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 Oífprint 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