Stimulation of lipid-linked oligosaccharide assembly during oviduct differentiation

Regulation of dolichyl phosphate-linked oligosaccharide assembly has been studied during the course of diethylstilbestrol-induced chick oviduct differentiation. Oviduct membranes from treated chicks form 4.6 times as much GlcNAc-P-P-Dol and GlcNAc2-P-P-Dol upon incubation with UDP-[14C]GlcNAc and MgCl2 than do membranes from untreated chicks. Assembly of oligosaccharide-lipid was studied by incubation of membranes with purified exogenous [14C]GlcNAc2-P-P-Dol and GDP-Man. Man transfer required a divalent cation (10 mM Mg2+) and detergent (0.5% Nonidet P-40 is optimal) and occurs in the presence of amphomycin (500 micrograms/ml). The apparent Km for GDP-Man is 1 microM and for [14C]GlcNAc2-P-P-Dol is 0.45 microM. The products are a series of sequentially formed dolichyl pyrophosphate-linked saccharides up to Man5GlcNAc2, the first of which is Man beta 1,4GlcNAc2. The same products are formed either in the presence or absence of amphomycin. Conversion of GlcNAc2-P-P-Dol to higher oligosaccharides is stimulated 3-fold by estrogen treatment of chicks. Similarly, the conversion of partially purified exogenously added Man beta-[14C]GlcNAc2-P-P-Dol is 4.6-fold higher after diethylstilbestrol treatment.     

Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.     

Alterations in lipid-linked oligosaccharide metabolism in human melanoma cells concomitant with induction of stress proteins

Challenge of human A375 melanoma cells with sodium arsenite induced the synthesis of stress proteins and stimulated [3H]mannose incorporation into a novel component migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 14 kDa (designated M14). Enhanced M14 expression was elicited by heavy metals (zinc, copper, cadmium, and nickel), thiol-reactive agents (iodoacetamide and auranofin), and hyperthermia. The kinetics of M14 induction and recovery from stress were similar to those of the stress proteins, but M14 half-life was only 15 min. Incorporation of [3H]mannose into M14 was inhibited by tunicamycin but not by cycloheximide or actinomycin D. M14 was metabolically labeled with [32P]orthophosphate but not by [35S] methionine or [3H]asparagine. Further studies revealed that M14 was selectively soluble in chloroform/methanol/water (10:10:3) and sensitive to both endo-beta-N-acetylglucosaminidase H digestion and mild acid hydrolysis. The latter released a water-soluble mannose-labeled moiety which eluted from Bio-Gel P-6 in a manner similar to Glc3Man9GlcNAc2. Together, these data suggest that M14 is a lipid-oligosaccharide intermediate of N-linked protein glycosylation and that enhanced expression of this class of molecule in response to chemical insults and hyperthermia is a newly described cellular reaction to stress.     

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc₃Man₉GlcNAc₂ (G₃M₉Gn₂) from the lipid-linked oligosaccharide (LLO) glucose₃mannose₉N-acetylglucosamine₂-P-P-dolichol (G₃M₉Gn₂-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2α (eIF2α) on Ser⁵¹ by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G₃M₉Gn₂-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2α Ser⁵¹ phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser⁵¹ kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.     

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.     

In vitro synthesis of a lipid-linked acetylated and pyruvilated oligosaccharide in Rhizobium trifolii

EDTA-treated Rhizobium trifolii cells (strain NA30) incorporate radioactivity from (14C) labeled UDP-Clc, UDP-ClcA, Acetyl-Coa and/or phosphoenol pyruvate into chloroform: methanol: water (1:2:0.3) extracts. The incorporation products have properties of prenyl-phospho-sugars; mild alkaline hydrolysis of these extracts produce cyclic phosphate esters suggesting the presence of a diphosphate bridge, and mild acid or catalytic reduction-alkaline phosphatase treatments release four main components a, b, c and d, as judged by paper electrophoresis and chromatography and gel filtration studies. The four components can be obtained (14C)acetyl-labeled, but only compound c and to a lesser degree compound b can be (14C)pyruvate-labeled. For the exopolysaccharide produced by this strain the following repeating unit has been proposed (Robertsen et al. (1981), Plant Physiol. 67, 389-400): (Formula: see text). The results obtained suggest that the octasaccharide repeating unit (compound a) with one (compound b) or two (compound c) ketal pyruvate residues are assembled on a lipid acceptor. All these compounds are assumed to be intermediates in the biosynthesis of R. trifolii exopolysaccharide.     

Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Detection of the lipid-linked precursor oligosaccharide of N-linked protein glycosylation in Drosophila melanogaster

The presence of a glycan of the same molecular size as the lipid linked precursor oligosaccharide (Glc₃Man₉GlcNAc₂) of the N-linked protein glycosylation pathway in mammalian cells has been detected in a glycolipid fraction of cultured Drosophila melanogaster cells. Oligosaccharide sequencing studies were consistent with the existence of a glucosylated high mannose containing structure, which may be the common precursor for N-linked protein glycosylation in insect cells.     

Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells.

Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.     

Regulation of TRP channels by N-linked glycosylation

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.     

The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation

The biosynthesis of the lipid-linked oligosaccharide substrate for N- linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid- linked Glc2Man9GlcNAc2. We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose- dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide. Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. This ensures that primarily completely assembled oligosaccharide is transferred to protein.      

Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.     

Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins

Hamster sarcoma virus (HSV) transformation of Nil-8 fibroblasts is associated with an increase in the average size of N-acetyllactosamine (complex) type N-linked glycans due to an increase in both the average number of branches/chain and in the fraction of N-linked glycans containing poly(GlcNAc(beta 1,3) Gal-(beta 1,4)) (polylactosaminylglycan) chains. Analysis of glycopeptides from the envelope glycoproteins of Sindbis virus and vesicular stomatitis virus (VSV) grown in Nil-8 and Nil/HSV cells indicated that the transformation-associated shift to larger N-linked oligosaccharides selectively affects some glycosylation sites far more than others. Glycosylation of the Sindbis virus glycoproteins and of Asn-179 of VSV G was similar in Nil-8 and Nil/HSV cells; oligosaccharide processing generally did not proceed beyond the biantennary complex stage. In contrast, Asn-336 of VSV G carried primarily biantennary complex glycans in Nil-8-grown virus (ratio, triantennary, and larger to biantennary complex glycans (tri+/bi) = 0.5) but more highly branched structures in Nil/HSV-grown virus (tri+/bi = 8.1). All of the triantennary or larger oligosaccharides from Asn-336 of Nil/HSV-grown VSV G bound to leukoagglutinating phytohemagglutinin-agarose, indicating the presence of a branch attached to the Man3GlcNAc2 core via a beta 1,6-linked GlcNAc residue and suggesting that increased UDP-GlcNAc:alpha-D-mannoside beta 1,6-N-acetylglucosaminyl transferase V (GlcNAc transferase V) activity accompanied transformation. At least 20% of these leukoagglutinating phytohemagglutinin-binding oligosaccharides were sensitive to an enzyme specific for polylactosaminylglycan chains, Escherichia freundii endo-beta-galactosidase.     

Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-Man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Chinese hamster ovary cells express a wide variety of glycoproteins with M_r ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate specificity and having no detectable type II kinase (mutant 10215); or (ii) an altered RI subunit and having no detectable type II kinase (mutant 10248); or (iii) exhibited the lowest level of total kinase with no detectable type I kinase but having a small amount of type II kinase (mutant 10260). Addition of 8Br-cAMP enhanced protein glycosylation index in wild type cells 10001 by 120% but only 7 to 23% in the mutant cells. The rate of lipid-linked oligosaccharide (LLO) biosynthesis was linear for 1 h in all cell types, but the total amount of LLO expressed was much less in PKA-deficient mutants. Pulse-chase experiments indicated that the t_1/2 for LLO turnover was also twice as high in PKA-deficient cells as in the wild type. Size exclusion chromatography of the mild-acid released oligosaccharide confirmed that both wild type and the mutant cells synthesized Glc₃Man₉GlcNAc₂-PP-Dol as the most predominating species with no accumulation of Man₅GlcNAc₂-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a K_m for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the k_cat for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the k_cat for DPMS in CHO cell mutants; however, in vitro protein phosphorylation with a cAMP-dependent protein kinase restored their kinetic activity to the level of the wild type. Published in 2004.     

Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells

The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation derived from hamster BHK21 cell line. It has a mutation in the DAD1 gene and enters apoptosis at the restrictive temperature of 39 degrees C. The defect of Dad1p causes a loss of N-linked glycosylation; therefore, it was thought that an inhibition of N-linked glycosylation induced apoptosis.However, tunicamycin, a potent inhibitor of N-linked glycosylation, had not caused apoptosis in wild-type BHK21 cells. In order to clarify this discrepancy, wild-type BHK21 cells treated with tunicamycin and tsBN7 cells incubated at 39.5 degrees C were examined by the annexin V staining and TUNEL methods. Both methods showed that tunicamycin induces apoptosis in wild-type BHK21 cells, similar to the defect of Dad1p. Thus, we concluded that loss of N-linked glycosylation causes apoptosis.