glycosylation in insect cells

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N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector.

N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector.

Most of the N-acetyl- b -glucosaminidases described previ- ously are lysosomal enzymes taking part in various degradation reactions (for a review, see reference 4). The enzyme identified in this study is clearly distinguishable from its lysosomal coun- terparts by its pronounced substrate specificity dependent on the prior action of a -mannosidase II and its definite involve- ment in HA N-glycan maturation, which points to the localiza- tion of this N-acetyl- b -glucosaminidase within the exocytotic pathway of Sf9 cells. However, the N-acetyl- b -glucosaminidase described here is probably identical to an enzyme that has been observed recently when lysates of three other lepidopteran cell lines were analyzed in vitro with synthetic oligosaccharides as the substrates (3). Our study extends this work by showing that this enzyme is involved in glycoprotein synthesis in intact cells and is therefore a constituent of the N-glycosylation pathway. Because of its crucial implications for the nature of insect cell- derived N-glycans, this enzyme can be considered the missing link between the intermediates of the conventional N-glyco- sylation cascade and the truncated oligosaccharides observed in insect cells. Accordingly, it becomes clear now that N glyco- sylation in Sf9 cells initially follows the classical route. After trimming of oligomannosidic side chains to Man 5 GlcNAc 2 , a
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Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells

Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells

Another possible explanation for the defect in AcSWT-1 was its ability to express enzymatically active glycosyltransferases during infection. As demonstrated above, glycosyltransferase expression by AcSWT-1 led to differential glycosylation of GP64 and, presumably, other viral and cellular glycoproteins produced during AcSWT-1 infection. GP64 has been impli- cated as a baculovirus attachment protein (7, 14) and is re- quired for baculovirus penetration (20, 31) and efficient release of budded virus from infected cells (21). Thus, there were many reasons to think that differential glycosylation of GP64 might adversely influence infectious budded virus progeny pro- duction by AcSWT-1. Alternatively, this defect might reflect the position of the ie1-hr5-glycosyltransferase cassette imme- diately upstream of the gp64 gene and/or the use of the p6.9 promoter to drive gp64 expression in AcSWT-1. Either of these latter two factors could subtly influence expression of the gp64 gene, which could decrease infectious budded virus progeny production by this virus. These possibilities were addressed, in part, by producing a third baculovirus expression vector, AcSWT-2c, in which the ie1-hr5-glycosyltransferase cassette was inserted within the v-cath gene instead of the p24-gp64 region and the rest of the viral genome is wild type (Fig. 1C). One-step growth experiments showed that AcSWT-2c and wild-type AcMNPV had very similar growth curves (Fig. 5B). Thus, expression of the glycosyltransferase activities was not responsible for the slight defect in infectious budded virus progeny production by AcSWT-1. We concluded that this de- fect might reflect the position of the ie1-hr5-glycosyltransferase cassette or the use of the basic protein promoter to express the gp64 gene in those viruses. However, considering the wild-type growth phenotype of AcSWT-2c, we made no further effort to determine the reason for the defect in AcSWT-1 and focused the remainder of our efforts on characterizing AcSWT-2c.
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Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice

Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice

High-yield production of soluble E2 glycoprotein in Drosophila S2 cells. Previous efforts to develop a recombinant HCV vaccine relied on expression of E2 or E1/E2 heterodimer in mammalian cells (27, 28, 38–40). It was reported that N-glycans associated with HCV E2 could modulate receptor-binding affinity, as well as neutralizing-epitope recognition (41). Thus, we hypothesized that glycosylation might influence the antigenicity and immunogenic- ity of sE2. To directly determine the contribution of glycosylation to the induction of bNAbs by sE2, we produced sE2 in mammalian and insect cell lines known to yield altered glycan structures on proteins (42). To produce the E2 protein of the HCV strain Con1 (genotype 1b) in insect cells, we used an established Drosophila S2 cell expression system (43). Transgenic cell lines expressing sE2 comprised of residues 384 to 661 were recovered following trans- fection of Drosophila S2 cells with pMT-sE2 and subsequent anti- biotic selection (Fig. 1A). The sE2 protein was secreted into the cell culture supernatant and remained stable for at least 9 days (Fig. 1B). sE2 was readily purified from the cell culture supernatant to near homogeneity (Fig. 1C) at high levels of up to 100 mg/liter of supernatant. The purified sE2 migrated at ⬃ 45 kDa (Fig. 1C), which is much higher than the predicted mass (⬃34 kDa) based on its amino acid sequence, suggesting possible glycosylation. To examine the extent and pattern of glycosylation, sE2 was digested with the endoglycosidases endo H and PNGase F. As shown in Fig. 1D, PNGase F treatment of sE2 generated a band at the calculated molecular mass of ⬃ 34 kDa, suggesting that sE2 is fully deglyco- sylated by PNGase F. Endo H digestion resulted in a band only slightly below that of untreated sE2, suggesting that sE2 con- tains endo H-resistant glycan types, such as paucimannose N- glycans, as previously reported for other S2 cell-produced gly- coproteins (42).
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Essential Role of Dengue Virus Envelope Protein N Glycosylation at Asparagine-67 during Viral Propagation

