Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of alpha-1,6-mannosyltransferase which initiates outer chain formation

A particulate fraction from the Saccharomyces cerevisiae mnn1 mutant was obtained after extracting a 115,000 x g pellet with 0.75% Triton X-100. Incubation of this preparation with labeled Man8GlcNAc and Man9GlcNAc in the presence of GDP-mannose followed by high pressure liquid chromatography showed the formation of Man9GlcNAc and Man10GlcNAc, respectively. Analysis by high resolution 1H NMR of the products indicates that, in each case, the mannose residue added is alpha-1,6-linked to the alpha-1,6-mannose residue of the substrate as follows (where M represents mannose and Gn represents N-acetylglucosamine): (Formula: see text). The mannosyltransferase therefore catalyzes the first step specific to the biosynthesis of the outer chain of yeast mannoproteins. The apparent Km values for both substrates are similar: 0.39 mM for Man8GlcNAc and 0.35 mM for Man9GlcNAc. The alpha-1,6-mannosyltransferase exhibits maximum activity between pH 7.1 and 7.6 in Tris maleate buffer, has an absolute requirement for Mn2+, and also requires Triton X-100. These results indicate that removal of the alpha-1,2-linked mannose residue from Man9GlcNAc is not essential for the alpha-1,6-mannosyltransferase which initiates outer chain synthesis, at least when oligosaccharides are used as substrates in a cell-free system.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions.     

Metabolism of cartilage proteins in cultured tissue sections.

The asparagine-linked oligosaccharides of the complex acidic-type from [3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined with extensive digestion with endo- and exo-glycosidases from bacterial and eukaryotic sources. The neutral products from the digestion with a mixture of exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral oligosaccharides that were all converted by digestion with eukaryotic beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small oligomannosyl core containing two alpha-linked mannose residues and a third mannose residue beta-linked to N-acetylglucosamine. These studies suggested that the complex acidic-type oligosaccharides from cellular and viral membrane glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of outer branch N-acetylglucosamine residues resulting in partial resistance to beta-N-acetylglucosaminidase from a bacterial source.     

Comparative study of lysosomal and cytosolic catabolisms of oligomannosidic-type glycans

In order to study the substrate specificities of the enzymes implicated in the catabolism of oligomannosidic-type glycans, the oligosaccharides Man9GlcNAc and Man5GlcNAc were incubated with rat liver lysosomal and cytosolic alpha-D-mannosidases and the hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Although they both occur in an ordered way, the two catabolic pathways are quite different. The lysomal pathway is realized in two stages: the first leads from Man9GlcNAc to Man5GlcNAc by preferential cleavage of the four alpha-1,2-linked mannose residues, and the second, Zn(2+)-dependent, leads from Man5GlcNAc to Man (beta 1-4) GlcN Ac by hydrolysis of alpha-1, 3- and alpha-1,6-linked residues. On the contrary, the cytosolic pattern leads by a pathway quite different to a unique hexasaccharide Man5GlcNAc which has, curiously, the same structure as one of the polyprenolic intermediates occurring in the cytosol during the biosynthesis of N-glycosylprotein glycans: Man (alpha 1-2) Man (alpha 1-2) Man (alpha 1-3) [Man (alpha 1-6)] Man (beta 1-4) GlcN Ac (beta 1-4) GlcNAc alpha 1-P-P-Dol.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Evaluation of the role of rat liver Golgi endo-alpha-D-mannosidase in processing N-linked oligosaccharides

