Inhibitor Of O-glycosylation Research Articles

Abstract 1264: Extracellular galectin-3 induces tumor-specific apoptosis through the interaction with highly glycosylated cell surface β1 integrin

Abstract Galectins are a family of animal lectins, which bind beta-galactose moieties. Gal3 is the only chimeric family member of Gals and consists of a collagen-like N-terminal region and a C-terminal carbohydrate recognition domain (CRD), which has binding affinity for galactose and N-acetyl-lactosamine. Gal3 is known to be secreted through Golgi apparatus-independent alternative pathway. Previously, we found that stimuli-induced p53 activation induced bystander killing effect against adjacent cells. This p53 bystander effect is mediated through the secretion of Gal3 with the requirement of p53-dependent induction of tumor suppressor activated pathway-6 (TSAP6). We found the apoptotic activity of extracellular Gal3: a variety of tumor cell types (malignant glioma, breast, colon, prostate, lung) are sensitive to Gal3-mediated killing, while normal cells (fibroblast, endothelial) are not affected. We demonstrated that conditional sGal3 induction (tet-on system) in glioma xenografts strongly inhibits tumor growth without overt toxicity in mice. We found that Gal3-mediated tumor cell killing was neutralized with lactose, a Gal3-CRD binding ligand, and Gal3-mediated apoptosis was inhibited by treatment with a caspase 9 inhibitor (LEHD) but not by a caspase 8 inhibitor (IETD). Gal3 interacts with several glycosylated cell surface proteins. We identified that Gal3-induced tumor specific apoptosis is mediated through Gal3-beta1 integrin binding through GST-Gal3 pulldown assay, cell membrane-specific Gal3-beta1 integrin co-immunoprecipitation, and beta1 integrin siRNA neutralization. Further, the treatment of N-glycosylation inhibitor (tunicamycin) interfered with Gal3-beta1 integrin interaction whereas, O-glycosylation inhibitor (benzyl-O-N-acetyl-D-galactosamine) increased N-glycosylation of beta1 integrin as well as consequent Gal3-beta1 integrin interaction and Gal3-mediated apoptosis. In summary, our data demonstrate that extracellular Gal3 interacts with tumor cells through highly-glycosylated cell surface beta1 integrin through its CRD binding and consequently induces apoptosis. These findings are important for our understanding of tumor-specific killing of Gal3 and suggest that Gal3 can be exploited as a therapeutic agent for cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1264.

Equatorin: Identification and Characterization of the Epitope of the MN9 Antibody in the Mouse1

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.

Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin β4

We previously found an inverse relationship between sialidase Neu1 expression and metastatic potential of murine cancer cells. To elucidate the mechanism underlying the cellular events, the human sialidase gene NEU1 was overexpressed or silenced in colon cancer HT-29 cells. When NEU1-overexpressing cells were injected transsplenically into mice, in vivo liver metastasis was significantly reduced. NEU1 suppressed cell migration, invasion and adhesion in vitro, whereas the silencing resulted in the opposite. One of the major molecular changes by NEU1 was decreased sialylation of integrin beta4, assessed by PNA- and MAL-II-lectin blotting of immunoprecipitates with anti-integrin beta4 antibody. The desialylation was accompanied by decreased phosphorylation of the integrin followed by attenuation of focal adhesion kinase and Erk1/2 pathway. Moreover, NEU1 caused downregulation of matrix metalloproteinase-7, overexpression of which is associated with cancer metastasis. Treatment of the cells with GalNAc-alpha-O-benzyl, an inhibitor of O-glycosylation, showed increased PNA-positive integrin beta4 with its decreased phosphorylation, indicating that sialic acid removal from the integrin O-glycans results in the decreased phosphorylation. Biotinylation and immunofluorescence staining exhibited some NEU1 molecules to be at the cell surface accessible to the integrin. These results suggest that NEU1 is important in regulation of integrin beta4-mediated signaling, leading to suppression of metastasis.

