Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.     

Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network.

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.     

The mannose 6-phosphate glycoprotein proteome

Most luminal lysosomal proteins are synthesized as precursors containing mannose 6-phosphate (Man6-P) and a number of recent studies have conducted affinity purification of Man6-P containing proteins as a step toward defining the composition of the lysosome. Approximately 60 known lysosomal proteins have been found in such studies as well as many other Man-6-P glycoproteins, some of which represent new lysosomal proteins. The latter are of considerable interest from cell-biological and biomedical perspectives, but differentiating between them and other proteins remains a significant challenge. The aim of this study was to conduct a global analysis of the mammalian Man6-P glycoproteome, implementing technical and biostatistical methods to aid in the discovery and validation of lysosomal candidates. We purified Man6-P glycoproteins from 17 individual rat tissues. To distinguish nonspecific contaminants (i.e., abundant or "sticky" proteins that are not fully removed during purification) from specifically purified proteins, we conducted a semiquantitative mass spectrometric comparison of protein levels in nonspecific mock eluates versus specific affinity chromatography eluates to identify those proteins that are specifically purified. We identified 60 known lysosomal proteins, representing nearly all that are currently known to contain Man-6-P. We also find 136 other proteins that are specifically purified but which are not known to have lysosomal function. This approach provides a list of candidate lysosomal proteins and also provides insights into the relative distribution of Man6-P glycoproteins.     

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.     

Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes

Varicella-zoster virus (VZV) codes for approximately eight glycosylated polypeptides in infected cell cultures and in virions. To determine the number of serologically distinct glycoprotein gene products encoded by VZV, we have developed murine monoclonal antibodies to purified virions. Of 10 monoclonal antibodies which can immunoprecipitate intracellular VZV antigens and virion glycoproteins, 1 (termed gA) reacted with gp105, 1 (termed gB) reacted with gp115 (intracellular only), gp62, and gp57, and 8 (termed gC) reacted with gp92, gp83, gp52, and gp45. The anti-gA monoclonal antibody neutralized VZV infectivity in the absence of complement. All eight anti-gC monoclonal antibodies neutralized only in the presence of complement. An anti-gB monoclonal antibody obtained from another laboratory also neutralizes in the absence of complement. Since the above reactivities account for all major detectable VZV glycoprotein species, the data strongly suggest that VZV has three major glycoprotein genes which encode glycosylated polypeptides with neutralization epitopes.     

Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells.

Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.     

Human herpesvirus-6 induces MVB formation, and virus egress occurs by an exosomal release pathway

The final envelopment of most herpesviruses occurs at Golgi or post-Golgi compartments, such as the trans Golgi network (TGN); however, the final envelopment site of human herpesvirus 6 (HHV-6) is uncertain. In this study, we found novel pathways for HHV-6 assembly and release from T cells that differed, in part, from those of alphaherpesviruses. Electron microscopy showed that late in infection, HHV-6-infected cells were larger than uninfected cells and contained many newly formed multivesicular body (MVB)-like compartments that included small vesicles. These MVBs surrounded the Golgi apparatus. Mature virions were found in the MVBs and MVB fusion with plasma membrane, and the release of mature virions together with small vesicles was observed at the cell surface. Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB/late endosome marker, and HHV-6 envelope glycoproteins. The viral glycoproteins also localized to internal vesicles in the MVBs and to secreted vesicles (exosomes). Furthermore, we found virus budding at TGN-associated membranes, which expressed CD63, adaptor protein (AP-1) and TGN46, and CD63 incorporation into virions. Our findings suggest that mature HHV-6 virions are released together with internal vesicles through MVBs by the cellular exosomal pathway. This scenario has significant implications for understanding HHV-6's maturation pathway.     

Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.     

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.     

The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.     

Catabolic pathway of oligosaccharide-diphospho-dolichol. Study of the fate of the oligosaccharidic moiety in mouse splenocytes

Metabolic labelling of mouse splenocytes with radioactive mannose indicates that the glycosylation process is accompanied by the release of soluble oligomannoside material. Chase experiments with an excess of unlabelled mannose indicate that the radioactivity is mainly chased from oligosaccharide-PP-Dol (PP-Dol = diphosphodolichol): 10% is recovered as (Man)9(GlcNAc)2-P, (Man)9(GlcNAc)2, (Man)9GlcNAc and (Man)5 GlcNAc, and 90% is rapidly degraded further. Tunicamycin inhibits both oligosaccharide-PP-Dol synthesis and the formation of the oligosaccharide material to the same extent. The results thus indicate that these soluble oligomannoside structures represent the main steps of the oligosaccharide-PP-Dol catabolic pathway, starting with the cleavage of the diphosphate bond. However, it cannot be excluded that part of this material is released from newly formed glycoproteins. The soluble oligomannoside material does not contain glucose residues despite the fact that part of the oligosaccharide-PP-Dol is glucosylated and it was shown, by the use of glucosidase I inhibitors (castanospermine, deoxynojirimycin) that, after cleavage, the glycan moiety of glucosylated oligosaccharide-PP-Dol is first rapidly deglucosylated. These experiments provide a physiological basis to our previous results obtained in vitro and allow the definition of further steps in the catabolic pathway of oligosaccharide-PP-Dol.     

Egress of varicella-zoster virus from the melanoma cell: a tropism for the melanocyte.

