Glycosyltransferase changes upon differentiation of CaCo-2 human colonic adenocarcinoma cells.

The spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture is associated with a decrease in polylactosaminoglycans, particularly those attached to the lysosomal membrane glycoprotein h-lamp-1 (Youakim et al., Cancer Res., 49:6889-6895, 1989). To elucidate the biosynthetic mechanisms leading to these alterations we have compared glycosyltransferase activities that are involved in the synthesis of polylactosaminoglycans and of the N- and O-glycan structures that provide the framework for the attachment of these chains. Glycosyltransferase activities in cell homogenates obtained from undifferentiated and differentiated CaCo-2 cells were assayed by high pressure liquid chromatography separation of enzyme products. The beta-galactosidase activities and extremely high pyrophosphatase activities in differentiated cells were effectively inhibited by 5 mM gamma-galactonolactone and 10 mM AMP, respectively. CaCo-2 cells contain most of the enzymes that are involved in N-glycan branching [N-acetylglucosamine (GlcNAc) transferases I to V] with the exception of GlcNAc transferase VI. The levels of GlcNAc transferase I activities were comparable in undifferentiated and differentiated cells, but GlcNAc transferase II to V activities were significantly increased upon differentiation. The enzyme activities that are directly involved in the synthesis of linear polylactosaminoglycans (Gal beta 4GlcNAc beta 3- repeating units), blood group i UDP-GlcNAc:Gal beta-R beta 3-GlcNAc transferase and UDP-Gal:GlcNAc beta 4-Gal transferase, were found at similar levels in undifferentiated and differentiated CaCo-2 cells. Since GlcNAc transferase III activity is known to inhibit further branching and galactosylation, these results suggest that its increased activity in differentiated CaCo-2 cells may be partly responsible for the decreased synthesis of fucosylated polylactosaminoglycans. Differentiated cells showed a 2-fold increase in O-glycan core 2 UDP-GlcNAc:Gal beta 3GalNAc alpha-R [GlcNAc to N-acetylgalactosamine (GalNAc)] beta 6-GlcNAc transferase activity. In contrast, O-glycan core 1 UDP-Gal:GalNAc alpha-R beta 3-Gal transferase activity was found decreased. Several enzymes that are found in homogenates from normal human colonic tissue are absent or barely detectable in CaCo-2 cells. These include blood group I UDP-GlcNAc:GlcNAc beta 3Gal beta-R (GlcNAc to Gal) beta 6-GlcNAc transferase, O-glycan core 3 UDP-GlcNAc:GalNAc alpha-R beta 3 GlcNAc transferase and O-glycan core 4 UDP-GlcNAc:GlcNAc beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-GlcNAc transferase.

Decrease in polylactosaminoglycans associated with lysosomal membrane glycoproteins during differentiation of CaCo-2 human colonic adenocarcinoma cells.

The proportion of labeled polylactosaminoglycans found in glycoproteins decreases during spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture (A. Youakim and A. Herscovics, Biochem. J., 247: 299-306, 1987). To identify polylactosaminoglycan-containing glycoproteins, CaCo-2 cells were incubated with [3H]glucosamine or [3H]fucose, for 24 h, and membrane glycoproteins solubilized with 0.5% Nonidet P-40 were fractionated by affinity chromatography on Datura stramonium (DSA)-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that a restricted set of glycoproteins with a molecular weight of about 100,000 bound to DSA-agarose. These labeled glycoproteins were shown to contain polylactosaminoglycans by DSA-agarose chromatography and endo-beta-galactosidase digestion of Pronase-derived glycopeptides. Immunoprecipitation of the [3H]glucosamine-labeled Nonidet P-40 extract with polyclonal antibodies to the lysosomal membrane proteins h-lamp-1 and h-lamp-2 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography also revealed a band with a molecular weight of about 100,000. The immunoprecipitates were digested with Pronase, and the resulting glycopeptides were first fractionated on Bio-Gel P-6 into excluded (Fraction I) and included (Fraction II) glycopeptides, and then by DSA-agarose affinity chromatography. A much greater proportion of labeled glycopeptides in undifferentiated cells (3 to 5 days in culture) than in differentiated cells (19 to 27 days in culture) was recovered in Fraction I; these glycopeptides were bound to DSA-agarose and were sensitive to endo-beta-galactosidase. This decrease in polylactosaminoglycans was associated primarily with h-lamp-1. These results indicate that h-lamp-1 of CaCo-2 cells contains polylactosaminoglycans and that it undergoes a change in glycosylation with differentiation.

