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1.
Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells 总被引:2,自引:0,他引:2
The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins. 相似文献
2.
The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins 总被引:20,自引:6,他引:14
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《The Journal of cell biology》1987,105(6):2665-2674
The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency. 相似文献
3.
The secretion of N-linked glycosylated alpha-lactalbumin was much higher in the expression system of yeast Pichia pastoris carrying goat alpha-lactalbumin cDNA than in mammalian milk. This is possibly because of the presence of N-linked glycosylation signal sequences, Asn(45)-Asp(46)-Ser(47) and Asn(74)-Ile(75)-Ser(76), in wild-type alpha-lactalbumin. Attempts to elucidate the mechanism of the higher secretion of glycosylated alpha-lactalbumin in P. pastoris were made. Mutant N45D that deleted the N-linked glycosylation signal sequence at position 45 predominantly secreted nonglycosylated protein. On the other hand, mutant D46N with another N-glycosylation signal site at position 46 only secreted N-linked glycosylated alpha-lactalbumin, i.e. not the nonglycosylated protein. The total secreted amount of mutant N45D was greatly enhanced, while the secreted amounts of the wild-type and mutant D46N were very low, suggesting that the increase in the number of glycosylation sites greatly reduced the secretion of alpha-lactalbumin. It seems likely that the glycosylated alpha-lactalbumin may be degraded by the quality control system. 相似文献
4.
C K?hne A Johnson S Tom D H Peers R L Gehant T A Hotaling D Brousseau T Ryll J A Fox S M Chamow P W Berman 《Journal of cellular biochemistry》1999,75(3):446-461
Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport. 相似文献
5.
J Aikawa T Yamashita M Nishiyama S Horinouchi T Beppu 《The Journal of biological chemistry》1990,265(23):13955-13959
The Mucor rennin gene encoding a prepro form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. The mature M. pusillus rennin secreted efficiently by yeast was a highly glycosylated protein. Analysis by a combination of site-directed mutagenesis of each of the three possible glycosylation sites and treatment of the secreted M. pusillus rennins with endo-beta-N-acetylglucosaminidase H revealed that the mature yeast M. pusillus rennin contained two asparagine-linked glycosylation sites among the three possible glycosylation sites. A mutation of the 2 glycosylated asparagine residues of M. pusillus rennin resulted in significant decreases in the level of secretion by yeast cells. In addition, the extent of glycosylation of M. pusillus rennin was found to affect the enzyme properties such as milk-clotting and proteolytic activities. 相似文献
6.
Synthesis and processing of the three major envelope glycoproteins of Epstein-Barr virus. 总被引:16,自引:12,他引:4
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In pulse-chase experiments, the three major Epstein-Barr virus envelope glycoproteins, gp350/300, gp250/200, and gp85, were shown to be synthesized from separate precursors of 190,000, 160,000, and 83,000 daltons, respectively. These three pulse-labeled species were chased into the mature forms of the glycoproteins between 1 and 3 h after transfer to nonradioactive medium. Digestion of precursor forms with endo-beta-N-acetylglucosaminidase H (endo H) yielded polypeptides of 160,000, 120,000, and 75,000 daltons. Comparison of these results with those from experiments with tunicamycin, which specifically blocks N-linked glycosylation, indicated that some other post-translational modification(s), probably O-linked glycosylation, contributes about 100,000 and 60,000 daltons of apparent molecular mass to gp350/300 and gp250/200, respectively. Experiments with endo H showed that mature gp350/300 and gp250/200 contain complex-type (endo H-resistant) N-linked glycosyl chains, whereas gp85 contains both high-mannose (endo H-sensitive)- and complex-type oligosaccharides. In contrast to the results obtained with the three envelope glycoproteins, no precursor forms of the two unglycosylated protein, p160 (the major Epstein-Barr virus capsid antigen) and p140 (an envelope protein), were detected. The partial proteolytic maps of gp350/300 and gp250/200 were quite similar, suggesting that polypeptide sequence homology could account for at least part of the observed serological cross-reactivity of the two proteins. Taken together, these results demonstrate that the polypeptide portions of gp350/300 and gp250/200 are closely related but not derived from a common precursor. Furthermore, the polypeptide portions comprise half or less of the apparent molecular weight of the mature glycoproteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
7.
Szakonyi G Klein MG Hannan JP Young KA Ma RZ Asokan R Holers VM Chen XS 《Nature structural & molecular biology》2006,13(11):996-1001
Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses. 相似文献
8.
