Asparaginyl-N-acetylgalactosamine. Linkage unit of halobacterial glycosaminoglycan

The cell surface glycoprotein of Halobacteria contains two different types of sulfated saccharides: hexuronic acid-containing oligosaccharides linked to the protein via asparaginylglucose, and a serially repeated saccharide unit containing amino sugars that resembles the animal glycosaminoglycans. Here we report that 1) the sulfated repeating unit saccharide is linked to the cell surface glycoprotein via asparaginyl-N-acetylgalactosamine, 2) the amino acid sequence surrounding this linkage region is -Asn-Ala-Ser-, and thus in agreement with the acceptor sequence ASN-X-Thr(Ser) common to all eucaryotic N-glycosidically bound saccharides determined so far; 3) in addition to galactose, galacturonic acid, N-acetylglucosamine, and N-acetylgalactosamine, the methylated hexuronic acid 3-O-methylgalacturonic acid occurs as a stoichiometric constituent of the sulfated building block of the glycosaminoglycan chain.     

Biosynthesis of sulfated saccharides N-glycosidically linked to the protein via glucose. Purification and identification of sulfated dolichyl monophosphoryl tetrasaccharides from halobacteria

A novel type of N-glycosidic linkage, asparaginyl glucose, occurs in the cell surface glycoprotein of halobacteria (Wieland, F., Heitzer, R., and Schaefer, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5470-5474). Sulfated oligosaccharides containing glucuronic acids are attached to the polypeptide chain via this linkage. Here we describe the isolation and chemical characterization of lipid-linked precursors of these saccharides, and these have the following new features. Rather than the bacterial undecaprenol, a C60-dolichol is the carrier lipid. The oligosaccharide is bound to this lipid via a monophosphate, rather than a pyrophosphate bridge. Sulfation of the saccharides is completed while they are linked to lipid and does not occur after transfer of the saccharides to protein.     

Transient methylation of dolichyl oligosaccharides is an obligatory step in halobacterial sulfated glycoprotein biosynthesis

Biosynthesis of sulfated saccharides that are linked to asparagine residues in the cell surface glycoprotein of Halobacterium halobium via a glucose residue involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates (Lechner, J., Wieland, F., and Sumper, M. (1985) J. Biol. Chem. 260, 860-866). During isolation and characterization of these lipid oligosaccharides we detected a group of related compounds containing additional unidentified sugar residues. Here we report that: 1) the unknown sugar residues were 3-O-methylglucose, linked peripherally to the lipid-saccharide intermediates; 2) the 3-O-methylglucose residues in the oligosaccharides occur only at the lipid-linked level but are absent at the protein-linked level; 3) cell surface glycoprotein biosynthesis in Halobacteria in vivo is drastically depressed when S-adenosylmethionine-dependent methylation is inhibited, indicating that methylation is an obligatory step during glycoprotein synthesis. We propose a mechanism for the transport of lipid oligosaccharides through the cell membrane, involving an intermediate stage in which the saccharide moieties are transiently modified with 3-O-methylglucose.     

Sequence of the halobacterial glycosaminoglycan

The cell-surface glycoprotein of halobacterium contains a sulfated repeating unit saccharide chain, similar to the mammalian glycosaminoglycans. The composition of a presumptive repeating pentasaccharide unit of this glycosaminoglycan is 1 GlcNAc, 1 GalNAc, 1 Gal, 1 GalA (where GalA represents galacturonic acid), 1 3-O-methyl-GalA, and 2 SO42-. Linkage to protein of this glycoconjugate involves the hitherto unique unit Asn-GalNAc, with the N-linked asparagine residue being the second NH2-terminal amino acid and part of the common N-linked glycosyl acceptor sequence Asn-X-Thr(Ser). Transfer of the completed, sulfated glycosaminoglycan from its lipid precursor to the protein occurs at the cell surface, and the presence of this sulfated saccharide chain in the cell-surface glycoprotein seems to be required to maintain the structural integrity of the rod-shaped halobacteria. In this paper, we report the complete saccharide structure of this N-linked glycosaminoglycan. This structure is deduced from chemical analyses of fragments that were isolated after hydrazinolysis and subsequent nitrous acid deamination or after mild acidic hydrolysis of purified Pronase-derived glycosaminoglycan-peptides. The halobacterial glycosaminoglycan consists, on the average, of 10 repeating pentasaccharide units of the following structure. (formula: see text) The reducing end N-acetylgalactosamine residue is linked directly to the asparagine, without a special saccharide linker region.     

