratories). Staining for
-2,3-sialylglycoconjugates was performed using
Maackia amurensis lectin II (MAA II; Vector Laboratories).
The middle 1 cm of tissues from each region of the gut was fixed for
2 h in 4% paraformaldehyde at 4°C, washed in ice-cold PBS
containing 30% sucrose overnight at 4°C, and embedded in OCT. Frozen
sections (6-7 µm thick) were blocked with PBS containing 2% BSA
and then stained with labeled lectin for 1 h (10 µg/ml).
Sections were then washed three times in cold PBS, mounted using
Anti-Fade (Vector Laboratories), and analyzed by confocal microscopy.
Statistical analysis. Results are means ± SE. Effects of age and treatment were analyzed by two-way ANOVA. After overall significance was confirmed, post hoc tests for individual variables were performed by a two-tailed unpaired t-test. Differences with P < 0.05 were considered significant.
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RESULTS |
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Age-dependent and region-specific changes in intestinal ST
activity.
To determine if there was a regional specificity in the developmental
expression of ST, enzyme activity was measured in the duodenum,
jejunum, ileum, and colon of mice from postnatal days 1 to
42 using asialofetuin as the acceptor, as it has broad
specificity. The results (Fig. 1) showed
that the highest ST activity was detected on day 1 in all
regions, with a proximal-to-distal increasing gradient with the lowest
activity in the duodenum (P < 0.01). During the first
3 wk (days 1 to 21), enzyme activities decreased to adult levels, with significant 1.4-, 3.4-, and 9.7-fold reductions recorded in the duodenum (P < 0.01), jejunum
(P < 0.001), and ileum (P < 0.001),
respectively. No developmental change was observed in the colon. Loss
of activity was gradual in all tissues of the small intestine from
days 1 to 21 and was not restricted to abrupt changes during the weaning period.
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Relative distribution of -2,3-ST and
-2,6-ST in mouse
intestine.
Enzymatic sialylation of asialofetuin was the measure of
2,3/6-ST,
the combined activity of
-2,3- and
-2,6-ST activities for both
N- and O-linked glycans. To investigate the
relative contribution of the specific ST activities to the above
changes in general ST activity, we used specific acceptors: asialo
submaxillary mucin (O-linked ST activity), asialo
1-acid glycoprotein (N-linked ST activity,
preference for
-2,6 linked), N-acetyllactosamine (N-linked
-2,6-ST activity), and
lacto-N-tetraose (N-linked
-2,3-ST activity)
to measure the specific ST activities in immature (2 wk old) and mature
(4 wk old) ileum and colon from the same mice as described above. The
results are shown in Fig. 2. In the
ileum, 80% of the ST activity was N-linked (Fig.
2B) rather than O-linked (Fig. 2C).
Most of the N-linked activity was accounted for by
-2,6-ST activity (Fig. 2D) with minimal contributions by
-2,3-linked ST (Fig. 2E). In the colon, there was an
appreciable contribution by N-linked ST activity (Fig.
2B), but there was a much higher relative contribution by
O-linked ST activity (Fig. 2C). Note that the
contribution by O-linked ST increases during colonic development whereas the contribution by N-linked ST
decreases in the colon during development, similar to
N-linked ST in the small intestine. As a result of these
opposing trends, the combined N- and O-linked
activity for colon (Fig. 2A) shows no significant change
with development. Furthermore, in the colon, unlike the small
intestine, there is a significant contribution to the
N-linked activity by the
-2,3-ST (Fig. 2E),
although the change in activity during development is again due largely
to the
-2,6-ST activity. These data are consistent with mRNA of all
-2,3-ST genes responsible for
-2,3-ST activities that are not
altered in the developing colon (24). These data
strongly support the conclusion that the loss of
-2,6-ST activity
during maturation of the small intestine in the mouse is primarily due
to a loss of
-2,6-ST activity specific for N-linked
glycans. There also seems to be a loss of this enzyme activity during
maturation of the colon that may be somewhat offset by an increase in
ST activity specific for O-linked glycans strongly expressed
in goblet cells.
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Age-dependent and region-specific changes in intestinal -2,6-ST
mRNA levels.
