Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.     

Variants of vaccinia virus hemagglutinin altered in intracellular transport.

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.     

Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.     

Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences

We investigated the requirements of the carboxyterminal sequence for surface expression of the influenza viral hemagglutinin (HA). Deletions in the cloned hemagglutinin gene were introduced at locations upstream from and spanning into the region that codes for the hydrophobic carboxyl terminus. Primate cells infected with recombinants of the deleted HA gene and an SV40 vector were negative for surface immunofluorescence and failed to adsorb erythrocytes. Polypeptide analysis showed that the mutant hemagglutinins lacking the normal hydrophobic carboxy-terminal sequences were secreted into the medium. These data provide evidence that these sequences of the influenza hemagglutinin are responsible for accumulation at the cell surface. During infection with each deletion mutant, a truncated HA polypeptide was found intracellularly. Both intracellular and extracellular HAs were glycosylated, since a third species representing the unglycosylated mutant hemagglutinin was detected in the presence of tunicamycin. Interestingly, the secreted and intracellular mutant HA polypeptides differ from the surface HA in their sensitivity to endoglycosidase H, indicating that an alteration of glycosylation has occurred.     

Structure and processing of the mouse mammary tumor virus glycoprotein precursor pr73env

The polyprotein precursor to the envelope glycoproteins of mouse mammary tumor virus was investigated by using subcellular fractionation procedures, pactomycin mapping techniques, tunicamycin inhibition of glycosylation, and endo-beta-N-acetyl glucosaminidase H-catalyzed removal of glycosylated residues in order to characterize the biosynthesis and processing of the precursor. The results suggest that the precursor (Pr73env) is synthesized on the rough endoplasmic reticulum as a transmembrane protein, with the carboxyl terminus remaining on the cytoplasmic side. The apoprotein as an estimated molecular weight of 60,000 and acquires five core oligosaccharide units during synthesis. Cleavage of the precursor precedes the secondary glycosylation steps and therefore probably occurs before transport to the plasma membrane. However, a minor population of Pr73env containing complex oligosaccharides was also found in the plasma membrane. The order of the glycoproteins in the precursor, as determined by pactomycin mapping, in NH2-gp52-gp36-COOH.     

Role of glycosylation in secretion of yeast acid phosphatase

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.     

A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.     

A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough endoplasmic reticulum

Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.     

Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.     

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.     

Glycosylation process in gonadotrophs: a quantitative electron microscope autoradiographic study with labeled glucosamine

Incorporation of [3H]glucosamine into dispersed anterior pituitary cells was studied by electron microscope autoradiography. Gonadotrophs were examined to determine the intracellular route and kinetic patterns of glycosylation. Studies were performed with cells from; (a) normal adult male rats; (b) rats orchidectomized 3 wk earlier; and (c) orchidectomized rats treated with tunicamycin. Our results show that incorporation of [3H]glucosamine first occurs in the rough endoplasmic reticulum (RER), then proceeds in the Golgi elements (where peripheral carbohydrates are attached). Treatment with tunicamycin results in a decrease in labeling of these 2 organelles. Comparison of the kinetic patterns in normal and castrated male rats shows that the accumulation of labeled glycosylated proteins in granules reaches a plateau within 2 hr post-pulse in normal rats, and rises during a 6-hr chase in castrated rats. However, because of the necessity for a rather long 15 min pulse, we cannot exclude the possibility that incorporation of glucosamine during the pulse may occur concomitantly in the RER and the Golgi saccules, to be followed by rapid transfer to the secretory granules.     

Comparative characterization of the glycosylation profiles of an influenza hemagglutinin produced in plant and insect hosts

The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts.     

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly.