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.     

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.     

Halobacterial flagellins are sulfated glycoproteins

The cell-surface glycoprotein of Halobacteria contains oligosaccharides of the type Glc4----1GlcA4----1GlcA4----1GlcA (where GlcA indicates glucuronic acid) with a sulfate group attached to each of the GlcA residues. We report here that in addition to this cell-surface glycoprotein, the halobacterial flagellar proteins (recently described by Alam, M., and Oesterhelt, D. (1984) J. Mol. Biol. 176, 459-475) also contain the same type of sulfated oligosaccharides. These flagellins have the following features. All of the individual flagellar proteins contain identical sulfated saccharide moieties linked to the amido nitrogen of Asn through a Glc residue (the novel type of N-glycosidic linkage that has been found in the cell-surface glycoprotein from Halobacteria (Wieland, F., Heitzer, R., and Schaefer, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5470-5474)). The amino acid sequence of one carbohydrate-binding region is Gln-Ala-Ala-Gly-Ala-Asp-Asn-Jle-Asn-Leu-Thr-Lys. This surrounding sequence CHO is consistent with the general formula Asn-X-Thr(Ser), common to all N-linked glycopeptides determined so far. Biosynthesis of flagellar glycoconjugates involved sulfated oligosaccharides linked to dolichol monophosphate. The individual glycoproteins making up the flagella are structurally closely related to one another.     

Carbohydrates and fertilization in animals

A frequently used mechanism for sperm-egg recognition in many species involves complementary protein-carbohydrate interaction. The usual paradigm includes complex glycoconjugates in reproductive tract fluids or on the eggs which are recognized by carbohydrate-binding proteins on the sperm surface. Various glycoconjugates are utilized in the steps of sperm capacitation, sperm binding to the egg extracellular matrix and vitelline membrane and induction of the acrosome reaction. Several types of complex glycoconjugates are involved in these processes, including proteoglycans, lactosaminoglycans, sulfated fucose-containing glycoconjugates, and glycoproteins. There appear to be some structural similarities between active glycoconjugates; they are large in molecular weight and complex, and they are often sulfated, fucosylated, and attached to a protein through serine or threonine residues. In some species, the protein core of the glycoconjugates also participates in the interaction by limiting the binding of carbohydrates to sperm only of the relevant species, likely by providing the proper steric arrangement for the interaction. In other cases the protein core seems to serve more as a crosslinker of the carbohydrate moieties. This review discusses the types of glycoconjugates implicated in fertilization and the complementary lectin-like proteins found on sperm.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

Novel N-glycosylation in eukaryotes: laminin contains the linkage unit beta-glucosylasparagine

The linkage unit to protein of N-linked carbohydrate in eukaryotic glycoproteins consists of N-acetylglucosamine, coupled to the amido nitrogen of asparagine. Additional N-glycosyl linkage units have been unequivocally proven to exist only in the cell surface glycoproteins of various bacteria. Based on immunological analyses, isolation and chemical characterization, we report that one of these units, namely glucose linked to asparagine, exists in the mammalian protein laminin, an extracellular basement membrane component. This finding and the occurrence of identical disaccharide structures in archaebacterial cell surface glycoproteins and mammalian basement membrane protein complexes points towards a conserved and distinct function of these extracellular structural elements. In addition, a method is described to uncover a masked epitope in fixed tissues by chemical O-deglycosylation. This has allowed to morphologically localize the antigen beta-Glc-Asn by immunofluorescence to the basement membranes of kidney glomeruli.     

Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos.

