Biomedical subjects

A Herscovics

Publications and source records attributed to A Herscovics.

At least 19 recordsLinked to original sources

PubMed1992

Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae.

The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.

Animals↗

PubMed1991

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase.

We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.

Amino Acid Sequence↗

PubMed1991

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.

Adenocarcinoma↗

PubMed1991

Multiple apomucin translation products from human respiratory mucosa mRNA.

Poly(A)-rich RNA was purified from a pool of five human tracheobronchial mucosa. After in vitro translation in a reticulocyte lysate and immunoprecipitation of the translated products, using either a polyclonal antiserum or a monoclonal antibody to deglycosylated respiratory mucin peptides, the products were characterized by SDS/PAGE. The respiratory mucin precursors migrated as a very large smear from almost the top of the resolving polyacrylamide gel to an area corresponding to a molecular mass of about 100 kDa. After hybridization with mucin cDNA probe TH 29 described by Crepin et al. [Crepin, M., Porchet, N., Aubert, J. P. & Degand, P. (1990) Biorheology 27, 471-484] respiratory mucin mRNAs also appeared polydisperse. Although degradation or incomplete translation of high-molecular-mass mRNA cannot be entirely ruled out, these results suggest that human respiratory apomucins consist of a family of peptides which share some common epitopes. This possibility is in agreement with (a) the diversity of mucin precursors observed previously with pulse/chase experiments performed with explants of human respiratory mucosa and (b) the polydispersity of secreted respiratory mucins observed by electron microscopy.

Animals↗

PubMed1991

Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae.

The in vitro specificity of the alpha 1-6 mannosyltransferase that initiates outer chain formation in Saccharomyces cerevisiae (Romero and Herscovics, J. Biol. Chem., 264, 1946-1950, 1989) was reassessed by fast atom bombardment mass spectrometry (FAB-MS). A particulate fraction from the mnn1 mutant was incubated with GDP-mannose and either Man9GlcNAc (M9T) isolated from thyroglobulin or Man8GlcNAc (M8Y) obtained by treatment of the M9T with the yeast specific mannosidase. The Man10GlcNAc (M10Y) and Man9GlcNAc (M9Y) oligosaccharides thus obtained, and the substrate oligosaccharides, were peracetylated or perdeuteroacetylated and submitted to FAB-MS using meta-nitrobenzylalcohol as the matrix. The latter was chosen as the matrix because it enhances the abundance of high-mass-fragment ions of peracetylated oligosaccharides and thereby facilitates the assignment of branching patterns. The results indicate that the alpha 1-6 mannosyltransferase catalyses the addition of mannose to the alpha 1-3 mannose residue, and thus provide additional new evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al. (J. Biol. Chem., 264, 11849-11856, 1989). [formula: see text] where Gn is N-acetylglucosamine, M is mannose and M is mannose added by the enzyme.

Carbohydrate Sequence↗

PubMed1989

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.

Adenocarcinoma↗

PubMed1989

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.

Glycoproteins↗

PubMed1989

Identification of human tracheo-bronchial mucin precursors.

Bronchial mucin peptide chains were obtained by performing a two-step chemical deglycosylation of the highly glycosylated regions (or glycopeptides) which are the most characteristic part of bronchial mucins. The deglycosylated preparation was used to prepare an antiserum directed against mucin peptide epitopes. This antiserum reacted with the area containing rough endoplasmic reticulum of goblet cells and of mucous gland of human bronchial mucosa but not with secretory vesicles. The antiserum was used for immunoprecipitation of radiolabelled mucin precursors in pulse-chase experiments with explants of human bronchial mucosa. SDS-polyacrylamide gel electrophoresis followed by fluorography revealed precursors with a molecular mass in the range of 200 to 400 kDa as early as after 10 min pulse labeling with [3H]threonine. These results suggest that the mucin polydispersity previously visualized by electron microscopy may be explained by the synthesis of several respiratory mucin peptide precursors with different molecular sizes and/or that precursors with different amounts of carbohydrate are rapidly formed.

Bronchi↗

PubMed1988

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc.

A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text). The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of about 60 kDa in the absence of reducing agent, and as two bands of about 44.5 kDa and 22.5 kDa in the presence of reducing agent. The apparent molecular weight of the soluble enzyme is about 75,000 by gel filtration on Sephacryl S-200. The specific alpha-mannosidase does not require the addition of divalent cation for activity, but it is inhibited by Tris, EDTA, Mn2+, Co2+, Zn2+, and Mg2+. The inhibition caused by EDTA can be reversed completely by Ca2+ and partially by Mg2+, but not by other divalent cations. The soluble alpha-mannosidase arises from a larger hydrophobic form of the enzyme which is found in the detergent phase during partition in Triton X-114. The formation of the soluble enzyme, which is recovered in the aqueous phase during partition in Triton X-114, is time- and temperature-dependent and is prevented by pepstatin, but not by other protease inhibitors. These results indicate that the purified soluble alpha-mannosidase represents the catalytically active domain of the enzyme which has been proteolytically released from its membrane-bound form.

Carbohydrate Sequence↗

PubMed1988

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.

