Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

Undersulfated proteoglycans are secreted by cultured chondrocytes in the presence of the ionophore monensin

The monovalent ionophore, monensin, inhibits secretion of many different proteins from a wide variety of cells. The site of blockage is at the golgi complex. We have exposed chick embryo chondrocytes in suspension culture to monensin, at concentrations ranging from 10(-8) to 10(-6) M. At the higher concentrations, between 10(-7) and 10(-6) M, monensin inhibited secretion of type II procollagen, which accumulated in the chondrocytes. At these concentrations of the ionophore, proteoglycan synthesis was inhibited, as measured by radioactive serine incorporation into core proteins and by radioactive glucosamine or SO4 incorporation into glycosaminoglycans. However, at a monensin concentration of 3 x 10(-8) M, the incorporations of serine and glucosamine were close to normal while SO4 incorporation was at 30% of control values. The ratio of glucosamine to serine in pronase-released glycosaminoglycans from culture media was unaffected by 3 x 10(-8) M monensin but the sulfate to serine ratio decreased to 29% of control values. Examination of the glycosaminoglycans by gel filtration showed a progressive increase in Kav values as sulfation decreased. Undersulfation was demonstrated by radiochromatographic analysis of the digestion products following incubation with chondroitinase ABC. The composite results show that monensin interferes with sulfation of newly synthesized proteoglycans.     

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.     

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.     

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.     

Effects of monensin on the synthesis, transport, and intracellular degradation of proteoglycans in rat ovarian granulosa cells in culture

Rat ovarian granulosa cells, isolated from immature female rats 48 h after stimulation with 5 IU of pregnant mare's serum gonadotropin, were maintained in culture. The effects of monensin, a monovalent cationic ionophore, on various aspects of proteoglycan metabolism were studied by metabolically labeling cultures with [35S]sulfate, [3H]glucosamine, or [3H]glucose. Monensin inhibited post-translational modification of both heparan sulfate (HS) proteoglycans and dermatan sulfate (DS) proteoglycans, resulting in decreased synthesis of completed proteoglycans [( 35S]sulfate incorporation decreased to 10% of control by 30 microM monensin, with an ED50 approximately 1 microM). Proteoglycans synthesized in the presence of monensin showed undersulfation of both DS and HS glycosaminoglycans and altered N-linked and O-linked oligosaccharides, suggesting that the processing of all sugar moieties is closely associated. Monensin caused a decrease in the endogenous sugar supply to the UDP-N-acetylhexosamine pool as indicated by an increased 3H incorporation into DS chains [( 3H]glucosamine as precursor) in spite of the decrease in glycosaminoglycan synthesis. Monensin reduced and delayed transport of both secretory and membrane-associated proteoglycans from the Golgi complex to the cell surface. It took 2-4 min for newly labeled proteoglycans to reach the main transport process inhibited by monensin. Monensin at 30 microM did not prevent internalization of cell surface 35S-labeled proteoglycans but almost completely inhibited their intracellular degradation to free [35S]sulfate (ED50 approximately 1 microM), resulting in intracellular accumulation of both DS and HS proteoglycans. Pulse-chase experiments demonstrated that one of the intracellular degradation pathways involving proteolysis of both DS and HS proteoglycans and limited endoglycosidic cleavage of HS continued to operate in the presence of monensin. These results suggest that the intracellular degradation of proteoglycans involve both acidic and nonacidic compartments with monensin inhibiting those processes that normally occur in such acidic compartments as endosomes or lysosomes by raising their pH.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.     

Effects of brefeldin A on the synthesis of chondroitin 4-sulfate by cultures of mouse mastocytoma cells.

Mouse mastocytoma cells were cultured with brefeldin A in medium containing [35S]sulfate and [3H]glucosamine in order to determine the effects of this fungal metabolite on the formation of chondroitin 4-sulfate by these cells. There was a marked reduction in the incorporation of [35S]sulfate into the glycosaminoglycan which was approximately equal to the reduction in the incorporation of [3H]hexosamine into the same molecule. The chondroitin 4-sulfate chain size was greatly diminished, while the number of chains appeared to remain relatively constant, indicating that the brefeldin A partially disrupted the polymerizing system, but had little effect upon movement of the nascent proteochondroitin to the site for chondroitin polymerization and sulfation.     

The effects of monensin on blood-borne arrest and glycosylation of BL/VL3 lymphoma cells.

We have previously shown that inhibitors of N-glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters. Arrest in the spleen of [111In]-labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0.1-1.0 microgram ml-1). We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio-Gel P6 and by affinity chromatography on immobilized lectins. After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N-acetyl-glucosamine). Similar findings were obtained with immunoprecipitated Thy-1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy-1 was not reduced in MON-treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON-treated cells.     