The influenza virus hemagglutinin (HA) glycoprotein synthesized from cloned DNA in a simian virus 40 vector is expressed on the surface of infected primate cells. Previously, it has been demonstrated that mutant HAs lacking the hydrophobic carboxy terminus fail to anchor on the cell surface and therefore are secreted extracellularly. During analysis of additional HA deletion mutants derived from an HA-simian virus 40 recombinant, we found a mutant with an altered hydrophobic carboxy terminus that exhibited another phenotype. This deletion mutant, dl-12, produced HA that was neither secreted nor expressed on the infected cell surface. The mutant HA was similar to the wild-type HA in apparent molecular weight and extent of glycosylation as assayed by endoglycosidase H sensitivity. The mutant HA localized near the perinuclear region of infected cells as indicated by an indirect immunofluorescence assay. Sequence analysis showed that a 5-base-pair deletion had occurred before the region encoding the hydrophobic carboxy terminus. Nevertheless, the physicochemical properties of the wild-type HA carboxy terminus were maintained in that the truncated HA carboxy terminus consisted of predominantly hydrophobic amino acids followed by several charged amino acids residues. This similarity in the carboxy terminus between the wild-type and mutant HAs may be responsible for the functional similarities observed. In spite of these similarities, the mutant HA failed to mature at the surface. These results suggest that the maturation of the mutant HA is blocked during a late stage in the transit to the cell surface.     

Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations

A hen oviduct membrane preparation that catalyzes both the N- and O-glycosylation of exogenous acceptor peptides was used to examine the possible involvement of lipid intermediates in enzymatic O-glycosylation. The results indicate that, under a variety of experimental conditions in which the dolichol-linked saccharides involved in N-glycosylation are readily observed, no lipid-linked intermediates for O-glycosylation could be detected. Whereas N-glycosylation is abolished by tunicamycin treatment and stimulated by dolichol phosphate addition, O-glycosylation is unaffected by such treatments. Further, the results of subcellular fractionation of oviduct membranes suggest that N-acetylgalactosaminyl:polypeptide transferase is localized primarily in membranes derived from the smooth endoplasmic reticulum and Golgi apparatus. This is in contrast to the subcellular site of N-glycosylation, which has previously been shown to be primarily the rough endoplasmic reticulum. These findings are discussed in relation to the function of dolichol phosphate in protein glycosylation.     

The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin

Eleven chimeric proteins were created in which the transmembrane, the cytoplasmic, or both topological domains of the influenza virus hemagglutinin (HA) were replaced with those from five other glycoproteins. All of the chimeric HAs reached the cell surface but appeared to differ in the degree to which they were stably folded. Comparisons of the rates of folding, passage into the Golgi, and arrival at the plasma membrane of wild-type HA and the chimeric proteins suggest that formation of a stable HA trimer is not an absolute requirement for export from the endoplasmic reticulum. In addition, there appear to be at least two steps at which the rate of transport can be altered during exocytosis, one occurring before and the other after the trimming of oligosaccharides by Golgi mannosidases. Certain of the chimeras differed from HA in their ability to pass through each of these steps. Replacement of the HA transmembrane domain with the analogous sequences from other proteins affected folding and transport of the chimeric HAs in ways that suggest that the HA transmembrane sequences form a specific structure in the membrane that differs from that formed by analogous sequences from the other proteins.     

Cytochemical localization of complex carbohydrates in the thyroid gland of normal and TSH-treated mice

Summary The silver methenamine method for the ultrastructural localization of carbohydrates and glycoproteins was applied to the thyroid glands of normal and TSH-treated mice. The majority of the cisternae of the rough endoplasmic reticulum showed a weak, but apparently positive reaction. These findings support the opinion that glycosylation of thyroglobulin occurs initially in the rough endoplasmic reticulum. By this method the Golgi apparatus was observed to display a staining gradient. The intermediate to inner saccules were intensely stained, whereas the outer saccules were not so heavily stained. This phenomenon indicates that the Golgi apparatus has a functional polarity for the addition of carbohydrates to thyroglobulin and other proteins. In the inner and/or the peripheral regions of the Golgi apparatus and in the apical cytoplasm, a large number of globules of various sizes, considered to be colloid droplets, lysosomes and apical secreting vesicles, showed a positive reaction. The luminal colloid was also positive with silver methenamine staining, with almost the same intensity as the globules and vesicles.This study was supported by a grant from the Japan Ministry of Education