Secretion of N-glycosylated human recombinant interleukin-1 alpha in Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
We have expressed fragments of the cDNA coding for mature human interleukin-1 alpha (hIL-1 alpha) in Saccharomyces cerevisiae. Mature hIL-1 alpha contains one potential N-linked glycosylation site that is not recognized in mammalian cells. Translational fusions to either one of three yeast signal sequences resulted in secretion of bioactive, N-glycosylated hIL-1 alpha. The extent of glycosylation was significantly reduced using the alpha-factor signal sequence, which itself contains three N-linked glycosylation sites known to be core glycosylated. N-glycosylation has no effect on biological specific activity. 相似文献
9.
Processing of the gp55-116 envelope glycoprotein complex (gB) of human cytomegalovirus. 总被引:22,自引:21,他引:1
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The processing pathway of the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus was studied using inhibitors of glycosylation and endoglycosidases. The results of these studies indicated that the mature gp55-116 is synthesized by the addition of both simple and complex N-linked sugars to a nonglycosylated precursor of estimated Mr 105,000. In a rapid processing step, the Mr 105,000 precursor is glycosylated to a protein of Mr 150,000 (gp150) which contains only endoglycosidase H-sensitive sugar linkages. The gp150 is then processed relatively slowly to a Mr 165,000 to 170,000 species (gp165-170), which is then cleaved to yield the mature gp55-116. Monensin prevented the final processing steps of the gp150, including cleavage, suggesting that transport through the Golgi apparatus is required for complete processing. Digestion of the intracellular forms of this complex as well as the virion forms confirmed the above findings and indicated that the mature virion form of gp55 contains 8,000 daltons of N-linked sugars. The virion gp116 contains some 52,000 to 57,000 daltons of N-linked carbohydrates and approximately 5,000 daltons of O-linked sugars. 相似文献
10.
Model for intracellular folding of the human immunodeficiency virus type 1 gp120. 总被引:18,自引:14,他引:4
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The intracellular folding of the human immunodeficiency virus type 1 gp120 has been assessed by analyzing the ability of the glycoprotein to bind to the viral receptor CD4. Pulse-chase experiments revealed that the glycoprotein was initially produced in a conformation that was unable to bind to CD4 and that the protein attained the appropriate tertiary structure for binding with a half-life of approximately 30 min. The protein appears to fold within the rough endoplasmic reticulum, since blocking of transport to the Golgi apparatus by the oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone did not appear to perturb the folding kinetics of the molecule. The relatively lengthy folding time was not due to modification of the large number of N-linked glycosylation sites on gp120, since inhibition of the first steps in oligosaccharide modification by the inhibitors deoxynojirimycin or deoxymannojirimycin did not impair the CD4-binding activity of the glycoprotein. However, production of the glycoprotein in the presence of tunicamycin and removal of the N-linked sugars by endoglycosidase H treatment both resulted in deglycosylated proteins that were unable to bind to CD4, suggesting in agreement with previous results, that glycosylation contributes to the ability of gp120 to bind to CD4. Interestingly, incomplete endoglycosidase H treatment revealed that a partially glycosylated glycoprotein could bind to the receptor, implying that a subset of glycosylation sites, perhaps some of those conserved in different isolates of human immunodeficiency virus type 1, might be important for binding of the viral glycoprotein to the CD4 receptor. 相似文献
11.
Synthesis, processing, and secretion of the Marek''s disease herpesvirus A antigen glycoprotein. 总被引:7,自引:7,他引:0
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The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption. 相似文献
12.
G P Livi J S Lillquist L M Miles A Ferrara G M Sathe P L Simon C A Meyers J A Gorman P R Young 《The Journal of biological chemistry》1991,266(23):15348-15355
Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus. 相似文献
13.
Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus. 总被引:16,自引:13,他引:3
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The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses. 相似文献
14.
Selective removal of alpha heavy-chain glycosylation sites causes immunoglobulin A degradation and reduced secretion. 总被引:6,自引:1,他引:5
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The importance of carbohydrate in the secretion of immunoglobulin A (IgA) has previously been suggested by results of studies with tunicamycin, which prevents N-linked glycosylation of all cell glycoproteins. To directly evaluate the role of individual oligosaccharides in the secretion of IgA, we have used site-directed mutagenesis to selectively eliminate the two N-linked attachment sites reported to be glycosylated in alpha heavy chains. Transfected wild-type and mutant alpha genes were expressed in kappa light-chain-producing MPC-11 variant myeloma cells, and secretion kinetics of the IgAs were compared. Removal of either or both glycosylation sites led to intracellular alpha heavy-chain degradation and a 90 to 95% inhibition of IgA secretion. These results reveal that both N-linked oligosaccharides of the alpha heavy chain are essential for intracellular stability and normal secretion of IgA. This suggests that the key function of carbohydrate here is to maintain proper conformation of the glycoprotein. We also found that when expressed in the MPC-11 variant cells, alpha heavy chains were glycosylated at a third, normally unused site. 相似文献
15.