Golgi membranes from rat liver have been shown to contain an endo-alpha-D-mannosidase which can convert Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1----3Man (Lubas, W. A., and Spiro, R. G. (1987) J. Biol. Chem. 262, 3775-3781). We now report that this enzyme has the capacity to cleave the alpha 1----2 linkage between the glucose-substituted mannose residue and the remainder of the polymannose branch in a wide range of oligosaccharides (Glc3Man9GlcNAc to Glc1Man4GlcNAc) as well as glycopeptides and oligosaccharide-lipids. Whereas the tri- and diglucosylated species (Glc3Man9GlcNAc and Glc2Man9GlcNAc), which yielded Glc3Man and Glc2Man, respectively, were processed more slowly than Glc1Man9GlcNAc, the monoglucosylated components with truncated mannose chains (Glc1Man8GlcNAc to Glc1Man4GlcNAc) were trimmed at an increased rate which was inversely related to the number of mannose residues present. The endomannosidase was not inhibited by a number of agents which are known to interfere with N-linked oligosaccharide processing by exoglycosidases, including 1-deoxynojirimycin, castanospermine, bromoconduritol, 1-deoxymannojirimycin, swainsonine, and EDTA. However, Tris and other buffers containing primary hydroxyl groups substantially decreased its activity. After Triton solubilization, the endomannosidase was observed to be bound to immobilized wheat germ agglutinin, indicating the presence of a type of carbohydrate unit consistent with Golgi localization of the enzyme. The Man8GlcNAc isomer produced by endomannosidase action was found to be processed by Golgi enzymes through a different sequence of intermediates than the rough endoplasmic reticulum-generated Man8GlcNAc variant, in which the terminal mannose of the middle branch is absent. Whereas the latter oligosaccharide is converted to Man5GlcNAc via Man7GlcNAc and Man6GlcNAc at an even rate, the processing of the endomannosidase-derived Man8GlcNAc stalls at the Man6GlcNAc stage due to the apparent resistance to Golgi mannosidase I of the alpha 1,2-linked mannose of the middle branch. The results of our study suggest that the Golgi endomannosidase takes part in a processing route for N-linked oligosaccharides which have retained glucose beyond the rough endoplasmic reticulum; the distinctive nature of this pathway may influence the ultimate structure of the resulting carbohydrate units.     

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.     

Characterization of lipid-linked octa- through undecasaccharides implicated in the biosynthesis of Saccharomyces cerevisiae mannoproteins.

The lipid-linked oligosaccharide Glc3-Man9(GlcNAc)2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) serves as a precursor for the biosynthesis of the inner core portion of the asparagine-linked polysaccharide of Saccharomyces cerevisiae mannoproteins. It has been shown previously that incubation of a microsomal preparation from this organism with UDP-N-acetylglucosamine and GDP-[14C]mannose gives rise to a series of lipid-linked oligosaccharides of the general structure Mann(GlcNAc)2, with n from 1 to 9. A structural characterization of Man1- to Man5(GlcNAc)2 oligosaccharides indicated that the major structures among these were identical to the intermediates proposed for the biosynthesis of animal glycoproteins (C. Prakash and I. K. Vijay, Biochemistry 21:4810-4818, 1982). In the present study, the structural characterization of the Man6- through Man9(GlcNAc)2 species was conducted. The Man6- through Man8(GlcNAc)2 species have two isomers, whereas Man9(GlcNAc)2 is monoisomeric. One isomer each of Man6- through Man8(GlcNAc)2 and the monoisomeric Man9(GlcNAc)2 are identical to the intermediates for the biosynthesis of asparagine-linked glycoproteins in animal systems. It is proposed that the steps of the lipid-linked assembly of the carbohydrate precursor for S. cerevisiae mannoproteins are identical to those of the major pathway in animal systems. A lack of acceptor substrate specificity by the mannosyltransferases, as observed with in vitro studies with animal systems, also might be responsible for the biosynthesis of multiple isomers reported here.     

Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

On the stringent requirement of mannosyl substitution in mannooligosaccharides for the recognition by garlic (Allium sativum) lectin. A Surface Plasmon Resonance Study

The kinetics of the binding of mannooligosaccharides to the heterodimeric lectin from garlic bulbs was studied using surface plasmon resonance. The interaction of the bound lectin immobilized on the sensor chip with a selected group of high mannose oligosaccharides was monitored in real time with the change in response units. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal alpha-1,2-linked mannose residues. An increase in binding propensity can be directly correlated to the addition of alpha-1,2-linked mannose to the mannooligosaccharide at its nonreducing end. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied, exhibited the greatest binding affinity (Ka = 1.2 x 10(6) m(-1) at 25 degrees C). An analysis of these data reveals that the alpha-1,2-linked terminal mannose on the alpha-1,6 arm is the critical determinant in the recognition of mannooligosaccharides by the lectin. The association (k1) and dissociation rate constants (k(-1)) for the binding of Man9GlcNAc2Asn to Allium sativum agglutinin I are 6.1 x 10(4) m(-1) s(-1) and 4.9 x 10(-2) s(-1), respectively, at 25 degrees C. Whereas k1 increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k(-1) decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with Allium sativum agglutinin I with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition.     

Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides.

