Effects of Inhibitors of Oligosaccharide Processing on P0Protein Synthesis and Incorporation into PNS Myelin

Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.     

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.     

Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma

Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L-fucose and the core sugar D-mannose turned over with much shorter half-lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life-span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.     

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes

The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.     

Biosynthesis and intracellular transport of the mouse macrophage Fc receptor

The membrane insertion, processing, and intracellular transport of the mouse macrophage Fc receptor for IgG1/IgG2b was studied using specific mono- and polyclonal anti-receptor antibodies. By immunoprecipitation from Triton X-114 lysates of radiolabeled J774 cells, we determined that the mature 60-kDa receptor is a transmembrane glycoprotein which is synthesized in the rough endoplasmic reticulum as a 53-kDa precursor. Digestion of the precursor with endo-beta-N-acetylglucosaminidase F demonstrated that the receptor consisted of a 37-kDa polypeptide to which four asparagine-linked oligosaccharides were attached. Proteinase K treatment of isolated microsomes indicated that the receptor also has a putative 15-kDa cytoplasmic domain apparently recognized by at least one anti-Fc receptor monoclonal antibody. An additional 15-kDa domain was found to be inaccessible to proteolysis from either side of the membrane. Pulse-chase experiments using [35S]methionine-, [3H]mannose-, and [3H]galactose-labeled cells showed that processing of the receptor's N-linked oligosaccharides occurred rapidly (t1/2 = 15 min) and resulted in the conversion of at least three of the chains to complex endo-beta-N-acetylglucosaminidase H-resistant forms. O-Linked oligosaccharides were not detected. Fc receptor was detected on the plasma membrane 30 min after its synthesis. Transport of newly synthesized receptors to the plasma membrane was slowed but not blocked by incubation of J774 cells at 20 degrees C or by the carboxylic ionophore monensin, although monensin completely inhibited the galactosylation of the receptor.     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport.

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.     

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Synthesis of sulfated glycoproteins by glial precursor cells

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [³H]glucosamine and [³⁵S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [³H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [³⁵S]sulfate into glycoproteins.     

The glycoprotein of measles virus. External radioactive labelling of its carbohydrate and partial characterization of the glycopeptide.

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3H after treatment with galactose oxidase/NaB3H4 or with [3H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79 000. After labelling with periodate/NaB3H4, which would result in specific labelling of sialic acid residues, the 79 000-mol.wt. glycoprotein was very weakly labelled. This suggests that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage.     

Biosynthesis of the sperm receptor during oogenesis in the mouse

During their growth phase, mouse oocytes synthesize and secrete three different glycoproteins, called ZP1, 2 and 3, that constitute the extracellular coat, or zona pellucida, of the oocyte. One of these glycoproteins, ZP3, exhibits properties expected for a sperm receptor. We have now used rabbit antisera that recognize ZP3 to immunoprecipitate [35S]methionine-labeled, intracellular precursors of this glycoprotein from growing oocytes cultured in vitro in the presence or absence of tunicamycin, a drug that prevents addition of N-linked oligosaccharides to nascent polypeptide chains. Electrophoretic analyses of these immunoprecipitates, as well as of immunoprecipitates digested with endo-beta-N-acetylglucosaminidase H (Endo H), indicate that ZP3 is synthesized as a 44,000 mol. wt. polypeptide chain to which either three or four high-mannose-type oligosaccharides are added, resulting in 53,000 and 56,000 mol. wt. ZP3 precursors, respectively. The latter species are converted to mature ZP3 (mol. wt. approximately 80,000) by processing of the high-mannose-type oligosaccharides (Endo H-sensitive) to complex-type oligosaccharides (Endo H-insensitive) prior to ZP3 secretion. The evidence presented reveals that the extreme heterogeneity of mature ZP3, with respect to both mol. wt. and isoelectric point, is partly a consequence of the N-linked oligosaccharides and not the polypeptide chain itself.     

Lipid-mediated glycosylation of a white matter membrane glycoprotein with an apparent molecular weight of 24,000.

White matter membrane preparations from pig brain catalyze the transfer of [¹⁴C]mannose from exogenous [¹⁴C]mannosylphosphoryldolichol into an endogenous oligosaccharide lipid. Under the same incubation conditions label is also incorporated into endogenous membrane glycoproteins. The enzymatic labeling of both classes of endogenous acceptors is stimulated by the addition of Ca²⁺. Several enzymatic properties of the mannosyltransferase activity responsible for the transfer of mannose from mannosylphosphoryldolichol into the oligosaccharide lipid intermediate have been examined. The [Man-¹⁴C] oligosaccharide lipid synthesized by this in vitro system has the solubility, hydrolytic and chromatographic characteristics of a pyrophosphate-linked oligosaccharide derivative of dolichol. The free [Man-¹⁴C]oligosaccharide liberated from the carrier lipid by mild acid treatment is estimated to contain 8 glycose units. All of the [¹⁴C]mannosyl units in the [Man-¹⁴C]oligosaccharide derived from exogenous [¹⁴C]mannosylphosphoryldolichol are released as free [¹⁴C]mannose by an α-mannosi-dase. No [¹⁴C]mannose is released during incubation with a β-mannosidase. The presence of an N,N′-diacetylchitobiose unit at the reducing end of the lipid-bound [Man-¹⁴C]oligosaccharide is indicated by its susceptibility to digestion by endo-β-N-acetylglucosaminidase H. Pronase digestion of the enzymatically labeled [Man-¹⁴C]glycoprotein yields a single [Man-¹⁴C]gly-copeptide fraction on Bio-Gel P-6 that appears to be slightly larger than the free [Man-¹⁴C]oligosac-charide released from the carrier lipid by mild acid hydrolysis. The [Man-¹⁴C]glycopeptide is cleaved by endo-β-N-acetylglucosaminidase H, and the neutral [Man-¹⁴C]oligosaccharide product appears to be identical to the product formed when the lipid-bound [Man-¹⁴C]oligosaccharide is degraded by the endoglycosidase. The glycopeptide linkage in the [Man-¹⁴C]glycoprotein is stable to mild alkali treatment. These results are consistent with the dolichol-linked [Man-¹⁴C]oligosaccharide, mannosy-lated via exogenous [¹⁴C]mannosylphosphoryldoiichol, being subsequently transferred en bloc from dolichyl pyrophosphate to asparagine residues in endogenous membrane polypeptide acceptors. SDS-polyacrylamide gel electrophoresis of the [Man-¹⁴C]glycoprotein, labeled when white matter membranes are incubated with [¹⁴C]mannosylphosphoryldolichol. revealed a major labeled polypeptide with an apparent mol wt of 24,000. A minor labeled membrane glycoprotein is also seen, having an apparent mol wt of 105,000.     

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

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

A source of error in the chromatographic study of 35S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis

HPLC combined with [35S]-sulfate/[3H]-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [35S] from 69.5 to 0.5% of the total recovered [35S]-sulfate with the remainder recovered as free [35S]-sulfate. The [3H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [35S]-or [3H]-labels under the uV peaks. No free [35S]-sulfate was obtained when [35S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [35S]-labeled glycoproteins may cleave the [35S]-sulfate ester linkages to the oligosaccharide chains. The [35S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.     

Structural glycoproteins from rabbit aortic media.

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.     

Endo-beta-N-acetylglucosaminidase H sensitivity of precursors to herpes simplex virus type 1 glycoproteins gB and gC.

The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation

Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)