To investigate possible control mechanisms for the above changes in
-2,6-ST activity, we examined developmental patterns of
-2,6-ST
mRNA accumulation. Of the three known genes whose products have
-2,6-ST activity specific for N-linked glycans, two are
known to be expressed in the small intestine. Quantitative RT-PCR was
applied to total RNA prepared from the duodenum, jejunum, ileum, and
colon from 2-, 4-, and 6-wk-old mice. The
-2,6-ST mRNA levels of one
of these two intestinal genes (24, 51) were expressed in a
pattern similar to that for enzyme activity, with a proximal-to-distal
increase in mRNA accumulation (Fig. 3). A
developmental decline in mRNA accumulation was observed in the
duodenum, jejunum, and ileum, but not in the colon. In the colon, loss
of specific N-linked
-2,6-ST activity may be partly
related to undetermined posttranslational modification or differential
promoter utilization. Despite this decline in mRNA levels, a similar
regional gradient was maintained in all ages examined in the mouse
small intestine.
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Effect of exogenous glucocorticoid on -2,6-ST mRNA accumulation
and enzyme activity in suckling mouse intestine.
Injection of CA on day 10 significantly accelerated the
developmental decline of
-2,6-ST activity by day 14 in
duodenum (2.5-fold), jejunum (2.3-fold), and ileum (13.5-fold) (Fig.
4A; P < 0.01). CA did not induce significant changes in the colon. The levels of
-2,6-ST mRNA accumulation after CA treatment paralleled
-2,6-ST activity (Fig. 4B). To confirm the general
maturational effect of CA in the developing small intestine, we
measured sucrase activity, which is a sensitive enzyme marker for small
intestinal development (31), in the same tissue extracts.
CA treatment significantly induced sucrase activity in the jejunum
(5.64 ±0.2 vs. 0.12 ± 0.05 µmol · h
1 · mg protein
1;
P < 0.001).
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SNA lectin staining to identify developmental and region-specific
intestinal sites of sialylated glycoconjugate expression.
To determine qualitative changes in the location of 2,6-linked
sialoglycosylation in tissue sections, SNA lectin fluorophore labeling
with visualization by Nomarski imaging was applied to intestinal
tissues (Fig. 5). Representative sections
of immature (day 1) ileum (Fig. 5, A,
B, E, and F) and colon (Fig. 5,
C, D, G, and H) compared
with 4-wk-old ileum (Fig. 5, I and J) and colon (Fig. 5, K and L) are depicted. These images show
that in immature 1-day-old gut,
2,6-linked sialglycosylation is
abundant and principally located in mucus-producing goblet cells in the
small and large bowel (Fig. 5, F and H). Weak
labeling was also observed in the mesenchyme and brush-border membrane.
A marked loss of staining was observed in the small intestine during
development, the most pronounced feature being an absence of
sialoglycosylation in the goblet cell population. This change was not
apparent in the colon where goblet cells were still labeled strongly
with FITC-conjugated SNA after 4 wk (Fig. 5L). CA treatment
appeared to precociously diminish the level of lectin labeling in the
small intestine, but not in the large intestine where goblet cells
still expressed sialyl
-2,6 glycoconjugates (data not shown).
Staining for
-2,3-sialylglycoconjugates was performed using MAA II
in 1-day-, 2-wk-, and 4-wk-old ileum and colon (data not shown). In
contrast with SNA staining, MAA II staining is not developmentally
altered and exhibits uniform labeling of crypt, villus, and colonic
cuff epithelium in all tissue. The goblet cell mucins were not labeled
with MAA II lectin staining. This data is consistent with the
N-linked specific
-2,3-ST activity (Fig. 2E).
These data suggest that although the developmental decline in
-2,6-linked sialylglycosylation was found in a number of different
cell types and extracellular matrix components throughout the
intestinal mucosa, the most prominent loss of expression in the small
intestine was in the goblet cell population.
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Sialyl 2,6-linked glycoconjugate expression in MVM during
development and after cortisone treatment of suckling mice.
Measurements of
-2,6-ST enzyme activity and mRNA accumulation in
mucosal scrapings reflect the combined changes in goblet cell and
mesenchymal expression, as well as expression in MVM. Because
2,6-linked sialylglycoproteins in the MVM of intestinal absorptive
epithelial cells constitute potential bacterial binding sites for
components of the gut microflora, we examined MVM from all the tissues
to measure any developmental and region-specific alterations in
sialylglycoconjugate content. A proximal-to-distal increase in
brush-border sialoglycoconjugate content was observed in all age groups
examined (Fig. 6), consistent with data
on whole mucosal scrapings. Likewise, a developmental decrease in
sialylglycoprotein content was observed in the small intestine and, in
contrast to the finding on whole scrapings, the colon. Similarly, these
developmental declines of
2,6-linked sialylglycoprotein in the MVM
fraction were accelerated by CA treatment in all tissues examined
(P < 0.001). Even though
-2,6-ST mRNA levels and
activity were not altered in the colon during development or by CA
(Figs. 1-3),
-2,6-linked sialylglycoprotein was significantly
reduced from 2 to 4 wk of age (P < 0.05). These age
and regional differences in
2,6-linked sialylated glycoconjugate
patterns may be due to changes in
-2,6-ST enzyme activity as well as
the availability and the turnover rate of MVM glycoproteins that
function as substrates for
-2,6-ST activity.