Lipid carriers in the synthesis of high-mannose glycoproteins in algae.

Particulate preparations from the chlorophyta Prototheca zopfii catalyze the incorporation of mannose and N-acetylglucosamine into glycolipids. These had been characterized as lipid monophosphate mannose, lipid pyrophosphate N,N'-diacetylchitobiose and various lipid-linked oligosaccharides containing two N-acetylglucosamine residues plus a variable number of mannose residues. The lipid moiety has the properties expected for dolichyl phosphate. The oligosacchride-linked lipids serve as precursors for the formation of a polymer sensible to pronase digestion. The oligosaccharide is linked by N-glycosidic linkage to an asparagine residue. In longer incubation periods, a polymer insensitive to pronase hydrolysis, but precipitable by copper salts such as cell wall mannans is formed. Polymer formation is inhibited by 1 mM bacitracin. The reactions leading to the formation of the mannoprotein were found associated to the rough endoplasmic reticulum. The synthesis of mannans was found to occur in the Golgi vesicles.

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization.

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture.

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells.

The effects of manno-1-deoxynojirimycin (ManDJN) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) were compared in IEC-6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N-linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H-sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7-9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7-9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP-treated cells were non-glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus-infected MDCK cells [(1984) J. Biol. Chem. 259, 12409-12413].

Effects of tunicamycin, N-methyl-1-deoxynojirimycin, and manno-1-deoxynojirimycin on the biosynthesis of lactosaminoglycans in F9 teratocarcinoma cells.

F9 teratocarcinoma cells were incubated with D-[2-3H]mannose or D-[6-3H]galactose, and the labeled glycopeptides obtained after exhaustive digestion by pronase were fractionated on Bio-Gel P-6 before and after treatment by endo-beta-N-acetylglucosaminidase H. Tunicamycin almost completely inhibited the synthesis of lactosaminoglycans found in excluded glycopeptides of large molecular weight. Manno-1-deoxynojirimycin greatly inhibited the incorporation of labeled mannose into both lactosaminoglycan and complex oligosaccharides, while it greatly increased that into Man8GlcNAc and Man9GlcNAc oligosaccharides. In contrast, N-methyl-1-deoxynojirimycin only partially inhibited the incorporation into lactosaminoglycan and complex oligosaccharides, and caused the accumulation of Glc3Man7-9GlcNAc oligosaccharides. These results demonstrate that, in these cells, lactosaminoglycans are N-linked, and suggest that there is transfer of both glucosylated and nonglucosylated oligosaccharides from lipid to protein.

Toxocara infection seroprevalence and its relationship with atopic features in a general adult population.