Essential Role of Dengue Virus Envelope Protein N Glycosylation at Asparagine-67 during Viral Propagation

bly/release in mammalian cells. In addition, E protein was undetectable in the media of BHK cells infected with the N67Q mutant virus (Fig. 2D). This phenotype could be due to alteration in the folding of E, assembly, or particle release. The replication defect appears to result from the lack of the N- linked glycan rather than the introduction of deleterious amino acid substitutions, because the E protein was fully functional in mosquito cells. All DV glycosylation mutants propagated in mosquito cells (Fig. 1). We provided evidence that the differ- ential requirements of E glycosylation for DV replication in mammalian and mosquito cells can be due to differing E se- cretion in the two host cells (Fig. 5). It is known that protein folding is dependent on temperature. Therefore, one possible explanation for this host-dependent phenotypic difference could be the ability of mosquito cells to properly fold and secrete the E at the low temperature used to grow C6/36 cells (28°C), while mammalian cells grown at 37°C are unable to secrete this protein. To analyze this possibility, we infected C6/36 and BHK cells, both grown at 32°C. The viruses lacking the glycosylation sites replicated efficiently at 32°C in C6/36 cells, while the same viruses were unable to propagate in BHK cells at the same temperature (data not shown). These results indicate that proper folding of E protein lacking a glycan at position 67 in mosquito cells is not a temperature-dependent phenomenon. Instead, it could be due to different protein folding pathways used by the two host cells. Insect and mam- malian cells are known to express different metabolic enzymes involved in processing N-glycans (43). N-linked carbohydrates play important roles in protein folding by serving as ligands of the ER chaperones calnexin and calreticulin. In addition, Erp57, a member of the PDI family, catalyzes correct disulfide bond formation specifically on glycoproteins bound to calnexin and calreticulin (34, 47). Erp57 could be involved in folding of the glycosylated E protein, because it contains 12 cysteines that form six highly conserved disulfide bonds. In light of our re- sults, it is likely that insect cells provide a glycosylation-inde- pendent pathway for E protein secretion, while in mammalian cells, host factors would be necessary to specifically recognize the glycan at Asn-67 for proper E-protein folding.
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Bioremediation of Bisphenol A by Glycosylation with Immobilized Marine Microalga Amphidinium  crassum  ——Bioremediation of Bisphenol a by Immobilized Cells

Bioremediation of Bisphenol A by Glycosylation with Immobilized Marine Microalga Amphidinium crassum ——Bioremediation of Bisphenol a by Immobilized Cells

tion of bisphenol A by A. crassum were carried out using eight flasks containing cultured cells (9 g) or immobi- lized cells, which included 9 g cells. In the case of the biotransformation by the cultured and immobilized C. roseus cells, cultured cells (70 g) or immobilized cells, which included 70 g cells, were partitionated to each flask. Substrate (0.2 mmol) was administered to each of flasks and the mixtures were incubated on a rotary shaker at 25˚C. At a day interval, one of the flasks was taken out from the rotary shaker, and the cells (or immobilized cells) and medium were separated by filtration. The ex- traction and analysis procedures were same as described above. The yield of the products was determined on the basis of the peak area from HPLC and expressed as a relative percentage to the total amount of the whole reac- tion products extracted.
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Polycaryocyte formation mediated by Sindbis virus glycoproteins.

Polycaryocyte formation mediated by Sindbis virus glycoproteins.