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DISCUSSION |
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This study provides the first evidence in the mouse of distinct
developmental and region-specific control of -galactoside
-2,6-ST
(N-linked
-2,6-ST) expression in the small and large intestine. In all tissues examined, regulation of combined ST (N- and O-linked
-2,6-ST) activity reflected
an anterior-to-posterior gradient, with the highest enzyme activity
recorded in the colon. In the small intestine, most of the ST activity
could be attributed to N-linked
-2,6-ST activity that
diminished during development. This decrease in N-linked
-2,6-ST activity was most likely regulated at the transcriptional
level. In contrast, developmental diminution of colonic
N-linked
-2,6-ST activity was not associated with a
similar alteration in mRNA accumulation, indicating possible posttranscriptional modification (34), differential
expression other ST genes (24, 36, 51, 53, 59), and/or
alternate promoter utilization (36, 58). However, a
clearer understanding of region-specific regulatory events of all
-2,3/6-ST genes is beyond the scope of this work. Importantly,
although a loss of N-linked
2,6 ST activity is evident
during development of the large bowel, nonspecific ST activity is not
altered. This is principally due to a developmental and tissue-specific
increase of O-linked
-2,3/6-ST activities most likely
being expressed in colonic goblet cell populations. However, further
investigation is required to examine whether measurement of cell
lineage-specific
-2,6-ST expression accurately reflects
developmental changes that are apparent in specific cell lineages such
as colonocytes. Exogenous glucocorticoid treatment of these mice
precociously induced gut maturation, as evidenced by a 47-fold increase
in intestinal sucrase activity, a classical indicator of development
(31). Treatment with CA in the small intestine
precociously accelerated the postnatal decline of
-2,6-ST expression
at the mRNA level, suggesting that regulation of
-2,6-ST represents
an important component of gut maturation. These changes partly coincide
with weaning, suggesting that luminal factors present in maternal milk
and the establishment of microflora in the gut may also contribute to
this developmental decline of
-2,6-ST expression.
Antibodies specific to -2,6-ST have demonstrated that this enzyme is
expressed predominantly in the intestinal epithelium in the rat
(55). In the mouse, we found that the glycosylation product of
-2,6-ST is also abundantly expressed in the epithelium, especially in goblet cells. This pattern of sialylation demonstrated a
clear regional specificity between the small and large bowel, the
latter site maintaining high levels of
-2,6-ST expression. In
contrast, MVM preparations isolated from colonocytes showed sialoglycoconjugates decreasing with maturation, indicating a possible
differential regulation of
-2,6-ST activity in absorptive and goblet
cells. The overall developmental decline of
-2,6-ST expression,
which seems to be primarily
-2,6-ST in the small intestine, may
likely be due to a combination of regulation in goblet cells and in enterocytes.
In contrast to the developmental decline of the IgG-Fc receptor
expression, which shows a high proximal-to-low distal gradient in
suckling rats (40, 47) or ileal bile acid binding and
absorbing proteins (42, 21) that are only expressed in the
distal small intestine during the third postnatal week, -2,6-ST
expression developed an increasing gradient along the cephalo-caudal
(anterior-posterior) axis of the gut. The developmental decline of
-2,6-ST activity in the small intestine is most likely controlled at
the transcriptional level, similar to the postweaning rise of sucrase
and trehalase activity (31, 33). The developmental decline
of IgG-Fc receptor expression in rat small intestine also relates to
its mRNA level (47) and in a similar manner to
-2,6-ST
expression precedes the weaning period. This differs markedly from the
postweaning decline of lactase activity where both transcriptional and
posttranscriptional processes are apparent (6, 30). These
new findings support previous suggestions (21, 31, 42)
that different, region-specific factors are involved in establishing
cephalocaudal gradients of gene expression in the developing small intestine.