Animals↗

PubMed1988

Inhibition of myoblast fusion by bromoconduritol.

It has recently been reported that the glucosidase I inhibitor, N-methyl-1-deoxynojirimycin (MDJN), inhibits myoblast fusion whereas the mannosidase inhibitor, 1-deoxymannojirimycin (ManDJN), has no effect on fusion. We now report that bromoconduritol, which is an active-site-directed covalent inhibitor of glucosidase II, also inhibits fusion at concentrations that have no effect on the plating efficiency or growth of rat L6 myoblasts. Significant inhibition of fusion was obtained at concentrations as low as 50 micrograms of bromoconduritol/mL, whereas inhibition of cell growth did not occur until concentrations of 250 micrograms/mL were reached. Rat L6 myoblasts were grown in the presence and absence of processing inhibitors and were surface labelled with 125I. Analysis of the iodinated proteins by two-dimensional gel electrophoresis demonstrated that a number of high-molecular-weight proteins (greater than 90,000) detected at the surface of control cells were absent from the surface of cells treated with MDJN or bromoconduritol. It is suggested that MDJN and bromoconduritol prevent the translocation of these proteins to the cell surface. The high-molecular-weight proteins detected at the surface of control cells were also detectable in ManDJN-treated cells, indicating that inhibition of N-linked complex oligosaccharide formation does not affect the translocation of these proteins to the myoblast cell surface.

1-Deoxynojirimycin↗

PubMed1987

Differentiation-associated decrease in the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonic adenocarcinoma cells.

CaCo-2 cells are human colonic adenocarcinoma cells which can differentiate spontaneously into enterocytes when maintained confluent for extended periods of time. Cells kept in culture for 4 days (rapidly growing), 7-9 days (early confluence) and 19-22 days (late confluence) were incubated for 24 h with L-[5,6-3H]fucose or D-[6-3H]glucosamine in order to examine the changes in glycoprotein carbohydrate structure that occur during this differentiation. Labelled glycopeptides obtained by exhaustive Pronase digestion of the cell-surface and cell-pellet fractions were fractionated on Bio-Gel P-6. A high-Mr glycopeptide fraction which was excluded from Bio-Gel P-6 was present in all cases. These glycopeptides were then fractionated by affinity chromatography on Datura stramonium agglutinin-agarose. The glycopeptides which were specifically bound to the lectin column were largely degraded by endo-beta-galactosidase, thereby indicating that they consisted of fucosylated polylactosaminoglycans. The proportion of labelled polylactosaminoglycans decreased with increasing time in culture, whereas sucrase activity, which is characteristic of differentiated enterocytes, increased. These results demonstrate that a relatively large decrease in the proportion of fucosylated polylactosaminoglycans occurs with differentiation of CaCo-2 cells.

Adenocarcinoma↗

PubMed1987

A rapid method for assay of glycosidases involved in glycoprotein biosynthesis.

A rapid procedure to measure processing glycosidases with labeled oligosaccharide as substrate is described, using assay of the specific processing alpha-mannosidase from Saccharomyces cerevisiae as an example. After incubation of [3H]mannose-labeled Man9GlcNAc with the mannosidase, a solution of concanavalin A is added, followed by polyethylene glycol to precipitate the oligosaccharide-lectin complex. The radioactivity present in the supernatant after centrifugation is then measured to determine the amount of labeled mannose released. It is shown that the results of this procedure are similar to those obtained previously using small columns of concanavalin A-Sepharose (B. Saunier, R. D. Kilker, Jr., J. S. Tkacz, A. Quaroni, and A. Herscovics (1982) J. Biol. Chem. 257, 14155-14161). The precipitation procedure, which can be applied to the assays of other processing enzymes, is much more convenient when a large number of samples must be analyzed.

Concanavalin A↗

PubMed1986

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.

1-Deoxynojirimycin↗

PubMed1986

Inhibition of myoblast fusion by the glucosidase inhibitor N-methyl-1-deoxynojirimycin, but not by the mannosidase inhibitor 1-deoxymannojirimycin.

The effects of N-linked-oligosaccharide-processing inhibitors on the fusion of rat L6 myoblasts to form myotubes were examined. The glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) greatly inhibited fusion, whereas the mannosidase inhibitor 1-deoxymannojirimycin (ManDJN) had relatively little effect, although both compounds prevented the formation of N-linked complex oligosaccharides. These results indicate that complex oligosaccharides on glycoproteins do not play a role in myoblast fusion. With MDJN, high-mannose oligosaccharides containing three glucose residues and seven to eight mannose residues were found at the cell surface, whereas with ManDJN, non-glucosylated high-mannose oligosaccharides with seven to nine mannose residues were obtained. These results indicate that the persistence of glucose residues on high-mannose oligosaccharides may be responsible for the inhibition of fusion. It is suggested that glucose either masks the cell-surface recognition process leading to fusion or prevents the cell-surface expression of specific glycoprotein(s) essential to the fusion process.

1-Deoxynojirimycin↗

PubMed1986

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.

1-Deoxynojirimycin↗

PubMed1985

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].

1-Deoxynojirimycin↗

PubMed1985

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.

1-Deoxynojirimycin↗