Poly N-acetyllactosamine substitutions on N- and not O-oligosaccharides or Thomsen–Friedenreich antigen facilitate lung specific metastasis of melanoma cells via galectin-3

Galectin-3 on vascular endothelium has been shown to facilitate lung specific metastasis. Metastatic variants of B16 melanoma were chosen to identify specific ligands that mediate lung colonization via galectin-3. Flow cytometry showed that, galectin-3 binding to cells correlates with surface expression of poly N-acetyllactosamine (polylacNAc) but not with other reported ligands, e.g. Thomsen-Friedenreich (T/Tn) antigen. Immobilized galectin-3 promoted adhesion of melanoma cells in a metastasis dependent manner. Moreover, adhesion and galectin-3 binding to cells were specifically inhibited with lactose. These properties together with lung metastasis were inhibited with N-glycosylation inhibitor Swainsonine (SW), whereas, O-glycosylation inhibitor Benzyl-alpha-N-acetylgalactosamine (BG) had no effect. BG treatment significantly increased expression of T/Tn antigen on low metastatic cells; however, had no effect on their metastatic potential. The studies very comprehensively demonstrate the importance of polylacNAc substitutions on N-oligosaccharides in galectin-3 mediated lung metastasis.

Mucin impedes cytotoxic effect of 5-FU against growth of human pancreatic cancer cells: overcoming cellular barriers for therapeutic gain

Mucins are high molecular weight glycoproteins expressed on the apical surface of normal epithelial cells. In cancer disease mucins are overexpressed on the entire cellular surface. Overexpression of MUC1 mucin in pancreatic tumours has been correlated with poor patient survival. Current chemotherapeutic approaches such as 5-fluorouracil (5-FU) has produced limited clinical success. In this study we investigated the role of mucin in cytotoxic drug treatment to determine whether the extracellular domain of mucin impedes cytotoxic drug action of 5-FU. Human pancreatic cancer cells revealed high and relatively moderate MUC1 levels for Capan-1 and HPAF-II, respectively, compared to MUC1 negative control (U-87 MG glioblastoma) that showed relatively non-specific anti-MUC1 uptake. Benzyl-α-GalNAc (O-glycosylation inhibitor) was used to reduce mucin on cell surfaces, and neuraminidase was used to hydrolyse sialic acid at the distal end of carbohydrate chains. Benzyl-α-GalNAc had no effect on cell morphology or proliferation at the concentrations employed. The inhibition of O-glycosylation resulted in significant 5-FU antiproliferative activity against Capan-1 and HPAF-II, but not against U-87 MG. However, the exposure of cells to neuraminidase failed to improve the cytotoxic action of 5-FU. Our experimental findings suggest that the overexpression of mucin produced by human pancreatic tumours might limit the effectiveness of chemotherapy.

Chondrocyte secreted CRTAC1: A glycosylated extracellular matrix molecule of human articular cartilage

Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-α-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

The influence of N- and O-glycosylation inhibitors on the glycosylation profile of cellular membrane proteins and adhesive properties of carcinoma cell lines

The effects of N- and O-glycosylation inhibitors on the expression of membrane proteins (MUC1 and some integrins) were evaluated in human endometrial (Ishikawa) and breast (MCF-7) cancer cells. Subconfluent cells were treated with 1-3 mg% concentration of tunicamycin and 2-10 mM of benzyl-N-acetyl-alpha-galactosaminide for 1-2 days, and used for flow cytometry, immunohistochemical staining, adhesion test and Western blotting. Benzyl-N-acetyl-alpha-galactosaminide inhibits MUC1 expression on the surface of breast more than endometrial cancer cells. Tunicamycin reduces MUC1 concentration on the cellular surface more than benzylglycoside, and greatly reduces glycosylation of glycoproteins, causing an increase in cell adhesion in both types of cancer cells. The expression of alpha2beta1 integrins on the surface of these cells was weak and decreased after treatment with inhibitors. Two different glycoforms of MUC1 proteins in endometrial cells and three in breast cancer cells were expressed and their molecular weights were reduced after treatment with glycosylation inhibitors. It was confirmed with lectin detection of carbohydrate epitopes (Tn and T) in MUC1 proteins. These observations show that glycosylation inhibitors altered the N- and O-glycan patterns in a sufficient manner, and positively modified the biological features of cancer cells.