In vitro studies on the mechanism of -2,6-ST control support a role
for transcriptional regulation of rat
-2,6-ST activity (49,
59). With the use of a hepatic cell line (H35 cells) and primary
hepatocytes, glucocorticoid treatment induced increased expression of
-2,6-ST via transcriptional control and not by altering mRNA
stability. Unlike hepatocytes, in the small intestine, steroids promote
a decline of
-2,6-ST expression. A number of other enzymes involved
in gluconeogenesis (e.g., phosphoenolpyruvate carboxykinase) are
induced by glucocorticoids in the liver but are repressed by the same
treatment in the small intestine (2, 16, 64). In contrast
to our in vivo data, Kolinska et al. (23) observed an
upregulation of
-2,6-ST mRNA levels and enzyme activity after
dexamethasone treatment of 7-day-old rat jejunum in organ culture
(18, 22). However, limitations in organ culture techniques
prevent elucidation of the precise control mechanism of
-2,6-ST
regulation. Also, distinct promoter utilization has been demonstrated
for
-2,6-ST expression in the liver and kidney (58),
but it is not known whether intestinal expression uses a comparable
hepatic promoter or a unique promoter specific for the
-2,6-ST gene.
However, availability of the genomic DNA clones for mouse and rat
-2,6-ST (17, 59) should help to delineate the
mechanisms of developmental and glucocorticoid-mediated regulation of
-2,6-ST expression in the small intestine, which is beyond the scope
of the current study.
The -2,6-ST is a membrane-bound protein, located almost exclusively
in the trans-Golgi network, whose catalytic domain faces into the lumen
of the Golgi cisternae. It is responsible for the addition of terminal
sialic acid residues on diverse membrane glycoproteins and glycolipids
and on secreted mucins. Sialylated N- and
O-glycans are important glycosylation products that regulate tissue development and disease processes. For example, appropriate cell-to-cell interactions during the development of the central nervous
system require that the neural cell adhesion molecule be highly
sialylated during embryogenesis (41). Aberrant sialylation may also adversely modify cellular-matrix interactions. Both
cell-to-cell and epithelial-matrix interactions have been implicated in
the development of the intestine (30), but developmental
changes in ST and its role in intestinal ontogenesis have not been
systematically evaluated. In extreme cases, aberrant sialylation may
promote metastasis of colorectal carcinomas (4). Enhanced
sialylation is also associated with c-Ha-ras oncogene
transformation, mediated by an increased gene expression and enzyme
activity of
-2,6-ST (28). Evidence is also mounting
that the product of
-2,6-ST activity is a necessary component of a
recognition epitope required for homotypic B cell interactions
(38). Moreover, sialylglycoproteins can also contain a
critical component of an epitope for cellular recognition such as the
sialyl-Lewis structures recognized by selectins (7, 27).
The presence of a highly sialylated intestinal surface at the time of birth may directly influence the binding of gut microflora on the epithelial surface of the small and large intestine. Attachment sites for several organisms may be masked by sialic acids, whereas other pathogenic and nonpathogenic bacterial strains secrete sialidase enzymes that enable them to overcome host defensive mechanisms and create novel binding sites for colonization (10). In contrast, many pathogenic bacteria and viruses recognize specific sialylated glycan structures as their adhesion receptors (11, 54, 63). For example, enterotoxigenic Escherichia coli K99 causes diarrhea in newborn piglets, calves, and lambs by expressing K99 fimbrial adhesins that bind to sialylated glycoproteins and glycolipids (44). N-glycolylneuraminyl-lactosyl-ceramide (NeuGc-GM3) has been identified as a major receptor for K99. The membrane content of NeuGc-GM3 is highest in newborn pigs and gradually decreases during development, as does the susceptibility to E. coli K99 enteritis (64). The regional and temporal variation in the expression of sialylated cell surface glycoconjugate receptors may explain the regional and temporal differences in susceptibility to pathogens that exist within a species, just as differential expression among species accounts for the species specificity of pathogens. Furthermore, developmental regulation of sialylation may contribute toward specificity of intestinal colonization.
Environmental factors may also regulate ST activity. Bacterial
colonization and its fermentation products such as short-chain fatty
acids (SCFA) may provide a direct luminal signal to switch off
-2,6-ST activity and mRNA. A major constituent of SCFA,
n-butyrate, is also a major fermentation product of
intestinal microflora and reduces up to 90% of
-2,6-ST mRNA in the
human hepatoma cell line Hep G2 (45). A dose- and
time-dependent effect of n-butyrate on glycosyltransferase
mRNA in T84 (human colonic adenocarcinoma) cells was demonstrated
(26) in which n-butyrate (3-5 mM) caused approximately an 80% inhibition of
-2,6-ST mRNA accumulation. The
effect on
-2,6-ST was near maximum by 6 h (26).