Tyrosine sulfation is not the last modification of entactin before its secretion from 3T3-L1 adipocytes

Tyrosine sulfation of entactin was studied by labeling of 3T3-L1 adipocytes with [35S]methionine or H2 35SO4 in the presence or absence of tunicamycin or monensin. Four precursors (EN1-4) at different steps of modification were detected in addition to mature entactin. Under normal conditions, EN2 and mature entactin were intracellular species, and the latter was sulfated and secreted. Inhibition of co-translational transfer of N-linked oligosaccharides by tunicamycin produced EN1 and EN3 as intracellular species, and EN3 was sulfated and secreted. Interruption of protein transport from medial to trans (distal) Golgi cisternae by monensin, and consequent blockage of terminal glycosylation caused intracellular accumulation of EN4. EN4 was sulfated and of different size compared to mature entactin. These facts suggested that tyrosine sulfation of entactin occurs in medial Golgi cisternae and is not the last modification before its secretion. Our results appeared inconsistent with recent observations by Baeuerle and Huttner [(1987) J. Cell Biol. 105, 2655-2664] that tyrosine sulfation of IgM occurred within the trans (distal) Golgi cisternae as the last modification before its exit from the Golgi complex.     

Helicobacter pylori lipopolysaccharide effect on the synthesis and secretion of gastric sulfomucin.

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion.     

CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa

We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [(3)H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [(3)H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in (3)H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.     

Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation

Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4-mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1 alpha, MIP-1 beta, IL-8) and cytokines (TNF-alpha, IL-1 beta, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.     

Intracoronary beta-irradiation enhances balloon-injury-induced tissue factor expression in the porcine injury model

Intracoronary brachytherapy (ICBT) effectively reduces restenosis but is associated with late thrombosis. Since tissue factor (TF) is an important mediator of arterial thrombosis, we tested the hypothesis that ICBT results in persistently augmented TF expression. Coronary arteries from 12 pigs were randomized to: control (C; no injury), oversized balloon injury (BI), or BI followed by ICBT. Animals were sacrificed at 1, 7, 14, or 60 days postprocedure, and coronary arteries collected for expression analyses and immunostaining. ICBT-treated arteries had higher TF antigen and activity at all time-points compared to BI arteries (Western blot: 16 571 +/- 2090 vs 10 135 +/- 2939 densitometric units, p = 0.001; ELISA: 0.42 +/- 0.13 nM vs 0.25 +/- 0.14 nM, p = 0.001; TF activity assay: 0.303 +/- 0.11 nM vs 0.18 +/- 0.07 nM, p = 0.01; immunohistochemical staining: 30.6 +/- 6.6% vs 11.5% +/- 3.2%, p = 0.01). TF expression increased following BI, increased further following ICBT, and persisted for the duration of the study. We conclude that TF expression increases after BI, but is further increased and persists for a longer duration following ICBT, suggesting that a TF-mediated mechanism may play a role in late thrombosis following ICBT.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.     

CFTR expression does not influence glycosylation of an epitope-tagged MUC1 mucin in colon carcinoma cell lines

The cause of the mucus clearance problems associated with cystic fibrosis remains poorly understood though it has been suggested that mucin hypersecretion, dehydration of mucins, and biochemical abnormalities in the glycosylation of mucins may be responsible. Since the biochemical and biophysical properties of a mucin are dependent on O-glycosylation, our aim was to evaluate the O-glycosylation of a single mucin gene product in matched pairs of cells that differed with respect to CFTR expression. An epitope-tagged MUC1 mucin cDNA (MUC1F) was used to detect variation in mucin glycosylation in stably transfected colon carcinoma cell lines HT29 and Caco2. The glycosylation of MUC1F mucin was evaluated in matched pairs of Caco2 cell lines that either express wild-type CFTR or have spontaneously lost CFTR expression. The general glycosylation pattern of MUC1F was evaluated by determining its reactivity with a series of monoclonal antibodies against known blood group and tumor-associated carbohydrate antigens. Metabolic labeling experiments were used to estimate the gross levels of glycosylation and sulfation of MUC1F mucin in these matched pairs of cell lines. Expression of CFTR in this experimental system did not affect the gross levels of glycosylation or sulfation of the MUC1F mucin nor the types of carbohydrates structures attached to the MUC1F protein.     

Chlorate: a reversible inhibitor of proteoglycan sulfation

Bovine aorta endothelial cells were cultured in medium containing [3H]glucosamine, [35S]sulfate, and various concentrations of chlorate. Cell growth was not affected by 10 mM chlorate, while 30 mM chlorate had a slight inhibitory effect. Chlorate concentrations greater than 10 mM resulted in significant undersulfation of chondroitin. With 30 mM chlorate, sulfation of chondroitin was reduced to 10% and heparan to 35% of controls, but [3H]glucosamine incorporation on a per cell basis did not appear to be inhibited. Removal of chlorate from the culture medium of cells resulted in the rapid resumption of sulfation.