47 research outputs found
Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking
We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactosamine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N-and/ or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures {[}Neu5Ac alpha 2-3Gal beta 1-3GalNAc-ol and Neu5Ac alpha 2-3Gal beta 1 -3(Neu5Ac alpha 2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells
Histochemical analysis of carbohydrate moieties and sugar-specific acceptors in the kidneys of the laboratory mouse and the golden spiny mouse (Acomys russatus)
The aims of this work were to histochemically
compare the pattern of lectin binding and
endolectin expression in different portions of nephrons
of two rodent species producing either normal
hyperosmotic urine (the laboratory mouse) or highly
concentrated urine (Acomys russatus, the golden spiny
mouse). A panel of biotinylated lectins and neoglycoproteins
and the avidin-biotin-peroxidase complex
technique were used on Bouin's fixed, paraffinembedded
sections. Various segments of the uriniferous
tubule in both species showed differential affinity for
labelled lectins and neoglycoproteins. Significant
differences were also evident between comparable
tubular segments in laboratory and golden spiny mouse
kidneys. Whether the histochemical expression of sugar
moieties of glycoconjugates as well as endolectins,
thus both sides of presumed protein-carbohydrate
interactions, may be correlated to the various
glycoproteins which would include constituents of the
glycocalyx and domains of a variety of transport
enzymes deserves further studies
Androgen status and expression of glycoconjugates and lectins in the epithelial cells of the mouse ventral prostate. A glycohistochemical approach
We investigated histochemically the
expression of glycans and endogenous sugar-binding
proteins (endolectins) in the ventral prostate of normal
and androgen-deprived mice. The avidin-biotinperoxidase
complex technique was used with a panel of
biotinylated lectins and neoglycoproteins, respectively,
on paraffin sections. Characteristic binding patterns
depending upon the type of lectin were observed in the
glandular epithelium and basement membranes. This
report also initiates the histochemical study of
endogenous glycan-binding proteins in this maleaccessory
sex organ. Remarkably, the endocrine status
was found to affect the distribution of lectin-reactive
determinants of glycoconjugates as well as endolectins.
The histophysiological significance of these
modifications in the glandular cells are discussed in the
light of previous biochemical findings obtained mainly
in humans and the rat
Contribution of carbohydrate histochemistry to glycobiology
The physiological irnportance of
carbohydrate biology has gradually emerged from a lot
of recent information on protein-carbohydrate and
carbohydrate-carbohydrate interaction in normal and
pathological conditions. After considering the
conventional methods which allowed researchers to
differentiate glycan-containing macromolecules from
other complex compounds (nucleic acids, proteins),
selected topics of intracellular and cellular organ
architecture are focused upon in which the use of lectins
and neoglycoproteins as histochemical reagents has
opened new horizons for the localization of glycoconjugates
in situ and the elucidation of their often still
enigmatic functions. The authors hope to place into
perspective that such glycohistochernical studies will
strongly contribute to the progress in the dynamically
growing field of glycobiology
The marriage of chemokines and galectins as functional heterodimers
Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein-protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine-galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena
Lectin-binding sites in the epithelium of normal
By using histochemical methods, the binding
pattern of various lectins in the epithelium of normal
human appendix vermiformis was assessed. In addition
to plant and invertebrate sugar receptors with nominal
monosaccharide specificity for a-L-Fuc (UEA-1), a-DMan
and a-D-Gluc (Con A), a-D-GalNAc (DBA), DGalNAc
(SBA, HPA) B-D-Gal (RCA-I) and D-Gal
(VAA), a mammalian B-galactoside-specific lectin (MW,
14 kDa) was included in the applied panel. The apical
surface of enterocytes presented binding sites for RCA-1
on al1 cells, binding sites of UEA-1, DBA, SBA, HPA
and VAA heterogeneously and no binding sites of Con A
and 14 kDa. Binding sites of DBA, SBA, HPA, VAA and
RCA-1 within enterocytes were located primarily focally
in a supranuclear position, whereas Con A and 14 kDa
bound to the cytoplasm both in apical and basa1 cell
parts.