BACKGROUND: The relationship of helminth infection with atopy is controversial. Toxocariasis is the most common helminth infection in industrialized countries. The study aimed to investigate the association between Toxocara exposure and atopic features. METHODS: The study is based on a cross-sectional survey of 463 subjects, randomly selected (stratified by decades of age) from a general adult population. Toxocara exposure was defined by the presence of serum Toxocara antibodies. Main outcome measures included total serum IgE levels, skin prick tests (SPT) to a panel of 13 relevant aeroallergens, specific IgE to aeroallergens (Phadiatop test), and respiratory symptoms evaluated by means of a questionnaire. RESULTS: A total of 134 subjects (weighted proportion 28.6%, 95% CI 26.5-30.7%) showed Toxocara exposure. Pet ownership, rural habitat, farming, and low educational level were associated with Toxocara exposure. Toxocara exposure was associated with both positive SPT (particularly to mites) and positive specific IgE (Phadiatop test) after adjusting for potential confounders. The effect of Toxocara exposure on total serum IgE levels and blood eosinophil count was different in SPT-positive subjects and SPT-negative individuals. In SPT-negative individuals, Toxocara exposure was associated with an increase in both serum IgE levels and eosinophil counts, whereas an opposite trend was observed in SPT-positive individuals. Toxocara exposure was not associated with respiratory symptoms. CONCLUSIONS: In this adult population, Toxocara exposure is associated with allergic sensitization, particularly to mites. There is evidence of an intriguing interaction between Toxocara exposure and allergic sensitization for both total serum IgE levels and blood eosinophil counts.

Transfer of nonglucosylated oligosaccharide from lipid to protein in a mammalian cell.

We have previously shown that the glucosidase inhibitor, N-methyl-1-deoxynojirimycin (MedJN), only partially inhibited N-linked complex oligosaccharide biosynthesis in F9 teratocarcinoma cells whereas the alpha-mannosidase I inhibitor, manno-1-deoxynojirimycin, completely prevented this synthesis (Romero, P. A. and Herscovics, A. (1986) Carbohydr. Res. 151, 21-28). In order to determine whether a pathway independent of processing glucosidases can occur, F9 cells were pulse-labeled for 2 min with D-[2-3H]mannose in the presence or absence of 2 mM MedJN. In control cells, Man7GlcNAc was identified in the protein-bound oligosaccharides released with endo-beta-N-acetylglucosaminidase H, in addition to the expected Glc1-3Man9GlcNAc and Man9GlcNAc arising from processing of Glc3Man9GlcNAc. MedJN completely prevented the removal of glucose residues from Glc3Man9GlcNAc, but did not greatly affect the appearance of Man7GlcNAc associated with protein. Labeled Man7GlcNAc was also found in the lipid-linked oligosaccharides of both control and treated cells. The 2-min pulse-labeled Man7GlcNAc obtained from both the lipid and protein fractions were shown to have identical structures by concanavalin A-Sepharose chromatography and by acetolysis and were clearly different from the Man7GlcNAc obtained from the usual processing pathway. These results demonstrate that transfer of a nonglucosylated oligosaccharide (Man7GlcNAc2) from dolichyl pyrophosphate to protein occurs in F9 cells.

Transfer of glucose-1-phosphate from UDP-glucose to lipid acceptors in plants.

Particulated preparations from pea seedlings formed lipid-linked glucose from UDP (14C)-glucose and endogenous acceptors at 0 C. The product has been identified as a polyprenyl pyrophosphate-glucose based on the following results: a) the glucolipid is labile to mild acid hydrolysis; b) when chromatographed in alkaline solvents, glucose-1,2-cyclic phosphate is obtained; c) the pattern elution of a DEAE-cellulose column fits in well with a pyrophosphate derivative; d) exogenous polyprenyl phosphate stimulated the incorporation of glucose into lipids; e) when incubated with (beta-32P)-UDP-glucose, radioactivity is incorporated into lipids; f) the reaction is reversed by 5 mM UMP. It is suggested that the glucolipid is an intermediate in glycan biosynthesis.

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of alpha-1,6-mannosyltransferase which initiates outer chain formation.