These data suggest that glycosylation of virus proteins is essential for Sindbis virus-infected cells to express FFWI, but that the conversion of PE2 to E2 or the binding of capsid to th[r]

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Inhibition of glucose metabolism prevents glycosylation of the glutamine transporter ASCT2 and promotes compensatory LAT1 upregulation in leukemia cells

Inhibition of glucose metabolism prevents glycosylation of the glutamine transporter ASCT2 and promotes compensatory LAT1 upregulation in leukemia cells

This study first provides the proof of concept that glucose deprivation by preventing the glycosylation of various proteins including those orchestrating cancer bioenergetics, may also alter glycolysis-independent metabolic routes in tumor cells. We showed indeed that inhibition of glucose metabolism in leukemia cells dramatically reduced the extent of glycosylation of ASCT2, a major transporter of glutamine, a key biosynthetic fuel in these cells [8, 9]. Although we failed to detect mislocalization of deglycosylated ASCT2, partial restoration of the cellular glycosylation pattern with mannose was associated with a significant increase in leukemia cell growth in the absence of glucose. Importantly, it was not only the complete glucose withdrawal but also the pharmacological inhibition of glycolysis by 2-deoxyglucose that led to a reduction in Table 1: Gln transporters
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Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1

Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1

How the β1 integrin traffics from ER to Golgi is still unclear. However, this transition indicates a potential tar- get for regulation of β1 integrin expression on cell sur- face. Our findings in Fig 5A showed that total amount of β1 subunit in Nm23/H7721 cells did not change, which was consistent with the results obtained by RT-PCR. But, the level of mature integrin isoform was decreased signif- icantly, while the level of partially glycosylated precursor was increased. It suggests that the expression of Nm23- H1 affects the glycosylation of integrin β1 precursor and the altered glycosylation of integrin β1 may contribute to the loss of cell surface integrin β1 in Nm23/H7721 cells. In previous studies by others, it was demonstrated that Nm23-H1 could down regulate the transcription of many glycosyltransferase genes, including GnT-V , α1,3 FucTs and ST3Gals and that they were correlated with anti- metastasis effect in tumor cells [15,37]. Accumulating evidence indicates that β1 integrin is an important target for GnT-V and ST6Gal. Therefore, it may be concluded that transfection of Nm23-H1 cDNA down regulates some key glycosyltransferase genes and then interferes the protein post-translational modification. In conse- quence, the glycosylation of β1 integrin precursor is impaired, leading to the loss of cell surface β1 integrin. However, the detailed mechanisms need to be further investigated. The mechanisms of regulating integrin- stimulated cell migration are very complex and the acti- vation of tyrosine kinases plays an important role in these events [4]. Emerging evidence supports the important role of FAK PTK in these processes. FAK activation has been linked to integrin clustering and is considered as a critical step in the initiation of cell migration. In cultured cells, overexpression of FAK can increase Fn-stimulated cell motility and this activity depends upon the integrity of the FAK Tyr-397 autophosphorylation site [38,39]. Our result showed that Nm23-H1 seemed to have no effect on the expression of FAK in H7721 cells, while it decreased the tyrosine phosphorylation of FAK, an important event in integrin-mediated signaling.
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Biosynthesis of the Sindbis Virus Carbohydrates

Biosynthesis of the Sindbis Virus Carbohydrates

To examine the extent of glycosylation of the intracellular forms of the virion glycoproteins and of PE2, Sindbis-infected chick cells were labeled with both a radioactive sugar and a mi[r]

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Characterisation of the adenovirus protease

Characterisation of the adenovirus protease

One of the substrates of the protease, the pre-terminal pTP protein was expressed in insect cells using a recombinant baculovirus.. Coinfections of cells with recombinant 23-kDa and pTP [r]

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Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