However, SCFA are mostly concentrated in the colon and substantially
lower levels are found in the proximal small intestine
(5), although it is feasible that these levels could
elevate significantly as a result of bacterial overgrowth in the small
bowel during various pathophysiological conditions. While it remains
possible that
-2,6-ST repression in adult enterocytes is mediated in
part by the n-butyrate pathway, the relative contribution of
this mechanism to the regulation of glycosyltransferase expression in
vivo remains to be investigated. Therefore, SCFA, fermentation
byproducts of microflora in the animals, might be one contributing
luminal factor in the developmental decline of
-2,6-ST in the mouse
gut. Their role in secondary regulation of ST activity may be examined
further under germ-free conditions.
Our findings in the developing mouse intestine agree with prior
observations on developing rat intestine with regard to the drop in
-2,3/6-ST in small intestine from birth to weaning, the premature
drop in activity with corticosteroid treatment, the persistence of the
activity in colon, and the proximal-to-distal differences in activity
in the gut. In the mouse, the
-2,6-ST activity predominates
over the
-2,3-ST activity in the small intestine; however, in the
mouse, the
-2,6-ST activity also comprises a significant portion of
the activity of the colon (12). These studies suggest that
such phenomena are general to mammals and that similar studies in
developing human intestine may produce information that will be helpful
in understanding normal functions in the gut, such as colonization, as
well as pathological enteric processes.
In the present study, we demonstrate that intestinal -2,6-ST mRNA
accumulation, enzyme activity, and the abundance of
-2,6-linked sialic acid residues on MVM glycoproteins are under developmental and
region-specific regulation and can be modified by cortisone treatment.
These results support the hypothesis that changes in the sialic acid
content in intestinal membrane glycoproteins are primarily due to
altered
-2,6-ST gene expression and that transcription may be
regulated by region-specific factors modulated by glucocorticoids.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grants HD-12437, HD-31852, AI-434472, HD-13021, PO1-DK-33506, and P30-DK-40561. D. Dai was supported by Forgarty Foundation/National Institute of Child Health and Human Development Training Grant D43-TW-01265 and by a grant from Wyeth Nutritionals International.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: W. A. Walker, Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 114 16th St. (114-3510), Charlestown, MA 02129 (E-mail: walker{at}helix.mgh.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00531.2000
Received 20 December 2000; accepted in final form 7 November 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Asano, M,
Furukawa K,
Kido M,
Matsumoto S,
Umesaki Y,
Kochibe N,
and
Iwakura Y.
Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells.
EMBO J
16:
1850-7,
1997
2.
Beale, EG,
Clouthier DE,
and
Hammer RE.
Cell-specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice.
FASEB J
15:
3330-3337,
1992.
3.
Beck, PL,
Xavier R,
Lu N,
Nanthakumar NN,
Dinauer M,
Podolsky DK,
and
Seed B.
Mechanisms of NSAID-induced gastrointestinal injury defined using mutant mice.
Gastroenterology
119:
699-705,
2000[ISI][Medline].
4.
Bresalier, RS,
Rockwell RW,
Dahiya R,
Duh QY,
and
Kim YS.
Cell surface sialoglycoprotein alterations in metastatic murine colon cancer cell lines selected in an animal model for colon cancer metastasis.
Cancer Res
50:
1299-1307,
1990[Abstract].
5.
Bugaut, M.
Occurrence, absorption, and metabolism of short chain fatty acids in the digestive tract of mammals.
Comp Biochem Physiol A Physiol
86:
439-472,
1987[ISI].
6.
Buller, HA,
Rings EH,
Pajkrt D,
Montgomery RK,
and
Grand RJ.
Glycosylation of lactase-phlorizin hydrolase in rat intestine during development.
Gastroenterology
98:
667-675,
1990[ISI][Medline].
7.
Butcher, EC.
Leukocyte-endothelial cell recognition: three or more steps to specificity and diversity.
Cell
67:
1033-1036,
1991[ISI][Medline].
8.
Chirgwin, JM,
Przybyla AE,
MacDonald RJ,
and
Rutter WJ.
Isolation of biologically-active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[ISI][Medline].
9.
Chu, SW,
and
Walker WA.
Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine.
Biochim Biophys Acta
883:
496-500,
1986[ISI][Medline].
10.
Corfield, T.
Bacterial sialidases: roles in pathogenicity and nutrition.
Glycobiology
2:
509-521,
1992[ISI][Medline].
11.
Dai, D,
Nanthkumar NN,
Newburg DS,
and
Walker WA.
Role of oligosaccharides and glycoconjugates in intestinal host defense.
J Pediatr Gastroenterol Nutr
30 Suppl:
S23-S33,
2000[ISI][Medline].
12.
Dall'Olio, F,
Malagolini N,
Di Stefano G,
Ciambella M,
and
Serafini-Cessi F.
