The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.     

Lipid and carbohydrate based adjuvant/carriers in immunology.

This review discusses various issues regarding vaccines; what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both.     

Chemoenzymatic synthesis of high-mannose type HIV-1 gp120 glycopeptides

A chemoenzymatic approach to the synthesis of glycoforms of HIV-1 gp120 glycopeptides is described. Thus, the high-mannose type glycopeptides [gp120 (336-342)] containing Man(9), Man(6) and Man(5) moieties, respectively, were synthesized in satisfactory yields via transglycosylation to the acetylglucosaminyl peptide, using the recombinant Arthrobacter Endo-beta-N-acetyl-glucosaminidase (Endo-A) as the key enzyme.     

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-beta-N-acetylglucosaminidase H

A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β-N-acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [¹²⁵I]concanavalin A or [¹²⁵I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [¹²⁵I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [¹²⁵I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N- and O-glycosidic linkages where the oligosaccharide is predominantly mannosyl.     

Glycosylation and posttranslational maturation of glycoproteins in embryonal carcinomas: identification of two distinct pools of high-mannose glycans

Embryonal carcinomas and early embryonic cells assemble a family of unusually large and complex carbohydrates. These glycans contain large amounts of the sugars galactose and N-acetylglucosamine and are decorated with fucose, sulfate, and sialic acids. We show that, by their sensitivity to inhibition by tunicamycin and by their resistance to cleavage by alkaline hydrolysis, in teratocarcinoma stem cells the expression of these glycans is on asparagine-linked cores. These glycans are part of the large spectrum of glycans that are assembled on mannose cores derived from a common, lipid-linked precursor glycan. We examined the fate of this precursor glycan after its transfer to protein and found that there are two distinct pools of protein-linked, high-mannose glycans, which can be distinguished on the basis of their rate of processing. One pool is processed rapidly to provide a wide spectrum of complex-type glycans. This processing occurs efficiently with little evidence of intermediate structures. The other, larger pool remains unprocessed, beyond glucose removal, at a time when complex-type glycans cease to accumulate. In contrast, high-mannose glycans are relatively minor components of the glycans labeled during long-term, continuous labeling, and in this situation they are processed to provide a spectrum of trimmed glycans.     

Role of mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins.

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.     

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.     

Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

Endo-β-N-acetylglucosaminidase H (endo H) is an enzyme which acts on asparagine- and lipid-linked oligosaccharides containing five or more mannose residues. Complex oligosaccharides and glycopeptides are completely resistant to the action of the enzyme. We have carried out pulse-chase experiments with ³⁵S-methionine and ³H-mannose in uninfected cells and in cells infected with Sindbis virus and vesicular stomatitis virus (VSV). In each case, the labeled materials were analyzed for sensitivity to endo H by polyacrylamide gel electrophoresis and gel filtration. We find that endo H releases all the labeled mannose from pulse-labeled proteins. Initially, the released material is nearly identical in size to the endo H cleavage product derived from lipid-linked oligosaccharides present in the same cells. During chase periods, ³⁵S-methionine and ³H-mannose protein becomes increasingly resistant to the enzyme. Moreover, the ³H-mannose-labeled material released from the protein during chase periods is smaller in size than the oligosaccharide from the lipid.On the basis of these results and results from other laboratories, we propose that during glycosylation of asparagine residues, a common oligosaccharide is transferred from the lipid carrier to protein and is subsequently processed to yield the so-called “high mannose” and “complex” oligosaccharides. Since, on the basis of present evidence, the lipid-linked oligosaccharide contains two N-acetylglucosamine, 8–12 mannose and 1–2 glucose molecules, it seems probable that the carbohydrate-processing systems remove half or more of the mannose and all of the glucose residues at sites destined to become complex glycopeptides. Removal of mannose and glucose residues may also occur at sites destined to become mature high mannose glycopeptides.     

Recent advances in the chemical synthesis of mucin-like glycoproteins

This purpose of this mini review is to familiarizereaders with the tools currently available for the synthesis of mucin-typeglycoproteins. The article will highlight recent approaches to thesynthesis of glycopeptide fragments bearing complex O-linkedglycans, as well as new strategies for the generation of full-lengthglycoproteins.     

Sulphated polysaccharide synthesis in brown algae

Summary Histochemical and autoradiographic techniques have been used to investigate the sites of synthesis, transport and location of sulphated polysaccharides in some larger brown seaweeds.The most rapid uptake of ³⁵SO₄ occurred when material was incubated in medium with 10^-4M carrier sulphate, negligable uptake occurring from seawater.Autoradiography using ³⁵SO₄ has shown that in Pelvetia sulphated material is synthesised by all cell types, particularly epidermal cells. In Laminaria spp. this activity is confined to specialized secretory cells which discharge into mucilage canals. In both instances the process of carbohydrate sulphation appears to occur in the Golgi-rich perinuclear region.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Lipid synthesis during the Escherichia coli cell cycle.

Lipid synthesis was examined in Escherichia coli cells at different stage of cell division. Exponentially growing cells were pulse-labeled with appropriate isotopes for 0.1 generation time, inactivated, and separated by size on a sucrose gradient. An abrupt increase in the rate of lipid synthesis occurred which was coincident with the initiation of cross walls. In contrast, the rate of protein synthesis during this same interval remained constant, resulting in an increased lipid/protein ratio in dividing cells. No changes in the composition of phospholipid head groups, fatty acids, or phospholipid molecular species were observed in cells at different stages of division. The observed increase in the rate of lipid synthesis may reflect a means by which the activities of membrane-associated enzymes are modulated during cross wall formation.     

Lipid synthesis in the adult dog heartworm, Dirofilaria immitis.

1. Incorporation studies with three labelled substrates--[14C]2-glycerol, [14C]1-acetate and [14C]1-oleic acid--demonstrated that adult dog heartworms can synthesize all classes of complex lipids present, including free cholesterol. 2. Diacylglycerols and phosphoglycerides were most rapidly labelled regardless of the precursor employed. 3. 14C from glycerol was found in the aqueous phase of saponified lipids, whereas that from oleic acid was in the fatty acid portion. 4. Tag from acetate was predominantly in the fatty acid portion of saponified lipids and also occurred in the unesterified fatty acids. 5. Acetate and unesterified fatty acids, as represented by oleic acid, were more readily used for lipid synthesis than was glycerol.     

Lipid synthesis during morphogenesis of Mucor racemosus.

Lipid synthesis increases coordinately with protein and RNA synthesis during morphogenesis of Mucor racemosus. The lipid synthesis inhibitor cerulenin can completely block morphogenesis under conditions in which cell growth continues. An increase in phospholipid turnover may be an important correlate to morphogenesis of Mucor spp., especially the turnover of phosphotidyl inositol and phosphatidyl ethanolamine. The increase in ornithine decarboxylase, which occurs during morphogenesis, is inhibited by the addition of cerulenin.