1. Walker LM, Phogat SK, Chan-Hui P-Y, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Kaminsky S, Zamb T, Moyle M, FIG 4 Influences of gp120 glycans on bnAb recognition. (A) Neutralization sensitivity of PGT135, PGT128, and b12 against BaL wild-type pseudovirus and glycan site mutants. bnAbs PGT128 and b12 are used as controls. To produce pseudoviruses, plasmids encoding Env were cotransfected with an Env-deficient genomic backbone plasmid (pSG3 ⌬ Env) in a 1:2 ratio with the transfection reagent polyethyleneimine (PEI) (1 mg/ml, 1:3 PEI-total DNA [Polysciences]) into HEK 293T cells. Pseudoviruses were harvested 72 h posttransfection. Neutralizing activity was assessed using a single-round replication pseudovirus assay with TZM-bl target cells, as described previously (2). Glycan sites N137, N295, N386, and N392 were removed using site-directed mutagenesis through an Asn-to-Ala mutation. Mutations were verified by DNA sequencing (MWG Eurofins, Germany). (B) Glycan profile of recombinant gp120 expressed in HEK 293T cells in the presence of kifunensine, analyzed as described in the legend to Fig. 2C. (C) Enzyme-linked immunosorbent assay (ELISA) data of PGT135 (blue) and 2G12 (black) binding to wild-type (continuous line) and kifunensine-treated (dashed line) gp120. ELISAs were performed as previously described (34).
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The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice

The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice

Whilst there have been two recent reports of the successful restoration of functional glycosylation and amelioration of the muscle pathology by AAV (Adeno-Associated Virus) vectors carrying either FKRP (41) or fukutin (42), one of the most promising forms of therapy proposed in recent years for the dystroglycanopathies is the up-regulation of LARGE; a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid to generate a heteropolysaccharide that confers α-dystroglycan with its ligand binding properties (43). This is based on observations showing that LARGE is able to restore α-dystroglycan glycosylation and functional laminin binding to cells taken from patients with congenital muscular dystrophy (FCMD, MEB and WWS), seemingly irrespective of the gene involved (44). Whilst this response may be dependent on the availability of O-mannosyl phosphate acceptor sites (32), this strategy is still considered as being potentially useful for a wide range of patients. We have previously shown that in mice, over-expression of LARGE on a wild type background induces no overt pathology and is only associated with a minor loss of force in response to eccentric exercise in older mice, supporting the idea that increasing levels of this glycosyltransferase may represent an important therapeutic approach (45). However, it remains crucial to test such a
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Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors

Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors

tors (38). Therefore, viruses containing complex glycans at the receptor-binding site would exhibit lower susceptibility to NA inhibitors than those lacking such glycans. To investigate this possibility, we utilized the influenza A viruses that belong to the H1N1 antigenic subtype as a model. Viruses of this subtype produced the devastating “Spanish flu” pandemic in 1918 (42), and antigenic drift variants of this virus remained in circulation in the human population until mid-1950s. In 1977, the virus reemerged in the human population, causing the “Russian flu” epidemic; since that time, antigenic drift variants have been in circulation in the human population. There is a considerable variance in the glycosylation patterns near the HA receptor- binding site among the human influenza A/H1N1 viruses, due to antigenic drift and host adaptation (20). For example, the so-called early laboratory-passaged virus A/WSN/33 (WSN) contains a single glycosylation site at Asn129, whereas the contemporary virus, A/Charlottesville/31/95 (CH/95), contains two additional glycosylation sites at Asn 94a and Asn 163) (Fig. 1) (numbering according to reference 52). In our previous studies, we demonstrated that both viruses were equally sus- ceptible to NA inhibitors by the enzyme inhibition assay, whereas in cell culture, the CH/95 virus was drug resistant and the WSN virus was drug susceptible (12). Moreover, reassor- tant virus that contained the HA gene from the CH/95 virus in the WSN virus genetic background also exhibited drug resis- tance in cell culture (12). In the present study, we investigated the contribution of HA glycans attached at Asn 94a, 129, and 163 to viral dependence on NA activity. Our findings provide further insights into molecular mechanisms underlying the hu- man virus resistance to NA inhibitors in cell culture.
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Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

VSV-infected HeLa cells were, therefore, labeled with one of four different sugar precursors 3H-labeled glucosamine, mannose, galactose, or fucose for periods ranging from 5 to 60 min, a[r]

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Detection of a Novel Unglycosylated Form of Hepatitis C Virus E2 Envelope Protein That Is Located in the Cytosol and Interacts with PKR