Postnatal development of rat colon epithelial cells is associated with changes in the expression of the beta 1,4-N- acetylgalactosaminyltransferase involved in the synthesis of Sda antigen of alpha 2,6-sialyltransferase activity towards N-acetyl-lactosamine.
Biochem J
270:
519-524,
1990[ISI][Medline].
13.
Fuchs, G,
Mobassaleh M,
Donohue-Rolfe A,
Montgomery RK,
Grand RJ,
and
Keusch GT.
Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin.
Infect Immun
53:
372-377,
1986[ISI][Medline].
14.
Furukawa, K,
Takamiya K,
Okada M,
Inoue M,
Fukumoto S,
and
Furukawa K.
Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes.
Biochim Biophys Acta
1525:
1-12,
2001[ISI][Medline].
15.
Gibson, UEM,
Heid CA,
and
Williams PM.
A novel method for real time quantitative RT-PCR.
Genome Res
6:
995-1001,
1996[Abstract].
16.
Girard, J,
Perdereau D,
Narkewicz M,
Coupe C,
Ferre P,
Decaux JF,
and
Bossard P.
Hormonal regulation of liver phosphoenolpyruvate carboxykinase and glucokinase gene expression at weaning in the rat.
Biochimie
73:
71-76,
1991[ISI][Medline].
17.
Hamamoto, T,
Kawasaki M,
Kurosawa N,
Nakaoka T,
Lee YC,
and
Tsuji S.
Two steps single primer mediated polymerase chain reaction: application to cloning of putative mouse -galactoside
2,6-sialyltransferase cDNA.
Bioorg Med Chem
1:
141-145,
1993[Medline].
18.
Hamr, A,
Delannoy P,
Verbert A,
and
Kolinska J.
The hydrocortisone-induced transcriptional down-regulation of -galactoside
2,6-sialyltransferase in the small intestine of suckling rats is suppressed by mifepristone (RU-38.486).
J Steroid Biochem Mol Biol
60:
59-66,
1997[ISI][Medline].
19.
Hamr, A,
Vlasakova V,
and
Kolinska J.
2,6-Sialyltransferase predominates in cultured jejunum of suckling rats: it is up-regulated by dexamethasone and secreted during cultivation.
Biochim Biophys Acta
1157:
285-289,
1993[ISI][Medline].
20.
Harduin-Lepers, A,
Recchi MA,
and
Delannoy P.
1994, the year of sialyltransferases.
Glycobiology
5:
741-758,
1995[ISI][Medline].
21.
Hwang, ST,
and
Henning SJ.
Hormonal regulation of expression of ileal bile acid binding protein in suckling rats.
Am J Physiol Regulatory Integrative Comp Physiol
278:
R1555-R1563,
2000
22.
Kolinska, J,
Baudysova M,
Zakostelecka M,
Kraml J,
and
Kadlecova L.
Regulation of sialylation of intestinal brush-border enzymes and of sialyltranferase activity in organ cultures by dexamethasone.
Biochem Int
22:
495-508,
1990[ISI][Medline].
23.
Kolinska, J,
Zakostelecka M,
Hamr A,
and
Baudysova M.
Coordinate expression of -galactoside
2,6-sialyltransferase mRNA and enzyme activity in suckling rat jejunum cultured in different media: transcriptional induction by dexamethasone.
J Steroid Biochem Mol Biol
58:
289-297,
1996[ISI][Medline].
24.
Kono, M,
Ohyama Y,
Lee YC,
Hamamoto T,
Kojima N,
and
Tsuji S.
Mouse beta-galactoside alpha 2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression.
Glycobiology
7:
469-479,
1997[Abstract].
25.
Kottgen, E,
Reutter E,
and
Gerok W.
Two different gamma-glutamyltransferases during development of liver and small intestine: a fetal and an adult glycoprotein.
Biochem Biophys Res Commun
72:
61-66,
1976[ISI][Medline].
26.
Li, M,
Andersen V,
and
Lance P.
Expression and regulation of glycosyltransferases for N-glycosyl oligosaccharides in fresh human surgical and murine tissues and cultured cell lines.
Clin Sci (Lond)
89:
397-404,
1995[ISI][Medline].
27.
Maly, P,
Thall A,
Petryniak B,
Rogers CE,
Smith PL,
Marks RM,
Kelly RJ,
Gersten KM,
Cheng G,
Saunders TL,
Camper SA,
Camphausen RT,
Sullivan FX,
Isogai Y,
Hindsgaul O,
von Andrian UH,
and
Lowe JB.
The alpha(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis.