A particulate fraction from the Saccharomyces cerevisiae mnn1 mutant was obtained after extracting a 115,000 x g pellet with 0.75% Triton X-100. Incubation of this preparation with labeled Man8GlcNAc and Man9GlcNAc in the presence of GDP-mannose followed by high pressure liquid chromatography showed the formation of Man9GlcNAc and Man10GlcNAc, respectively. Analysis by high resolution 1H NMR of the products indicates that, in each case, the mannose residue added is alpha-1,6-linked to the alpha-1,6-mannose residue of the substrate as follows (where M represents mannose and Gn represents N-acetylglucosamine): (Formula: see text). The mannosyltransferase therefore catalyzes the first step specific to the biosynthesis of the outer chain of yeast mannoproteins. The apparent Km values for both substrates are similar: 0.39 mM for Man8GlcNAc and 0.35 mM for Man9GlcNAc. The alpha-1,6-mannosyltransferase exhibits maximum activity between pH 7.1 and 7.6 in Tris maleate buffer, has an absolute requirement for Mn2+, and also requires Triton X-100. These results indicate that removal of the alpha-1,2-linked mannose residue from Man9GlcNAc is not essential for the alpha-1,6-mannosyltransferase which initiates outer chain synthesis, at least when oligosaccharides are used as substrates in a cell-free system.

N-methyl-1-deoxynojirimycin, a novel inhibitor of glycoprotein processing, and its effect on fowl plague virus maturation.

The glucose analogue N-methyl-1-deoxynojirimycin was found to be a specific inhibitor of the trimming of the outermost glucose residue of the N-linked precursor-oligosaccharide Glc3Man9GlcNAc2, and therefore of oligosaccharide processing, in fowl plague virus-infected chicken-embryo cells. The fowl plague virus glycoproteins in N-methyl-1-deoxynojirimycin-treated cells contain oligosaccharides of the composition Glc3ManxGlcNAc2 (x = 7, 8, and 9). Inhibition of trimming of the outermost glucose residues does not prevent release of infectious virus with oligosaccharides of the composition Glc3Man7(GlcNAc)2. On the other hand inhibition of the trimming of the innermost glucose residue does inhibit release of infectious virus (Datema, R., Romero, P. A., Legler , G., and Schwarz, R. T. Proc. Nat. Acad. Sci. USA 79, 6787-6791 (1982) ).

Comparison between 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin as inhibitors of oligosaccharide processing in intestinal epithelial cells.

The alpha-glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) inhibits the synthesis of N-linked complex oligosaccharides in rat intestinal epithelial cells to the same extent as reported previously for 1-deoxynojirimycin (DJN) [Saunier, Kilker, Tkacz, Quaroni & Herscovics (1982) J. Biol. Chem. 257, 14155-14161]. Analysis of each of the endo-beta-N-acetylglucosaminidase H (endo H)-sensitive oligosaccharides separated by h.p.l.c. with yeast glucosidase I, which specifically removes the terminal glucose residue from oligosaccharides containing three glucose residues, and with jack-bean (Canavalia ensiformis) alpha-mannosidase, indicates that both inhibitors cause the accumulation of a mixture of glucosylated oligosaccharides containing one to three glucose residues and seven to nine, and even possibly six, mannose residues. About 70% of the endo H-sensitive oligosaccharides formed in the presence of MDJN contain three glucose residues, compared with only about 20% of the corresponding oligosaccharides of the DJN treated cells. It is concluded that both compounds inhibit the formation of N-linked complex oligosaccharides by interfering with the processing glucosidases. These compounds are valuable in the study of the role of oligosaccharides in glycoproteins.

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Partial purification of the alpha-1,6-mannosyltransferase that initiates outer chain synthesis.

The alpha-1,6-mannosyltransferase (alpha-1,6-ManT) that initiates outer chain synthesis in Saccharomyces cerevisiae was partially purified along with an alpha-1,2-mannosyltransferase (alpha-1,2-ManT) that acts on alpha-methylmannoside. The enzymes were solubilized by extracting a 145,000 g pellet of S.cerevisiae mnn1 mutant with 1% Triton X-100. The extract was then passed through a concanavalin A-Sepharose column and the bound material was eluted with alpha-methylmannoside. After exhaustive dialysis, the fractions containing both mannosyltransferase activities were chromatographed on DEAE-Trisacryl which removed approximately 90% of the alpha-1,2-ManT. The fractions containing alpha-1,6-ManT and residual alpha-1,2-ManT were further purified by sequential chromatography on Sephacryl S-200 and CM-Trisacryl. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of individual fractions eluted from Sephacryl S-200 and from CM-Trisacryl, followed by silver staining of the gels, showed two major bands whose intensity corresponded to the enzyme activities. A protein band of approximately 62 kDa corresponded to the alpha-1,6-ManT and another band of approximately 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.