Apical Transport and Folding of Prostate-specific Membrane Antigen Occurs Independent of Glycan Processing

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.

Action of a library of O-glycosylation inhibitors on the growth of human colorectal cancer cells in culture

O-glycosylation is thought to play a significant role in the regulation of cell growth. However, only limited information is available, and few specific and selective inhibitors have been found. We have synthesized a library of O-glycosylation inhibitors based on benzyl-O-N-acetyl-D-galactosamine. These inhibitors were tested with an established series of human colorectal cancer cell lines, which model the adenoma-carcinoma sequence. Cancer cells were incubated with the inhibitors, and examined for cell growth patterns, and cellular and subcellular glycosylation using a range of lectins with confocal microscopy. The specificity of O-glycan inhibition was confirmed for the library, relative to other forms of glycosylation. All inhibitors tested resulted in smaller cell yields. However, a differential effect on O-glycosylation was detected using the lectins showing variation of localization at a subcellular level in the various cell lines. Further differential action of the inhibitor library was observed for apoptosis and on the cell cycle with the cell lines tested. This work demonstrates that O-glycosylation is closely involved in the regulation of cell growth in colorectal cancer cells and that the generation of a library of low-molecular-mass inhibitors offers a valuable means of examining this regulation at the molecular level.

Characterization of nuclear ferritin and mechanism of translocation

Ferritin, normally considered a cytoplasmic iron-storage protein, is also found in cell nuclei. It is an established fact that H-ferritin is the major form of nuclear ferritin, but little is known about the roles of ferritin in nuclei or about the mechanisms that control its appearance within the nuclear volume. In the present study, we show that, for human SW1088 astrocytoma cells, the nuclear and cytoplasmic forms of H-ferritin are products of the same mRNA. Histochemical and biochemical evidence is presented showing that ferritin is distributed non-randomly within the nuclear volume and that it preferentially associates with heterochromatin. Both cytoplasmic and nuclear populations of H-ferritin contain mixtures of non- and O-glycosylated forms, but the nuclear population is enriched in O-glycosylated forms. Cells treated with alloxan, a potent inhibitor of O-glycosylation, contained significantly less nuclear ferritin compared with cells grown in control media. Alloxan inhibited the reappearance of H-ferritin in nuclei of cells released from conditions of iron depletion, but did not prevent its disappearance from nuclei of cells undergoing iron depletion. These results suggest that O-glycosylation accompanies the transfer of ferritin from the cytoplasm to the nucleus, but does not influence the reverse process. The picture that emerges is one in which ferritin translocation between the cytoplasm and the nucleus is post-translationally regulated and responds to environmental and nutritional cues.

Regulatory roles of altered N- and O-glycosylation of CD45 in galectin-1-induced cell death in human diffuse large B cell lymphoma

To clarify the functions of CD45 N- and O-glycans in diffuse large B cell lymphoma (DLBCL), we analyzed the antiproliferative effects of bovine galectin-1 (beta-galactoside-binding lectin-1), which reacts with CD45 N-glycans and O-glycans, on human lymphoma cells. Bovine galectin-1 induced cell death of the DLBCL cell line HBL-2 in vitro. Swainsonine (SW) is a potent inhibitor of alpha-mannosidase II which catalyzes the synthesis of complex type N-linked oligosaccharides, and benzyl-GalNAc (BZGalNAc) is a potent inhibitor of O-glycosylation. Treatment with SW or BZGalNAc prevented cell death of HBL-2 cells by galectin-1. Western blot analysis revealed SW treatment reduced the molecular weight by about 5 kDa of one isoform at 190 kDa among three isoforms of CD45 which have N-linked oligosaccharide ligands for galectin-1. BZGalNAc treatment reduced the molecular weight of another isoform by about 15 kDa. These data suggest that the amount of CD45 N-glycans or O-glycans was reduced by SW and BZGalNAc treatment, respectively, and that reduction of CD45 N-glycans or O-glycans may prevent the interaction between CD45 and galectin-1. Alteration in CD45 N-glycans or O-glycans may regulate cell death of lymphoma cells through the interaction between CD45 N-glycans or O-glycans and galectin-1 in DLBCL.

Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells.