The pathway of envelopment and egress of the varicella-zoster virus (VZV) and the primary site of viral production within the epidermal layer of the skin are not fully understood. There are several hypotheses to explain how the virus may receive an envelope as it travels to the surface of the monolayer. In this study, we expand earlier reports and provide a more detailed explanation of the growth of VZV in human melanoma cells. Human melanoma cells were selected because they are a malignant derivative of the melanocyte, the melanin-producing cell which originates in the neural crest. We were able to observe the cytopathic effects of syncytial formation and the pattern of egress of virions at the surfaces of infected monolayers by scanning electron microscopy and laser-scanning confocal microscopy. The egressed virions did not appear uniformly over the syncytial surface, rather they were present in elongated patterns which were designated viral highways. In order to document the pathway by which VZV travels from the host cell nucleus to the outer cell membrane, melanoma cells were infected and then processed for examination by transmission electron microscopy (TEM) at increasing intervals postinfection. At the early time points, within minutes to hours postinfection, it was not possible to localize the input virus by TEM. Thus, viral particles first observed at 24 h postinfection were considered progeny virus. On the basis of the TEM observations, the following sequence of events was considered most likely. Nucleocapsids passed through the inner nuclear membrane and acquired an envelope, after which they were seen in the endoplasmic reticulum. Enveloped virions within vacuoles derived from the endoplasmic reticulum passed into the cytoplasm. Thereafter, vacuoles containing nascent enveloped particles acquired viral glycoproteins by fusion with vesicles derived from the Golgi. The vacuoles containing virions fused with the outer plasma membrane and the particles appeared on the surface of the infected cell. Late in infection, enveloped virions were also present within the nuclei of infected cells; the most likely mechanism was retrograde flow from the perinuclear space back into the nucleus. Thus, this study suggests a role for the melanocyte in the pathogenesis of VZV infection, because all steps in viral egress can be accounted for if VZV subsumes the cellular pathways required for melanogenesis.     

Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L-cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases

During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.     

Glycoproteins secreted from suspension-cultured tobacco BY2 cells have distinct glycan structures from intracellular glycoproteins.

Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular glycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35-39]. Extracellular glycoproteins have six distinct N-glycans (marked by *) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40% (Man5GlcNAc2, 28.8%; Man6GlcNAc2*, 6.4%; and Man7GlcNAc2*, 3.8%), while the plant-complex-type structures account for nearly 60% (GlcNAc2Man3Xyl1GlcNAc2*, 32.1%; GlcNAc1Man3Xyl1GlcNAc2 (containing two isomers)*, 6.2%; GlcNAc2Man3GlcNAc2*, 4.9%; Man3Xyl1Fuc1GlcNAc2, 8.3%; and Man3Xyl1GlcNAc2, 3.7%).     

Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.     

Glycoprotein E of varicella-zoster virus enhances cell-cell contact in polarized epithelial cells

Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca(2+) binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts.     

Temporal morphogenesis of herpes simplex virus type 1-infected and brefeldin A-treated human fibroblasts

BACKGROUND: Insights in the herpesvirus-cell interactions are of general cell biology interest, especially to studies of intracellular transport, and of considerable significance in the efforts to generate drugs, vaccines, and gene therapy. However, the pathway of virus particle egress and maturation is a contentious issue. MATERIALS AND METHODS: The intracellular transport was inhibited in cultured herpes simplex virus type 1 (HSV-1) infected human fibroblasts by brefeldin A (BFA). The virus-cell interactions including the viral envelopment, transport of HSV-1 virions, and transport of viral glycoprotein D (gD-1) and glycoprotein C (gC-1) were studied by titration assay, immunoblot, immunofluorescence light microscopy, and immunogold electron microscopy of cryosections. RESULTS: gD-1 and gC-1 were synthesized and normally transported to the plasma membranes of untreated HSV-1 infected host cells. BFA (1 microg/ml medium) effectively blocked the transport of the glycoproteins to the plasma membranes and affected the tubulin and vimentin of the cytoskeleton. Viral particles and glycoproteins accumulated in the perinuclear space and the endoplasmic reticulum of BFA treated cells. Withdrawal of BFA influence up to 9 hr resulted in restored tubulin and vimentin, transport of glycoproteins to the plasma membranes, and steady release of infectious viral particles to the extracellular space superior to the cellular assembly of new virions. The ultrastructural data presented support that the primary envelopment of viral particles occur at the nuclear membranes containing immature glycoproteins followed by multiple de-envelopments and re-envelopments of the virions during the transport and maturation in the endoplasmic reticulum and the Golgi complex. CONCLUSIONS: BFA-induced changes include the cytoskeleton with significant effect on HSV-1 maturation and egress. The data support a multiple-step envelopment of HSV-1 in a common pathway of glycoprotein synthesis and virion egress.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Intracellular lectins associated with N-linked glycoprotein traffic

The vectorial intracellular transport of N-glycan-linked glycoproteins is indispensable for biological functions. In order to sort these glycoproteins to the correct destination, animal intracellular lectins play important roles as sorting receptors. The roles of such lectins in the biosynthetic pathway from the endoplasmic reticulum (ER) to the cell surface are addressed in this review. Calnexin and calreticulin function via specific carbohydrates in quality control of newly synthesized glycoproteins in the ER, and ERGIC-53 seems to function in the transport of glycoproteins from ER to the Golgi complex. In addition to the well-understood role of mannose 6-phosphate receptor in lysosomal protein sorting, the vesicular integral protein of 36 kDa (VIP36) functions as a sorting receptor by recognizing high-mannose type glycans containing alpha1-->2Man residues for transport from Golgi to the cell surface in polarized epithelial cells.