Glucosylation of membrane-bound proteins by lipid-linked glucose.

Particulate preparations from Pisum sativum. were able to incorporate [(14)C]glucose from UDP-[(14)C]glucose into oligosaccharide-linked lipids was formed by an oligosaccharide chain containing 7-8 glucose residues linked to dolichol, presumably via a pyrophosphate. The polymer was identified as a membrane-bound glucoprotein that could be solubilized by Triton X-100. SDS gel electrophoresis showed that a polypeptide with an apparent molecular weight of 13,000 could be glucosylated from dolichyl-phosphate-glucose. This was coincident with the electrophoretic mobility of the ß subunit of the pea lectin in the same system. The glucosylated protein was solubilized from the membranes by sonication and showed the same carbohydrate-binding ability as pea lectins. These results strongly suggest that pea lectins can be glucosylated by the lipid intermediate pathway.

The nucleotide sequence of TTP1, a gene encoding a predicted type II membrane protein.

The DNA sequence of a 2967 bp fragment located near the centromere of chromosome II, between the CEN2 and FUR4 genes, was determined. The segment contains a new open reading frame of 1794 bp. The product encoded by the gene, designated TTP1, is a predicted type II membrane protein of 597 amino acid residues with a short cytoplasmic NH2-terminus, a membrane-spanning region and a large COOH-terminal region containing three potential N-glycosylation sites. Gene disruption indicated that TTP1 is not essential for cell growth.

Effects of inhibitors of N-linked oligosaccharide processing on the biosynthesis and function of insulin and insulin-like growth factor-I receptors.

We have used specific inhibitors of oligosaccharide processing enzymes as probes to determine the involvement of oligosaccharide residues in the biosynthesis and function of insulin and insulin-like growth factor-I receptors. In a previous study (Duronio, V., Jacobs, S., and Cuatrecasas, P. (1986) J. Biol. Chem. 261, 970-975) swainsonine was used to inhibit mannosidase II, resulting in the production of receptors containing only hybrid-type oligosaccharides. These receptors had a slightly lower molecular weight and were much more sensitive to endoglycosidase H, but otherwise behaved identically to normal receptors. In this study, we used two compounds that inhibit oligosaccharide processing at earlier steps: (i) N-methyl-1-deoxynojirimycin (MedJN), which inhibits glucosidases I and II and yields glucosylated, high mannose oligosaccharides, and (ii) manno-1-deoxynojirimycin (MandJN), which inhibits mannosidase I and yields high mannose oligosaccharides. In the presence of MandJN, HepG2 cells synthesized receptors of lower molecular weight, which were cleaved into alpha and beta subunits and were able to bind hormone and autophosphorylate. These receptors were as sensitive to endoglycosidase H as receptors made in the presence of swainsonine. In the presence of MedJN, receptors of only slightly lower molecular weight than normal were synthesized and were shown to contain some glucosylated high mannose oligosaccharides. These receptors were able to bind hormone and retained hormone-sensitive autophosphorylation activity. In both cases, the incompletely processed receptors could be detected at the cell surface by cross-linking of iodinated hormone and susceptibility to trypsin digestion, although less receptor was present in cells treated with MedJN. Studies of receptor synthesis using pulse-chase labeling showed that the receptor precursors synthesized in the presence of MedJN were cleaved into alpha and beta subunits at a slower rate than normal receptors or those made in the presence of MandJN. Inhibition of oligosaccharide processing had no effect on the association of the receptor subunits into disulfide-linked oligomeric complexes.