G L Firestone 《The Journal of biological chemistry》1983,258(10):6155-6161
The relationship of protein glycosylation to compartmentalization and processing of mouse mammary tumor virus (MTV) glycoproteins has been examined in M1.54, a cloned line of MTV-infected rat hepatoma tissue culture cells. Previous work established that full maturation of MTV glycoproteins in this cell line requires dexamethasone, a synthetic glucocorticoid (Firestone, G. L., Payvar, F., and Yamamoto, K. R. (1982) Nature (Lond.) 300, 221-225). The ability to regulate production of the full complement of five mature membrane-associated and secreted viral glycoproteins from one initially synthesized precursor has been used to advantage in the present work. At concentrations of tunicamycin that specifically inhibit N-linked protein glycosylation, incorporation of [35S]methionine into total cellular and secreted protein is not detectably affected, MTV-specific mRNAs are produced normally, and the nonglycosylated form of the glycosylated viral precursor polyprotein accumulates within the cells. However, tunicamycin inhibits the site-specific cleavage of the glycosylated polyprotein and distribution of MTV polypeptides to the cell surface and extracellular fractions. Thus, when tunicamycin-treated cultures of M1.54 are exposed to dexamethasone and [35S]methionine, no labeled viral antigens are detected in the culture medium. Similarly, tunicamycin prevents the appearance of membrane-associated viral antigens that can be labeled externally by lactoperoxidase-mediated iodination and it protects the cells against the cytolytic effects of MTV-specific antiserum and complement. Taken together, these results are consistent with the view that while glycosylation of some proteins may be unessential for their compartmentalization and processing, it does appear to be correlated with proper maturation of others. The hormone-dependent maturation of MTV glycoproteins in M1.54 may be particularly useful for study of this latter class since glycosylation is stringently associated with their compartmentalization and cleavage. 相似文献
16.
Structural analysis of the varicella-zoster virus gp98-gp62 complex: posttranslational addition of N-linked and O-linked oligosaccharide moieties. 总被引:32,自引:29,他引:3
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Varicella-zoster virus specifies the formation of several glycoproteins, including the preponderant gp98-gp62 glycoprotein complex in the outer membranes of virus-infected cells. These viral glycoproteins are recognized and precipitated by a previously described monoclonal antibody designated monoclone 3B3. When an immunoblot analysis was performed, only gp98 was reactive with monoclone 3B3 antibody; likewise, titration in the presence of increased concentrations of sodium dodecyl sulfate during antigen-antibody incubations caused selective precipitation of gp98 but not gp62. Further structural analyses of gp98 were performed by using the glycosidases endo-beta-N-acetylglucosaminidase H (endoglycosidase H) and neuraminidase and two inhibitors of glycosylation (tunicamycin and monensin). In addition to gp98, antibody 3B3 reacted with several intermediate products, including gp90, gp88, gp81, and a nonglycosylated polypeptide, p73. Since gp98 was completely resistant to digestion with endoglycosidase H, it contained only complex carbohydrate moieties; conversely, gp81 contained mainly high-mannose residues. Polypeptide p73 was immunodetected in the presence of tunicamycin and designated as a nascent recipient of N-linked sugars, whereas gp88 was considered to contain O-linked oligosaccharides because its synthesis was not affected by tunicamycin. The ionophore monensin inhibited production of mature gp98, but other intermediate forms, including gp90, were detected. Since the latter product was similar in molecular weight to the desialated form of gp98, one effect of monensin treatment of varicella-zoster virus-infected cells was to block the addition of N-acetylneuraminic acid. Monensin also blocked insertion of gp98 into the plasma membrane and, as determined by electron microscopy, inhibited envelopment of the nucleocapsid and its transport within the cytoplasm. On the basis of this study, we reached the following conclusions: the primary antibody 3B3-binding epitope is located on gp98, gp98 is a mature product of viral glycoprotein processing, gp98 contains both N-linked and O-linked oligosaccharide side chains, gp90 is the desialated penultimate form of gp98, gp88 is an O-linked intermediate of gp98, gp81 is the high-mannose intermediate of gp98, and p73 is the unglycosylated precursor of gp98. 相似文献
17.
Dos Reis Almeida FB Carvalho FC Mariano VS Alegre AC Silva Rdo N Hanna ES Roque-Barreira MC 《PloS one》2011,6(12):e29216
The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology. 相似文献
18.
Subcellular localization of glycoproteins encoded by the viral oncogene v-fms 总被引:43,自引:6,他引:37
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The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gp120fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steady-state measurements showed that most gp120fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gp140fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gp140fms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fms-coded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane. 相似文献
19.
Dudich E Dudich I Semenkova L Benevolensky S Morozkina E Marchenko A Zatcepin S Dudich D Soboleva G Khromikh L Roslovtceva O Tatulov E 《Protein expression and purification》2012,84(1):94-107
Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP. 相似文献
20.
The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted. 相似文献