Because adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP), we inquired whether HBP activation affects pancreatic beta-cell survival. Exposure of human islets to high glucose resulted in increased apoptosis of beta-cells upon serum deprivation that was reversed by azaserine. Also, glucosamine, a direct precursor of the downstream product of the HBP, increased human beta-cells apoptosis upon serum deprivation, which was reversed by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADGP), an inhibitor of protein O-glycosylation. These results were reproduced in RIN rat beta-cells. Glucosamine treatment resulted in inhibition of tyrosine-phosphorylation of the insulin receptor (IR), IRS-1, and IRS-2, which was associated with increased O-glycosylation. These changes caused impaired activation of the PI 3-kinase/Akt survival signaling that resulted in reduced GSK-3 and FOXO1a inactivation. BADGP reversed the glucosamine-induced reduction in insulin-stimulated phosphorylation of IR, IRS-1, IRS-2, Akt, GSK-3, and FOXO1a. Impaired FOXO1a inactivation sustained expression of the pro-apoptotic protein Bim, without affecting Bad, Bcl-XL, or Bcl-2 expression. These results indicate that hyperglycemia may increase susceptibility to apoptosis of human and rat beta-cell through activation of the HBP. Increased routing of glucose through this metabolic pathway results in impaired activation of the IR/IRSs/PI3-kinase/Akt survival pathway by induction of O-glycosylation of signaling molecules.

Inhibition of the O-glycan elongation limits MUC1 incorporation to cell membrane of human endometrial carcinoma cells

We have studied how benzyl-N-acetyl-alpha-D-galactosaminide, O-glycosylation inhibitor, affects the polymorphism and shedding of membrane-bound MUC1 mucin, and change in adhesive properties of cancer cells. In endometrial adenocarcinoma cells (Ishikawa line), high molecular weight MUC1 mucin was shed from cellular membrane and could be detected in culture medium 24 h after [14C]threonine labelling. Short-time (2 days) exposure of these cells to benzyl-N-acetyl-alpha-D-galactosaminide was associated with a reduction in sialic acid level and increase in T antigen content in cellular MUC1 mucin. These changes could be inverted after removal of the inhibitor. A longer, 6-day action of the inhibitor induced a decrease in sialic acid and T antigen levels in cellular MUC1 mucin. Benzyl-N-acetyl-alpha-D-galactosaminide treatment caused the occurrence of a few incompletely glycosylated glycoforms of MUC1 in cells, but not in culture medium. Adhesion of endometrial cells to ECM compounds (type I collagen) was increased by benzyl-N-acetyl-alpha-D-galactosaminide treatment, indicating that glycosylation of extracellular domain of MUC1 can modulate adhesive properties of cells.

O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon.

Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine (O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti-O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 microU/ml) or glucagon (1.5 x 10(-5) M). We also assessed subcellular localization of the native and glycosylated Sp1 in H411E cells treated with either hormone in the presence of deoxynorleucine (DON, an indirect inhibitor of O-glycosylation) or streptozotocin (STZ, an indirect stimulator of O-glycosylation). Insulin stimulated both total and O-GlcNAc-modified Sp1 primarily in the nucleus and induced CaM gene transcription (P < 0.0001). In contrast, glucagon promoted accumulation of Sp1 in the cytoplasm but not the nucleus, without significantly stimulating (P = not significant) either its O-glycosylation or transcription of the CaM gene. DON inhibited O-glycosylation of Sp1 and its ability to migrate to the nucleus and transactivate CaM gene transcription. In contrast, cotreatment of cells with STZ and glucagon enhanced O-glycosylation of Sp1, promoting its migration to the nucleus and resulting in increased CaM gene transcription. Thus O-glycosylation of Sp1 by insulin, but not glucagon, apparently enhances its (Sp1) nuclear recruitment and results in activation of CaM gene transcription.

The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl- N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.

Induction of a storage phenotype and abnormal intracellular localization of apical glycoproteins are two independent responses to GalNAcalpha-O-bn.