Cell
86:
643-653,
1996[ISI][Medline].
28.
Marer, NL,
Laudet V,
Svensson EC,
Cazlaris H,
Van Hille B,
Lagrou C,
Stehelin D,
Montreuil J,
Verbert A,
and
Delannoy P.
The c-Ha-ras oncogene induces increases expression of 2,6-sialyltransferase in rat fibroblast (FR3T3).
Glycobiology
2:
49-56,
1992[Abstract].
29.
Mobassaleh, M,
Koul O,
Mishra K,
McCluer RH,
and
Keusch GT.
Developmentally regulated Gb3 galactosyltransferase and -galactosidase determine Shiga toxin receptors in intestine.
Am J Physiol Gastrointest Liver Physiol
267:
G618-G624,
1994
30.
Montgomery, RK,
Mulberg AE,
and
Grand RJ.
Development of the human gastrointestinal tract: twenty years of progress.
Gastroenterology
116:
702-731,
1999[ISI][Medline].
31.
Nanthakumar, NN,
and
Henning SJ.
Ontogeny of sucrase-isomaltase gene expression in rat intestine: responsiveness to glucocorticoids.
Am J Physiol Gastrointest Liver Physiol
264:
G306-G311,
1993
32.
Neutra, MR,
and
Forstner JF.
Gastrointestinal mucus: synthesis, secretion and function.
In: Physiology of the Gastrointestinal Tract (2nd ed), edited by Johnson LR.. New York: Raven, 1987, p. 975-1009.
33.
Oesterreicher, TJ,
Nanthakumar NN,
Winston JH,
and
Henning SJ.
Rat trehalase: cDNA cloning and mRNA expression in adult rat tissues and during intestinal ontogeny.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R1220-R1227,
1998
34.
O'Hanlon, TP,
Lau KM,
Wang XC,
and
Lau JY.
Tissue-specific expression of beta-galactoside alpha-2,6-sialyltransferase transcript heterogeneity predicts a divergent polypeptide.
J Biol Chem
264:
17389-17394,
1989
35.
Pang, KY,
Bresson JL,
and
Walker WA.
Development of gastrointestinal surface. VIII. Lectin identification of carbohydrate differences.
Am J Physiol Gastrointest Liver Physiol
252:
G685-G691,
1987
36.
Paulson, JC,
Weinstein J,
and
Schauer A.
Tissue-specific expression of sialyltransferase.
J Biol Chem
264:
10931-10934,
1989
37.
Paulson, JC,
Weinstein J,
and
de Souza-e-Silva U.
Identification of a Gal beta 1 goes to 3GlcNAc alpha 2 goes to 3 sialyltransferase in rat liver.
J Biol Chem
257:
4034-4037,
1982
38.
Powell, LD,
Sgroi D,
Sjoberg ER,
Stamenkovic I,
and
Varki A.
Natural ligands of the B cell adhesion molecule CD22b carry N-linked oligosaccharides with 2,6-linked sialic acids that are required for recognition.
J Biol Chem
268:
7019-7027,
1993
39.
Reglero, A,
Rodriguez-Aparicio LB,
and
Lueno JM.
Polysialic acid.
Int J Biochem
25:
1517-1527,
1993[ISI][Medline].
40.
Rodewald, R.
Distribution of immunoglobulin G receptors in the small intestine of young rat.
J Cell Biol
85:
18-32,
1980[Abstract].
41.
Rutishauser, U,
Acheson A,
Hall AK,
Mann DM,
and
Sunshine J.
The neural cell adhesion molecule (N-CAM) as a regulator of cell-cell interactions.
Science
240:
53-57,
1988[ISI][Medline].
42.
Sacchettini, JC,
Hauft SM,
Van Camp SL,
Cistola DP,
and
Gordon JI.
Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium.
J Biol Chem
265:
19199-19207,
1990
43.
Schauer, R.
Chemistry, metabolism, and biological functions of sialic acids.
Adv Carbohydr Chem Biochem
40:
131-234,
1982[ISI][Medline].
44.
Seignole, D,
Mouricout M,
Duval Iflah Y,
Quintard B,
and
Julien R.
Adhesion of K99 fimbriated Escherichia coli to pig intestinal epithelium: correlation of adhesive and non-adhesive phenotypes with the sialoglycolipid content.
J Gen Microbiol
137:
1591-1601,
1991[ISI][Medline].
45.
Shah, S,
Lance P,
Smith TJ,
Berenson CS,
Cohen SA,
Horvath P,
Lau JTY,
and
Baumann H.
n-Butyrate reduces the expression of -galactoside
2,6-sialyltransferase in Hep G2 cells.