Detection of a Novel Unglycosylated Form of Hepatitis C Virus E2 Envelope Protein That Is Located in the Cytosol and Interacts with PKR

and immunoprecipitated using three different anti-E2 mono- clonal antibodies of different origins. Two major HCV-specific products were immunoprecipitated with all three antibodies used (Fig. 1A). One (⬇68 kDa) corresponds to the glycosy- lated form of E2, with various bands representing different degrees of glycosylation (7), and the other, with an apparent molecular mass of 38 kDa, corresponds in size to that pre- dicted for the unglycosylated E2. The same results were ob- tained in HeLa cells, although the level of E2 expression in this cell line was lower (Fig. 1B). To confirm that the low-molec- ular-weight product corresponds to an unglycosylated form of E2, transfected cells were labeled in the presence of tunicamy- cin, a glycosylation inhibitor, and immunoprecipitated with an anti-E2 antibody, or alternatively, the immunoprecipitated E2 was digested with glycosidases: peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H) (Fig. 1C). Inter- estingly, after tunicamycin treatment, two distinct bands, of 38 and 42 kDa, were found. The lower band corresponds to p38, indicating that p38 is likely an unglycosylated form of E2 (E2-p38). The nature of p42 is not known, but it has also been previously described by Grakoui et al. (9). One possible inter- pretation is that p42 represents the product of another post- translational modification of E2 that is not inhibited by tuni- camycin. Complete removal of carbohydrates by PNGase F yielded the same two bands of 38 and 42 kDa as with the tunicamycin treatment, consistent with the interpretation that E2 may have another posttranslational modification in addi- tion to N-glycosylation. The intensity of the band correspond- ing to E2-p38 was reduced after PNGase F treatment, probably due to dilution of the sample after digestion. Since E2-p42 was observed only after treatments that inhibit glycosylation or remove glycans and its presence corresponds to a decrease of E2-gp68, these results suggest that E2-p42 is the true deglyco- sylated form of the mature E2, E2-gp68. The absence of E2- p42 in untreated cells suggests either that E2-p42 is a transient conformation that is rapidly glycosylated or, more likely, that the putative additional posttranslational modification of E2- p42 occurs simultaneously with glycosylation. Endo H diges- tion yielded a third product, of 44 kDa, which probably repre- sents a partially deglycosylated E2, since Endo H cleaves after the first N-acetylglucosamine of glycans but does not remove them completely. The same results were obtained in HeLa cells (data not shown). As an ultimate confirmation, all the
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Espirito Santo Virus: a New Birnavirus That Replicates in Insect Cells

Espirito Santo Virus: a New Birnavirus That Replicates in Insect Cells

the virions, and internally, VP3 forms a ribonucleoprotein com- plex with the genomic RNA (32), and VP1 is found both free and covalently attached to the genomic RNA (19). There is no descrip- tion of entomobirnaviruses that infect mosquitoes or mosquito- derived cell lines. The only common contaminants of mosquito cell lines described are the small densoviruses, 20-nm viruses be- longing to the family Parvoviridae (4). Among them, a new denso- virus from Aedes albopictus C6/36 cells that were chronically infected was recently isolated and characterized (9). Aedes albopic- tus, along with Aedes aegypti, is considered to be one of the most important dengue virus (DENV) vectors, with an even higher level of susceptibility to dengue virus than A. aegypti (37). The Aedes albopictus C6/36 cell line has become very important in the study of arboviruses because of its wide range of susceptibilities to dif- ferent viruses and its ability to produce plaques with a number of them (16, 44). It was described in early studies that some Aedes albopictus cell lines developed in the late 1960s presented contam- ination with multiple viruses, such as parvovirus, togavirus, and orbivirus-like particles (26). These persistent and innocuous viral infections were described to be common in insects and crusta- ceans as single, dual, or multiple coinfections (27).
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Susceptibility to hepatotoxicity in transgenic mice that express a dominant negative human keratin 18 mutant

