Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells.

The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Studies of acceptor site specificities for three members of UDP-GalNAc:N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC.

The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP) containing clusters of threonine residues mimicking the mucin tandem repeat unit of MUC5AC. The O-glycosylated products obtained after in vitro reactions were fractionated by capillary electrophoresis and the purified glycopeptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalNAc residues was transferred into the MUC5AC motif peptide and the preferential order of incorporation for each GaNTase isoform was determined. Our results suggest that clusters of threonine appear to be essential for site recognition of peptide backbone by the ubiquitous GaNTases and also support the notion that the different GaNTase isoforms with varying substrate specificities are involved in a hierarchical order of O-glycosylation processing of the mucin-type O-glycoproteins.

Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides.

The recombinant proteins of the two novel UDP-N-acetylgalactosamine (GalNAc) glycopeptide:N-acetylgalactosaminyltransferases (designated gpGaNTase-T7 and gpGaNTase-T9) were assayed with O-glycosylated products obtained from the prior action of the ubiquitous transferases (GaNTase-T1 and GaNTase-T2) towards MUC5AC mucin motif peptides (GTTPSPVPTTSTTSAP and peptides with single amino acid substitutions, GTTPSAVPTTSTTSVP and GTTPSPVPTTSITSVP, that are a reflection of mucin molecule polymorphism). gpGaNTase-T9 is known to be expressed differentially and more abundantly than gpGaNTase-T7 in some tissues; the results of in vitro glycosylation also indicates a difference in acceptor substrate specificities between the gpGaNTase isoforms. With the use of capillary electrophoresis, MS and Edman degradation, our study suggests that, in the O-glycosylation of mucin-type proteins, approach and recognition signalling by gpGaNTase-T7 and gpGaNTase-T9 depend largely on the peptide's primary structure (for example the presence of multiple clusters of hydroxy amino acids and the number of GalNAc residues attached to the peptide backbone). O-glycosylation in terms of sites of attachment seems to be less random than previously described and, if sequential reactions are ordered throughout the Golgi stack, the complete O-glycosylation of the mucin molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.

Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity.

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide.

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.

Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.

Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry.

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.

Dipeptidyl aminotransferase activity and in vitro O-glycosylation of MUC5AC mucin motif peptides by human gastric microsomal preparations.

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.

Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s).

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamineto human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

O-Glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line.

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performing in vitro reactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype. As acceptor for the UDP-N-acetylgalactosamine:polypeptide Nacetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used. A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different pattern of galactosaminylation in pre- and postconfluent cells. Core 1 UDP-galactose:N-acetyl-alpha-galactosaminyl-R 3-beta-galactosyltransferase (3-beta-galactosyltransferase) activityalso decreased with the differentiation, whereas CMP-neuraminic acid:galactose-beta-1, 3-N-acetyl-alpha-galac- tosaminyl-R 3-alpha-sialyltransferase activity increased. In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-beta-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an alpha-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation.

Identification of hyaluronic acid and chondroitin sulfates in human follicular fluid and their effects on human sperm motility and the outcome of in vitro fertilization.

This study measured glycosaminoglycans (GAGs) in human follicular fluid (hFF) obtained from patients undergoing hormonal stimulation with combined GnRH agonist followed by gonadotropin hormone (hMG or FSH) for in vitro fertilization (IVF). The GAGs were partially characterized through the use of various mucopolysaccharidases and then their effects tested on human sperm motility. In hFF, the GAG and protein concentrations were 4.4 +/- 1.3 mg/L and 32.6 +/- 3.2 g/L respectively. Chondroitins (CS) and hyaluronic acid (HA) significantly stimulate sperm motion in comparison to the control. The oocytes inseminated with GAGs-pretreated spermatozoa showed a significantly higher rate of cleavage and pregnancy or delivered number. The present results suggest that hFF is a rich source of sulfated glycosaminoglycan (CS), and non-sulfated glycosaminoglycan (HA). The stimulatory effects of hFF on human sperm motility may well depend on CS and HA. GAGs pretreated sperm may enhance the fertilizing ability of spermatozoa and thus the IVF outcome.

Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation.

HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high performance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-alpha-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-alpha-O-benzyl was metabolized by the cells to Gal beta 1-3GalNAc-alpha-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan alpha-2,3-sialyltransferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sialylation.

Analysis by electrospray mass spectrometry of glycopeptides from the in vitro O-glycosylation reaction using human mucin motif peptide.

A mucin-motif peptide in the one-letter code T T T P S P P M T T P I T P P A, representative of the human intestinal mucin tandem repeat sequence (MUC2), containing several threonine residues in clusters, was used as an acceptor substrate to investigate the effect of peptide structure on the activity of crude preparation of human gastric UDP-GalNAc:polypeptide N-acetyl galactosaminyltransferases. High-performance liquid chromatography was performed to separate the different products of the in vitro O-glycosylated reaction. The electrospray mass spectrometry was used to identify the different masses (m/z) of these products. Although the m/z of glycopeptide(s) could be higher than the detection limits of the spectrometer, an accurate study of the doubly charged ions allowed us to demonstrate the linkage of more than two sugars. Hence, the peptide MUC2 will accept at least four carbohydrate residues but the exact substituted positions should be confirmed by further sequence determination.

Combination of high-performance anion-exchange chromatography and electrospray mass spectrometry for analysis of the in vitro O-glycosylated mucin motif peptide.

Reversed-phase high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection were developed for the study of products obtained from the in vitro O-glycosylation of a mucin motif peptide, TTSAPTTS, the most representative sequence encoded by the human gene MUC5C. After incubation of the peptide, which is rich in clustered hydroxyamino acids, by both human colonic and gastric microsomal homogenates, the glycosylated products were separated by HPLC and HPAEC and analysed by electrospray mass spectrometry (ES-MS). The combination of HPAEC and ES-MS was the approach used for evaluating the differences between the polypeptide N-acetylgalactosaminyltransferase activity in different digestive tissues.