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.     

Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids

The assembly of most of the ceramide-linked glycolipids (GSLs) in eukaryotic cells occurs in Golgi bodies. At least 18 different glycolipid:glycosyltransferases (GSL:GLTs) have been characterized, 10 of which have been solubilized. These GLTs can be classified into 2 distinct groups: 1) GLTs dedicated to either Dol-P-P-sugar(s) or ceramide-linked sugar(s); and 2) GLTs with dual loyalties (i.e., they compete with glycolipid- and glycoprotein-bound oligosaccharides). Studies with solubilized and purified GalNAcT-1 and GalNAcT-2 from embryonic chicken brains prove that GalNAcT-1 (UDP-GalNAc:GM3 beta 1-4GalNAcT) is specific for GSL, whereas GalNAcT-2 (UDP-GalNAc:Gb3 beta 1-3GalNAcT) can transfer to an oligosaccharide containing the alpha-linked terminal galactose. Similarly, GalT-3 (UDP-Gal:GM2 beta 1-3GalT) is more specific for ganglio-oligosaccharide and GalT-4 (UDP-Gal:Lc3 beta 1-4GalT) can transfer galactose to N-acetylglucosamine linked to p-nitrophenol, glycolipid or glycoprotein. Both GalT-3 and GalT-4 have been separated and purified from embryonic chicken brains. Studies with solubilized SAT-4 and SAT-3, from bovine spleen and embryonic chicken brains, respectively, suggest the existence of 2 different gene-expressed alpha 2-3SATs. The newly discovered FucT-3 (GDP-Fuc:NeuGc-iLc6-alpha 1-3FucT) from human colon carcinoma (Colo-205) has also been solubilized and separated from other GSL:GLTs. Using a new activity gel-Western blot combined technique, the molecular mass of this FucT-3 was determined to be 105 kDa.     

Polyisoprenoid epoxides stimulate the biosynthesis of coenzyme Q and inhibit cholesterol synthesis

In our search for compounds that up-regulate the biosynthesis of coenzyme Q (CoQ), we discovered that irradiation of CoQ with ultraviolet light results in the formation of a number of compounds that influence the synthesis of mevalonate pathway lipids by HepG2 cells. Among the compounds that potently stimulated CoQ synthesis while inhibiting cholesterol synthesis, derivatives of CoQ containing 1-4 epoxide moieties in their polyisoprenoid side chains were identified. Subsequently, chemical epoxidation of all-trans-polyprenols of different lengths revealed that the shorter farnesol and geranylgeraniol derivatives were without effect, whereas the longer derivatives of solanesol enhanced CoQ and markedly reduced cholesterol biosynthesis. In contrast, none of the modified trans-trans-poly-cis-polyprenols exerted noticeable effects. Tocotrienol epoxides were especially potent in our system; those with one epoxide moiety in the side-chain generally up-regulated CoQ biosynthesis by 200-300%, whereas those with two such moieties also decreased cholesterol synthesis by 50-90%. Prolonged treatment of HepG2 cells with tocotrienol epoxides for 26 days elevated their content of CoQ by 30%. In addition, the levels of mRNA encoding enzymes involved in CoQ biosynthesis were also elevated by the tocotrienol epoxides. The site of inhibition of cholesterol synthesis was shown to be oxidosqualene cyclase. In conclusion, epoxide derivatives of certain all-trans-polyisoprenoids cause pronounced stimulation of CoQ synthesis and, in some cases, simultaneous reduction of cholesterol biosynthesis by HepG2 cells.     

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi

Many microorganisms secrete surface‐active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis‐targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross‐talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.     

The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids

The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.     

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.     

Pictet-Spenglerase involved in tetrahydroisoquinoline antibiotic biosynthesis

Nonribosomal peptide synthetase (NRPS) is a programmable modular machinery that produces a number of biologically active small-molecule peptides. Saframycin A is a potent antitumor antibiotic with a unique pentacyclic tetrahydroisoquinoline scaffold. We found that the nonribosomal peptide synthetase SfmC catalyzes a seven-step transformation of readily synthesized dipeptidyl substrates with long acyl chains into a complex saframycin scaffold. Based on a series of enzymatic reactions, we proposed a detailed mechanism involving the reduction of various peptidyl thioesters by a single R domain followed by iterative C domain-mediated Pictet-Spengler reactions. This shows that NRPSs possess a remarkable capability to acquire novel function for diversifying structures of peptide natural products.     

Enzymes involved in jasmonic acid biosynthesis

(+)-7-epi-Jasmonic acid has attracted much attention as an inducible plant signal that stimulates the expression of an array of wound-inducible and defence-related genes as well as playing a role in various developmental responses. This review focuses on the enzymology of jasmonate biosynthesis and addresses open questions with respect to the regulation and subcellular localisation of the jasmonic acid pathway. The apparent analogy of jasmonate biosynthesis from a -linolenic acid in plants with that of the arachidonate-derived signalling molecules of the leukotriene- and prostaglandin-type in animals make the jasmonic acid cascade one of general biological interest.     

Application of oil refinery waste in the biosynthesis of glycolipids by yeast

Candida antarctica or Candida apicola synthesized surfactants (glycolipids) in the cultivation medium supplemented with oil refinery waste, either with soapstock (from 5.0% to 12.0% v/v) or post-refinery fatty acids (from 2.0% to 5.0% v/v). The efficiency of glycolipids synthesis was determined by the amount of waste supplemented to the medium and was from 7.3 to 13.4 g/l and from 6.6 to 10.5 g/l in the medium supplemented with soapstock and post-refinery fatty acids, respectively. The studied yeast also synthesized glycolipids in the non-supplemented medium however, by the enrichment of medium with the oil refinery waste, a 7.5-8.5-fold greater concentration of glycolipids was obtained in the post-culture liquid then in the medium without addition of oil refinery waste. The yeast synthesized from 6.6 to 10.3 g dry biomass/l and the intra-cellular fat content was from 16.8% to 30.2%. The efficiency of glycolipids synthesis was determined by yeast species, medium acidity and culture period. The surface tension of the post-culture liquid separated from yeast biomass was reduced to 35.6 mN/m, which corresponded to the surface tension obtained at the critical micelle concentration of glycolipids.     

Epididymal glycoprotein involved in rat sperm association

Recently, a new head-to-head sperm association was described in the rat during epididymal transit. This association was called a rosette and a filamentous and PAS-positive material was also described joining the sperm heads. The begining of rosette formation in the epididymis and the linking material between heads have remained unclear. Epididymides of adult rats were fixed by vascular perfussion and thin sections of the principal regions were studied by transmission electron microscopy (TEM). The first evidence of rosette formation was observed in the distal corpus. Rosettes were isolated from the distal corpus and processed for immunogold and immunofluorescence microscopy to detect an epididymal glycoprotein called DE. This glycoprotein is secreted by the corpus epididymis and appears to be involved in sperm maturation. Colloidal gold marks and fluorescence were observed in the linking material between the sperm heads. The results presented here show that rosettes begin to appear following the sites of DE secretion and permit us to postulate that DE is involved in rosette formation and constitutes another example of gamete-epididymal interaction. © 1994 Wiley-Liss, Inc.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.