J Biol Chem
267:
10652-10658,
1992
46.
Shibuya, N,
Goldstein I,
Broekaert WF,
Nsimba-Lubaki M,
Peeters B,
and
Peumans WJ.
The elderberry (Sambucus nigra L) bark lectin recognizes the Neu5Ac (2,6) Gal/GalNAc sequence.
J Biol Chem
262:
1596-1601,
1987
47.
Simister, NE,
and
Rees AR.
Isolation and characterization of a Fc receptor from neonatal rat small intestine.
Eur J Immunol
15:
733-738,
1985[ISI][Medline].
48.
Slomiany, BL,
Murty VL,
and
Slomiany A.
Isolation and characterization of oligosaccharides from rat colonic mucus glycoprotein.
J Biol Chem
255:
9719-9723,
1980
49.
Svensson, EC,
Soreghan B,
and
Paulson JC.
Organization of the -galactoside
2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation.
J Biol Chem
265:
20863-20868,
1990
50.
Taatjes, DJ,
and
Roth J.
Selective loss of sialic acid from rat small intestinal epithelial cells during postnatal development: demonstration with lectin-gold techniques.
Eur J Cell Biol
53:
255-266,
1990[ISI][Medline].
51.
Takashima, S,
Tachida Y,
Nakagawa T,
Hamamoto T,
and
Tsuji S.
Quantitative analysis of expression of mouse sialyltransferase genes by competitive PCR.
Biochem Biophys Res Commun
260:
23-27,
1999[ISI][Medline].
52.
Torres-Pinedo, R,
and
Mahmood A.
Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine. I. Evidence of a developmental shift from terminal sialylation to fucosylation.
Biochem Biophys Res Commun
125:
546-553,
1984[ISI][Medline].
53.
Tsuji, S.
Molecular cloning and functional analysis of sialyltransferases.
J Biochem (Tokyo)
120:
1-13,
1996[Abstract].
54.
Varki, A.
Biological role of oligosaccharides: all of the theories are correct.
Glycobiology
3:
97-130,
1993[Abstract].
55.
Vertino-bell, A,
Ren J,
Black JD,
and
Lau JY.
Developmental regulation of -galactoside
-2,6-sialyltransferase in small intestinal epithelium.
Dev Biol
165:
126-136,
1994[ISI][Medline].
56.
Vockley, J,
D'Souza MP,
Foster CJ,
and
Hariss H.
Structural analysis of human adult and fetal alkaline phosphatase by cyanogen bromide peptide mapping.
Proc Natl Acad Sci USA
81:
6120-6123,
1984[Abstract].
57.
Wang, XC,
O'Hanlon TP,
and
Lau JTY
Regulation of -galactoside
2,6-sialyltransferase gene expression by dexamethasone.
J Biol Chem
264:
1854-1859,
1989
58.
Wang, XC,
O'Hanlon TP,
Young RF,
and
Lau JTY
Rat -galactoside
2,6-sialyltransferase genomic organization: alternate promoters direct the synthesis of liver and kidney transcripts.
Glycobiology
1:
25-31,
1990[Abstract].
59.
Wang, XC,
Smith TJ,
and
Lau JTY
Transcriptional regulation of the liver -galactoside
2,6-sialyltransferase by glucocorticoids.
J Biol Chem
265:
17849-17853,
1990
60.
Weinstein, J,
de Souza-e-Silva W,
and
Paulson JC.
Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver.
J Biol Chem
257:
13835-13844,
1982
61.
Weinstein, J,
Lee EU,
McEntee K,
Lai PH,
and
Paulson JC.
Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor.
J Biol Chem
262:
17735-17743,
1987
62.
Wiegandt, H.
Glycolipids. Amsterdam: Elsevier Science, 1985, p. 199-260.
63.
Yolken, RH,
Peterson JA,
Vonderfecht SL,
Fouts ET,
Midthun K,
and
Newburg DS.
Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.
J Clin Invest
90:
1984-1991,
1992[ISI][Medline].
64.
Yuyama, Y,
Yoshimatsu K,
Ono E,
Saito M,
and
Naiki M.
Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae.
J Biochem (Tokyo)
113:
488-492,
1993[Abstract].
65.
Zabala, MT,
Lorenzo P,
Alvarez L,
Berlanga JJ,
and
Garcia-Ruiz JP.
Serotonin increases the cAMP concentration and the phosphoenolpyruvate carboxykinase mRNA in rat kidney, small intestine, and liver.
J Cell Physiol
150:
451-455,
1992[ISI][Medline].