Our previous studies on an inhibitor of O-glycosylation of glycoproteins, GalNAcalpha-O-bn, in the model of enterocytic HT-29 cells, have shown at the cellular level an alteration of the normal localization of apical glycoproteins, and at the biochemical level an in situ synthesis and storage of sialylated GalNAcalpha-O-bn oligosaccharides. The purpose of this study was to examine if a relation existed between these two events, using different cell lines. Intracellular storage of GalNAcalpha-O-bn metabolites occurred in HT-29 and CAPAN-1 cells but not in Caco-2 cells. On the other hand, an accumulation of endosomal/lysosomal compartments was observed in HT-29 and CAPAN-1 cells but not in Caco-2 cells. These data focused on a GalNAcalpha-O-bn-derived storage phenotype in HT-29 and CAPAN-1 cells. The apical membrane glycoproteins MUC1 and CEA showed an abnormal localization inside intracytoplasmic vesicles in HT-29 cells, whereas they kept their normal localization in Caco-2 and CAPAN-1 cells. Studies on the glycosylation of these apical glycoproteins showed that GalNAcalpha-O-bn inhibited the glycosylation in a cell-specific manner. The alteration in the apical targeting of glycoproteins, and the appearance of a GalNAcalpha-O-bn-derived storage phenotype are two independent and cell type-specific events. The former depends on the inhibition pattern of the glycosylation of endogenous glycoproteins, whereas the latter is connected to the intracellular accumulation of GalNAcalpha-O-bn metabolites.

MUC1 as a human tumor marker

MUC1 mucin serves as a tumor-associated antigen in various carcinomas. We have reported the expression of MUC1 in multiple myeloma (MM) cells and the establishment of an HLA-unrestricted cytotoxic T lymphocyte (CTL) that recognized MUC1 from peripheral lymphocytes in a patient with MM. In this study, we successfully induced MUC1-specific CTLs from bone marrow cells in two MM patients. Both CTLs lysed MUC1-positive cell lines with different kinds of HLA, and the cytotoxicity was inhibited by anti-CD3, anti-αβ T cell receptor and anti-MUC1 monoclonal antibodies. The treatment with O-glycosylation inhibitor augmented the cytotoxicity of the CTLs. These data showed that HLA-unrestricted CTLs that recognize the underglycosylated form of MUC1 could be induced from MM patients. MUC1 is also an important factor in cancer metastasis. We previously reported that MUC1 mucin has anti-adhesion effects that contribute to the separation of tumor cells from the primary site. We next investigated the involvement of MUC1 in tumor invasion that is a second step of metastasis. MUC1-transfectants transplanted subcutaneously into nude mice invaded the muscular layer. The treatment with O-glycosylation inhibitor abolished these effects of MUC1. Our data indicated that MUC1 mucin increases the invasive ability of tumor cells, while O-glycan plays an essential role.

Differential effect of GalNAcalpha-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern.

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.

Apical transport of osteopontin is independent of N-glycosylation and sialylation.

Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl-alpha-GaINAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin.

Apical Secretion and Sialylation of Soluble Dipeptidyl Peptidase IV Are Two Related Events

The role of glycans in the apical targeting of proteins in epithelial cells remains a debated question. We have expressed the mouse soluble dipeptidyl peptidase IV (DPP IV ectodomain) in kidney (MDCK) and in intestinal (Caco-2) epithelial cell lines, as a model to study the role of glycosylation in apical targeting. The mouse DPP IV ectodomain was secreted mainly into the apical medium by MDCK cells. Exposure of MDCK cells to GalNac-α-O-benzyl, a drug previously described as an inhibitor of mucin O-glycosylation, produced a protein with a lower molecular weight. In addition this treatment resulted in a decreased apical secretion and an increased basolateral secretion of mouse DPP IV ectodomain. When expressed in Caco-2 cells, the mouse DPP IV ectodomain was secreted mainly into the basolateral medium. However, BGN was still able to decrease the amount of apically secreted protein and to increase its basolateral secretion. Neuraminidase digestion showed that the most stricking effect of BGN was a blockade of DPP IV sialylation in both MDCK and Caco-2 cells. These results indicate that a specific glycosylation step, namely, sialylation, plays a key role in the control of the apical targeting of a secreted DPP IV both in MDCK and Caco-2 cells.