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

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

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.     

The formation of oligoglucans linked to lipid during synthesis of beta-glucan by characterized membrane fractions isolated from peas.

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble beta-glucans, which like the oligosaccharides contained beta(1leads to 3) and beta-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in beta-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.     

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.     

Formation of unusual mannosamine-containing lipid-linked oligosaccharides in Madin-Darby canine kidney cell cultures.

Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is the major lipid-linked oligosaccharide (LLO) produced by Madin-Darby canine kidney cells in culture. However, when these cells are incubated in the presence of millimolar concentrations of mannosamine and labeled with [2-3H]mannose, they accumulate various LLO that have smaller-sized oligosaccharides with unusual structures and the Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is not detected. Thus in the presence of 10 mM mannosamine, more than 80% of the oligosaccharides are eluted from concanavalin A-Sepharose with 10 mM alpha-methylglucoside, indicating that they no longer have the tight-binding characteristics of control oligosaccharides. In addition, 20-40% of these oligosaccharides bind to Dowex 50-H+, indicating the presence of mannosamine in these structures. Interestingly enough, these abnormal oligosaccharides are still transferred to protein. The mannosamine-induced oligosaccharides were separated into neutral and basic fractions on a cation exchange resin. The neutral oligosaccharides ranged in size from hexose3(GlcNAc)2 to hexose10(GlcNAc)2 with the major species being Man5(GlcNAc)2 to Man7(GlcNAc)2. These oligosaccharides were almost completely susceptible to digestion by alpha-mannosidase and by endoglucosaminidase H. The basic oligosaccharides showed anomolous behavior on the Bio-Gel P-4 columns and appeared to be of small size on the standard columns, ranging from hexose2 to hexose4. However, most of these oligosaccharides were susceptible to digestion by endoglucosaminidase H as well as by alpha-mannosidase, suggesting that they were of different size and structure than would be predicted from the gel filtration patterns. Significantly, when the basic oligosaccharides were subjected to chemical N-acetylation, or when the gel filtration columns were run at high pH rather than at the usual pH of 3.0, the basic oligosaccharides migrated like much larger oligosaccharides. These data provide strong evidence to indicate that some mannosamine can be incorporated into the LLO, and that these mannosamine-containing oligosaccharides exhibit unusual properties. Preliminary studies indicated that Madin-Darby canine kidney cells do incorporate label from [3H]mannosamine into the LLO.     

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.     

The substrate specificity of bovine and feline lysosomal alpha-D-mannosidases in relation to alpha-mannosidosis

Lysosomal alpha-mannosidases were partially purified from bovine and feline liver and employed to digest a large number of oligosaccharides with structures corresponding to the oligomannosyl parts of complex, hybrid, and high-mannose glycans. The incubation products were identified by high pressure liquid chromatography with reference compounds of defined structure and by acetolysis. For all classes of substrates, the lysosomal alpha-mannosidases displayed a high degree of in vitro specificity with regard to the hydrolysis of mannose residues. Thus, in each case, 1 or at most 2 residues were always preferentially cleaved so that the degradative process proceeded down a well defined pathway. A comparison of the relative efficiency with which lysosomal alpha-mannosidases catalyzed the hydrolysis of particular oligosaccharides and of the structures of the resulting intermediates with those of the compounds accumulated in alpha-mannosidosis allows conclusions to be drawn regarding the nature of the enzymatic defect. In bovine alpha-mannosidosis, the oligosaccharides are those expected for a partial deficiency of normal lysosomal alpha-mannosidase, so that they correspond to intermediates in the normal catabolic pathway. In feline alpha-mannosidosis, in which the alpha-mannosidase deficiency is more severe than in cattle, the accumulated oligosaccharides primarily represent intact oligomannosyl moieties of N-linked glycans rather than the products of residual alpha-mannosidase activity.     

Studies on the effect of mevinolin (lovastatin) and mevastatin (compactin) on the fusion of L6 myoblasts

The effect of mevastatin and mevinolin on the fusion of L6 myoblasts was studied. Both compounds were potent inhibitors of myoblast fusion at concentrations as low as 0.25 M, but fusion was restored when the inhibitors were removed. Both compounds resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing they were causing a reduction in N-linked cell surface glycoproteins. There was a reduction in creatine phosphokinase activities in the presence of both compounds showing that they were affecting biochemical differentiation. The presence of both compounds inhibited the incorporation of labeled mannose from GDP-mannose into lipid-sugar and N-linked glycoprotein, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies is that mevinolin and mevastatin are inhibiting myoblast fusion by affecting the synthesis of fusogenic cell surface N-linked glycoproteins probably by affecting the synthesis of dolichol phosphate containing oligosaccharides that are required as intermediates in N-linked glycoprotein biosynthesis.Abbreviations HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A - Dol dolichol - Dol-P dolichol phosphate - Man mannose - GlcNAc N-acetylglucosamine - Glc glucose - conA concanavalin A - WGA wheat germ agglutinin - CPK creatine phosphokinase     

Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum

The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi.     

Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.     

Erythroglycan biosynthesis in K-562 cells. Inhibition of synthesis by tunicamycin and lack of attachment to the G-protein of vesicular-stomatitis virus.

K-562 cells, which express foetal erythroglycan, are shown to synthesize the lipid-linked oligosaccharide intermediates commonly found in tissues and cultured fibroblasts. The addition of tunicamycin, which blocks the formation of these intermediates and thus of asparagine-linked oligosaccharides, inhibits the synthesis of erythroglycan (Mr 7000-11 000). Vesicular-stomatitis-virus infection of K-562 cells results in the glycosylation of the G-protein with the transferrin-type oligosaccharide (Mr 3000), but not with the larger erythroglycan. These results suggest that, in K-562 cells, the early stages of erythroglycan biosynthesis are the same as those of the transferrin-type oligosaccharides. However, maturation of the oligosaccharide is influenced by protein structure such that erythroglycan is only expressed on specific glycoproteins.     

Structures of galactose-containing oligosaccharides of alpha-mannosidase from porcine kidney

Oligosaccharides of a non-oligomannoside type were released from porcine alpha-mannosidase by hydrazinolysis, and were fractionated into at least 15 homogeneous oligosaccharides. Most of them are oligosaccharides with galactose and N-acetylglucosamine residues attached to a common core, alpha Man2 beta Man beta GlcNAc(+/- alpha-L-Fuc)beta GlcNAc. About 50% of the oligosaccharides contain one or two outer chains composed of one beta-linked N-acetylglucosamine and two beta-linked galactose residues attached to the core portions, and the others seem to be metabolic intermediates. Based on the results of studies on the binding of alpha-mannosidase to RCA (Ricinus communis agglutinin) I-agarose and MBP (mannan-binding protein)-Sepharose, which are specific for glycoproteins possessing N-acetyllactosamine-type and oligomannoside-type (including oligomannosides with N-acetylglucosamine at the reducing termini) oligosaccharides, respectively, about 85% of the enzyme molecules were found to have both types of oligosaccharides. Similarly, it was shown that of the several acid hydrolases present in the lysosomes purified from rat liver, only alpha-mannosidase has both types of oligosaccharides, and the greater parts of beta-glucuronidase, acid phosphatase and beta-N-acetylhexosaminidase seem to have only oligomannoside-type oligosaccharides.     

A mouse submandibular sialomucin containing both N- and O-glycosylic linkages

A sialomucin from mouse submandibular glands was treated with mild base-Me₂SO. This treatment cleaves O-glycosylically linked oligosaccharides, but preserves the integrity of the protein core. After treatment with mild base-Me₂SO, 49.2% (by weight) of the oligosaccharides were removed from the polypeptide; they were composed of residues of 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, sialic acid, and d-galactose. These oligosaccharides were linked O-glycosylically via 2-acetamido-2-deoxy-d-galactose. Chromatography of the base-Me₂-SO-treated mucin on Sephacryl S-300 indicated that the protein core, with its base-resistant oligosaccharides, is a single, high-molecular-weight species. The mild-base-resistant linkages remaining on the protein core (50.8% of the total carbohydrates by weight) also contained d-mannose. The presence of these mild-base-resistant linkages, and the formation of 2-acetamido-2-deoxy-d-glucitol following treatment with m NaOH-m NaBH₄, confirmed the presence of N-glycosylic linkages.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.     

Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors

Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence.     

Involvement of Oligosaccharides in Adaptation of Winter Wheat Seedlings to Subzero Temperatures

We studied the dynamics of endogenous content of biologically active oligosaccharides in the roots of winter wheat seedlings. Previously, these oligosaccharides proved to mediate the development of frost resistance during the first days of hardening (Zabotinaet al., 1998). The changes in their endogenous content can be described by a curve with a single peak observed 6 h after the onset of frost hardening. The capacity of these polysaccharides to increase frost resistance (LT₅₀was evaluated by leakage of electrolytes) when added to growth medium did not depend on the pretreatment duration (from 1.5 to 18 h) but decreased if they were introduced in the course of the adaptive response. Inhibition of the adaptive response by inhibitors of RNA and protein synthesis ceased in the presence of the oligosaccharides. We believe that the oligosaccharides that are products of metabolism of the cell wall polysaccharides are involved in adaptation to low temperature.     

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)     

Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.     

Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.