Embryos of the sea urchin, Stronglyocentrotus purpuratus, synthesize several classes of sulfated and non-sulfated glycoproteins during gastrulation. The antibiotic tunicamycin, which is a specific inhibitor of the N-glycosylation of proteins, inhibits the synthesis of lipid-linked oligosaccharides in these embryos at concentrations which have little effect on the biosynthesis of other classes of glycolipids or on protein synthesis. As a consequence of this inhibition, glycoproteins with oligosaccharide side chains of the general type (Man)5-7-(GlcNAc)2 are not synthesized. In addition, the biosynthesis of a novel class of sulfated glycoproteins is inhibited. In contrast, no effect upon the synthesis of sulfated glycosaminoglycans is seen. The morphogenetic consequence of tunicamycin treatment is that development of embryos from the mesenchyme blastula to the gastrula stage is arrested. The results provide evidence that during development glycoproteins containing both unsulfated and sulfated N-glycosidically linked oligosaccharide chains are synthesized via the lipid-linked pathway. The biosynthesis of these molecules appears to be a prerequisite to the differentiation and morphogenesis that occurs during gastrulation.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In the accompanying paper (Green, E.D., and Baenziger, J.U. (1987) J. Biol. Chem. 262, 25-35), we elucidated the structures of the anionic asparagine-linked oligosaccharides found on the bovine, ovine, and human pituitary glycoprotein hormones. In this study, we determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear alpha-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by greater than 10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (alpha 2,3 versus alpha 2,6). In addition to differences in the proportion of sulfated and sialylated structures on LH and FSH, there were site-specific variations in the amount of mono- and disulfated oligosaccharides at different glycosylation sites on LH alpha-beta dimers. The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.     

Characterization of the major sulfated protein of mouse pancreatic acinar cells: A high molecular weight peripheral membrane glycoprotein of zymogen granules

The major sulfated protein of the mouse pancreatic acinar cell, gp300, hsa been identified and characterized with monoclonal and polyclonal antibidies. gp300 is a glycoprotein of M_r = 300,000 which contains ～40% of metabolically incroporated [³⁵S] sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis. demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with [^3H]glucosamine and [³⁵H]sulfate was chemically and enzymaticlly treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycan. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released ～17% of [₃S]. Mild alkaline borohydride treatment after removal of N-linked sugar relased the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicating the presence of sulfated O-linked oligosaccharides. Biosunynthetic studies and PNGase digestion F digestion indicate that the core protein is ～210 KDa, with apparent contrinution of ～35 KDa N-linked sugar, and ～55 KDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presece of Galβ(1–3)GalNAc and sialic acid α(2–3)Gal in O-linked oligosaccharide, and Galβ(1–4)GLcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral memberane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in reponse to hormone stimulation ofacini, so it is not a secertroy product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule bigeneses.     

Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.     

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.     

Isolation and characterization of low sulfated heparan sulfate sequences with affinity for lipoprotein lipase

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.     

Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.     

Formation of mannosyl-lipids by an ectomannosyltransferase in suspension of BALB/c fibroblasts

A mannosyltransferase has been detected in suspensiosn of BALB/c fibroblasts incubated with GDP-[¹⁴C]mannose. The experimental evidence indicates the cell surface as the most likely site for the enzyme. The transferase synthesizes both glycolipids and glycoproteins. The lipid compounds have properties suggestive of lipid-linked mono- and oligosaccharides which can function as intermediates in glycoprotein synthesis. The formation of these compounds by a cell surface enzyme suggests that lipid-linked intermediates may play an important role in the glycosylation of membrane components.     

Isolation of a temperature-sensitive FM3A mutant deficient in asparagine-linked glycosylation by selecting for resistance to tritiated mannose suicide

Protein glycosylation mutants in the mouse mammary carcinoma cell line FM3A were selected for ability to withstand exposure to [2-3H]mannose at 39 degrees C. G258 , one of the mutant cells isolated, has been characterized. G258 cells were temperature-sensitive for cell growth. Moreover, G258 cells showed temperature sensitivity for [3H]mannose incorporation into the TCA-insoluble fraction. To study the biochemical basis of the defect in glycoprotein biosynthesis, the formation of lipid-linked saccharides was examined. The results showed that the formation of lipid-linked oligosaccharides was severely inhibited in G258 cells at 39 degrees C. At 33 degrees C, G258 cells synthesized Glc3Man9GlcNAc2-PP-Dol, the fully assembled lipid-linked oligosaccharides, but at 39 degrees C, G258 cells were able to synthesize merely the smaller lipid-linked oligosaccharides (approximately up to Man3GlcNAc2 -PP-Dol), but were unable to synthesize the larger lipid-linked oligosaccharides.