In the follicle-associated epithelium more enterocytes
expressed SBA- and VAA-binding sites than in the crypt
epithelium. No differences between the lectin-binding
pattern of M-cells and enterocytes were found in the
follicle-associated epithelium. Intraepithelial macrophages
were heterogeneously positive for the full panel
of applied lectins. In contrast, intraepithelial lymphatic
cells expressed binding sites only for RCA-1 and less
prominently for Con A, VAA and 14 kDa. Goblet cell
mucus contained lectin-binding sites in a heterogeneous
manner: binding sites for Con A were not detected in
goblet cells for DBA, SBA, VAA and 14 kDa in less
than 20%, for UEA-1 in 20-40%, for HPA in 40-60% and
for RCA-1 in 60-100% of the goblet cells. Secreted
mucus differed in its lectin-binding capacity from intracellular
goblet cell mucus selectively by an increase of
UEA-1, SBA- and RCA-1-binding sites and a lack of 14
kDa-binding sites. Comparative study of lectin binding
to goblet cell mucin in another region of the large
intestine, namely the rectosigmoid, demonstrated that
DBA, SBA and 14 kDa bound mainly to the dista1 colon,
while UEA-1 and VAA labelling was selectively found in
OĂfprint reguests lo: Dr. Ulrich Brinck, M.D., Department of Pathology,
University of Gottingen, Robert-Koch-Str. 40, D-37075 Gottingen,
Germany
apendiceal goblet cell mucin.
Comparing the lectin-binding pattern in normal
appendix epithelium and in appendicitis, the percentage
of goblet cells expressing DBA- and SBA-binding sites
in mucus globules was found to be about 4 times higher
in appendicitis than in normal appendix. These results
demonstrate that the expression of lectin-binding sites in
appendiceal goblet mucin is specifically altered in
appendicitis, indicating that there are selective changes
of glycosylation of mucin in goblet cells mainly of the
lower and middle crypt segment. Changes of lectinbinding
pattern in appendicitis are discussed in
connection with histochernical findings in inflammatory
bowel disease
Histochemical study of expression of lectin-reactive carbohydrate epitopes and glycoligand-binding sites in normal human appendix vermiformis, colonic mucosa, acute appendicitis and colonic adenoma
In a glycohistochemical analysis of human
appendix vermiformis we report the assessment of lectin
binding in cells of the Gut Associated Lymphoid Tissue
of normal samples and in acute appendicitis using a
panel of plant, invertebrate and mammalian lectins with
specificity for a-L-Fuc (UEA-I), a-D-Gluc and a-DMan
(Con A), a-D-GalNAc (DBA), GalNAc (SBA,
HPA), B-Gal (RCA-I, 14 kDa=galectin-l) and a-, B-Gal
(VAA). Moreover, we initiate the study of expression of
carbohydrate-binding sites in this tissue and in colonic
mucosa, employing several types of carrier-immobilized
carbohydrate ligands as suitable probes for this purpose.
Within the three populations of macrophages intralsubepithelial
macrophages of the dome region, the
lamina propria of the intercryptal region and the follicleassociated
epithelium were apparently reactive with
most of the lectins and also with mannose and fucose
residues of the tested neoglycoproteins. Distinguishing
features of germinal center macrophages in relation to
intra-/subepithelia1 phagocytes were the lack of binding
of UEA-I and DBA. In comparison to all other types of
phagocytes, macrophages of the T-region displayed a
rather restricted binding capacity only to Con A and
RCA-I. Labeling of macrophages with SBA, HPA and
VAA in this location was only rarely found. With respect
to dendritic cells no consistently positive reaction was
seen for follicular cells, whereas interdigitating cells of
the T-region bound Con A, HPA and RCA-I, and, less
frequently, SBA.
Lymphocytes in all anatomical subsites of the Gut
Associated Lymphoid Tissue, centrocytes, centroblasts and plasma cells had binding sites for Con A and RCA-I
in common. Notably, a small number of lymphocytes
mostly in the T-region but also in B-cell-rich areas expressed intranuclear binding sites for fucose and
mannose residues. Intraepithelial lymphocytes and
lymphatic cells of the T-region differed from lymphocytes
in other regions by a more frequent expression of
VAA-binding sites.
The epithelium of appendix vermiformis and colonic
mucosa not only presents lectin binding sites, but also
has the capacity to bind carbohydrate structures, as
shown by labeled glycoligand-exposing neoglycoproteins.
In normal mucosa the extent of binding
appeared to be associated with maturation of cells, the
surface epithelium showing the most intense staining
reaction. This pattern is not detectable in colonic
adenoma which reveal increased intensity, when
compared to normal mucosa. In contrast to development
of hyperplasia, acute inflammation in appendicitis
caused no detectable changes of neoglycoprotein
binding. Taking our previous assessment on lectin
binding in appendicitis into account, we conclude that
glycosylation of goblet cell mucus, but not the capacity
to bind certain sugar epitopes responds to inflammatory
processes, whereas tumorigenesis of colonic adenoma
can also affect the binding of neoglycoproteins