The genome of Saccharomyces cerevisiae contains five genes that encode type II transmembrane proteins with significant amino acid similarity to the alpha-1,3-mannosyltransferase Mnn1p. The roles of the three genes most closely related to MNN1 were examined in mutants carrying single and multiple combinations of the disrupted genes. Paper chromatographic analysis of [2-3H]mannose-labeled O-linked oligosaccharides released by beta-elimination showed that the MNT2 (YGL257c) and MNT3 (YIL014w) genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth alpha-1,3-linked mannose residues to form Man4 and Man5 oligosaccharides whereas MNT4 (YNR059w) does not appear to be required for O-glycan synthesis.     

The role of vitamin A in the glycosylation reactions of glycoprotein synthesis in an ''in vitro'' system.

Microsomal membrane preparations from rat livers, when incubated with labelled sugar-nucleotides, were shown to synthesize labelled oligosaccharide-lipids in the presence of excess exogenous dolichyl phosphate. Under the incubation conditions defined in the present study, dolichyl pyrophosphoryl(DolPP)GlcNAc2-Man5, DolPPGlcNAc2Man9 and DolPPGlcNAc2Man9Glc3 were the principal oligosaccharide-lipids formed by both control and vitamin A-deficient membranes. However, deficient membranes synthesized 3.2 +/- 0.8 times as much oligosaccharide-lipids and 2.6 +/- 0.7 times as much dolichyl phosphate mannose (DolPMan) and dolichyl phosphate glucose (DolPGlc) as the controls. The transfer of the oligosaccharide chain from the dolichol carrier to the endogenous protein acceptors in vitamin A-deficient microsomes (microsomal fractions) was only 57.5 +/- 9.5% of that of controls. After endo-beta-N-acetylglucosaminidase treatment, only one oligosaccharide species was isolated from both control and vitamin A-deficient microsomal glycoproteins, and was characterized as GlcNAcMan9Glc3. We conclude that the decreased incorporation of labelled mannose and glucose from sugar-nucleotides into the glycoproteins must be due to decreased transfer of GlcNAc2Man9Glc3 from the dolichol carrier to the protein acceptors. This conclusion was further substantiated by the finding that control membranes transferred 4-6 times as much labelled oligosaccharides from exogenously added dolichol-linked substrate (DolPPGlcNAc2Man9Glc3) to endogenous microsomal protein acceptors as compared with the vitamin A-deficient membranes. Attempts to reverse this defect by addition of retinol or retinyl phosphate (a source of retinyl phosphate mannose) to the incubations were unsuccessful.     

Detection of a conformational change in G gamma upon binding G beta in living cells

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N-linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1(+) gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated alpha-1,6-linked mannose into pyridylaminated Man(9)GlcNAc(2), indicating that och1(+) encodes an alpha-1,6-mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa

We have previously reported that the oligosaccharides transferred in vivo from dolichol-P-P derivatives in protein N-glycosylation in trypanosomatids are devoid of glucose residues and contain 2 N-acetylglucosamine and 6, 7, or 9 mannose units depending on the species. In this respect trypanosomatids differ from wild type mammalian, plant, insect, and fungal cells in which Glc3Man9GlcNAc2 is transferred. We are now reporting that incubation of Glc1-3Man9GlcNAc2-P-P-dolichol and Man7-9GlcNAc2-P-P-dolichol with membranes of Trypanosoma cruzi, Leptomonas samueli, Crithidia fasciculata, and Blastocrithidia culicis and an acceptor hexapeptide leads to the transfer of the six above mentioned lipid-linked oligosaccharides at the same rate. Control experiments performed under similar conditions but with rat liver and Saccharomyces cerevisiae membranes showed that, as already known, Glc3Man9GlcNAc2 is preferentially transferred in the latter systems. We have also previously reported that, once transferred to protein, the oligosaccharides become transiently glucosylated in trypanosomatids. Depending on the species, protein-linked Glc1Man5-9GlcNAc2 have been transiently detected in cells incubated with [14C] glucose. We are now reporting that glucosidase activities degrading both Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 were detected in T. cruzi, L. samueli, and C. fasciculata. The enzymatic activities were associated with a membrane fraction; they had a neutral optimum pH value, and similarly to mammalian glucosidase II, the enzyme acting on the monoglucosylated substrate showed a decreased affinity when the latter contained fewer mannose residues. No glucosidase I-like enzyme acting on Glc3Man9GlcNAc2 was detected in any of the three above-mentioned protozoan species. This result is consistent with the fact that no oligosaccharides containing 3 glucose units occur in trypanosomatids.     

Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha- linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8- oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP- treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.     

Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides

The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.