Susceptibility to hepatotoxicity in transgenic mice that express a dominant negative human keratin 18 mutant

eases is accumulating at a rapid pace (reviewed in references 4–7). The diversity of the IF associated human diseases reflects the diversity and tissue-specific expression of the large cyto- plasmic IF protein family. Examples of the tissue-specific ex- pression of IF proteins include keratins in epithelia, desmin in muscle, and neurofilaments in neuronal cells. To date, muta- tions in 11 of the more than 20 keratins (K1-K20) are known to cause human disease, as in epidermolysis bullosa simplex (EBS) (K5, K14); epidermolytic hyperkeratosis and epidermal nevi (K1/K10 and K10, respectively); epidermolytic pal- moplantar keratoderma (K9); ichthyosis bullosa of Siemens (K2e); pachyonychia congenita (K6, K16, and K17); and white sponge nevus (K4 and K13) (see references 8 and 9 for K16/17 and K6, respectively; 10 and 11 for K4 and K13, respectively; and 4–7 for K5, K14, K1, K10, K9, K2 mutations). Furthermore, mutations in neurofilament-H have been identified in a small number of sporadic cases of amyotrophic lateral sclerosis (12). In contrast to the above mentioned epidermal keratins, mutations in simple epithelial-type keratins have not been as- sociated with a human disease to date. These latter keratins in- clude K8, K18, K19, and K20, which are expressed in variable combinations in glandular tissues such as liver, exocrine pan- creas and intestine (1–3). Despite the lack of a human disease association with simple epithelial keratins, several animal models as well as the epidermal keratin diseases identified to date suggest that one or more disease are likely to be identified as simple epithelial keratin human disease(s). The relevance and importance of animal models in identifying IF related hu- man diseases is demonstrated by the expression of a truncated K14 in transgenic mice (13) with a resultant phenotype that ul- timately led to the identification of epidermolysis bullosa sim- plex as the first described human IF-related disease (14). In the case of simple epithelial keratin animal models, K8-null mice bleed into their livers and die at a late embroynic stage (15) or develop colitis, colonic hyperplasia, and rectal prolapse (16) depending on the genetic background of the mice. Fur- thermore, ectopic expression of epidermal keratins in pancre- atic islet cells (17) or in hepatocytes (18) results in keratin fila- ment disruption in these cells and development of diabetes or chronic hepatitis, respectively. In addition, we recently de- scribed the generation of transgenic mice that express human K18 that is mutated at a highly conserved arginine residue (arg89 to cys) (19). This arg is conserved among many IF pro- teins, and is a hot mutation spot in several of the epidermal keratin associated human diseases described to date (4–7). Mice expressing the arg mutant human K18, but not wild-type Address reprint requests to Nam-On Ku and all other correspondence
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Processing of the glycoprotein of feline immunodeficiency virus: effect of inhibitors of glycosylation.

Processing of the glycoprotein of feline immunodeficiency virus: effect of inhibitors of glycosylation.

To determine whether the processing of the FIV glycoprotein was altered in the presence of inhibitors of N-linked glycosylation or glycoprotein transport, FIV-infected CRFK cells were tr[r]

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Growth as a solid tumor or reduced glucose concentrations in culture reversibly induce CD44 mediated hyaluronan recognition by Chinese hamster ovary cells

Growth as a solid tumor or reduced glucose concentrations in culture reversibly induce CD44 mediated hyaluronan recognition by Chinese hamster ovary cells

The density, molecular isoform, and posttranslational mod- ifications of CD44 can markedly influence growth and met- astatic behavior of tumors. Many CD44 functions, including some involving tumors, have been attributed to its ability to recognize hyaluronan (HA). However, only certain CD44- bearing cells bind soluble or immobilized HA. We now show that CD44 made by wild-type Chinese hamster ovary (CHO-K1) cells and a ligand-binding subclone differ with re- spect to N-linked glycosylation. While both bear CD44 with highly branched, complex-type glycoforms, CD44 expressed by the wild type was more extensively sialylated. CHO-K1 cells which failed to recognize HA when grown in culture gained this ability when grown as a solid tumor and reverted to a non–HA-binding state when returned to culture. The ability of CHO-K1 cells to recognize HA was also reversibly in- duced when glucose concentrations in the medium were re- duced. Glucose restriction influenced CD44-mediated HA binding by many but not all, of a series of murine tumors. Glucose concentrations and glycosylation inhibitors only partially influenced CD44 receptor function on resting mu- rine B lymphocytes. These observations suggest that glucose levels or other local environmental conditions may mark- edly influence glycosylation pathways used by some tumor cells, resulting in dramatic alteration of CD44-mediated functions. ( J. Clin. Invest. 1997. 100:1217–1229.) Key words: cell adhesion • glycosylation • extracellular matrix • blood
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The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus.

The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus.

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membrane- bound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipi- tated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83- to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N -glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal micros- copy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.
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