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1
Garbutt, Michael, Ryan Liebscher, Victoria Wahl-Jensen, Steven Jones, Peggy Möller, Ralf Wagner, Viktor Volchkov, Hans-Dieter Klenk, Heinz Feldmann, and Ute Ströher. "Properties of Replication-Competent Vesicular Stomatitis Virus Vectors Expressing Glycoproteins of Filoviruses and Arenaviruses." Journal of Virology 78, no. 10 (May 15, 2004): 5458–65. http://dx.doi.org/10.1128/jvi.78.10.5458-5465.2004.
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ABSTRACT Replication-competent recombinant vesicular stomatitis viruses (rVSVs) expressing the type I transmembrane glycoproteins and selected soluble glycoproteins of several viral hemorrhagic fever agents (Marburg virus, Ebola virus, and Lassa virus) were generated and characterized. All recombinant viruses exhibited rhabdovirus morphology and replicated cytolytically in tissue culture. Unlike the rVSVs with an additional transcription unit expressing the soluble glycoproteins, the viruses carrying the foreign transmembrane glycoproteins in replacement of the VSV glycoprotein were slightly attenuated in growth. Biosynthesis and processing of the foreign glycoproteins were authentic, and the cell tropism was defined by the transmembrane glycoprotein. None of the rVSVs displayed pathogenic potential in animals. The rVSV expressing the Zaire Ebola virus transmembrane glycoprotein mediated protection in mice against a lethal Zaire Ebola virus challenge. Our data suggest that the recombinant VSV can be used to study the role of the viral glycoproteins in virus replication, immune response, and pathogenesis.
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Jorgenson, Rebecca L., Volker M. Vogt, and Marc C. Johnson. "Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping." Journal of Virology 83, no. 9 (February 18, 2009): 4060–67. http://dx.doi.org/10.1128/jvi.02425-08.
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ABSTRACT Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites.
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Quinn, Derek J., Neil V. McFerran, John Nelson, and W. Paul Duprex. "Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology." Bioscience Reports 32, no. 3 (March 29, 2012): 333–43. http://dx.doi.org/10.1042/bsr20110100.
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Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) – haptoEGFPs – which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein–haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.
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Lay Mendoza, Maria Fernanda, Marissa Danielle Acciani, Courtney Nina Levit, Christopher Santa Maria, and Melinda Ann Brindley. "Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins." Viruses 12, no. 12 (December 17, 2020): 1457. http://dx.doi.org/10.3390/v12121457.
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Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.
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Joshua, G. W. P., L. J. S. Harrison, and M. M. H. Sewell. "Developmental changes in proteins and glycoproteins revealed by direct radio-iodination of viable Taenia saginata larvae." Parasitology 99, no. 2 (October 1989): 265–74. http://dx.doi.org/10.1017/s0031182000058728.
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SummaryDirect surface I radio-isotope labelling techniques and SDS—PAGE analysis were used to compare the proteins and lentil—lectin adherent glycoproteins of the bovine stage of viable Taenia saginata larvae at three points in their development, the invasive oncospheres, immature (4-week-old) and mature (12 to 16-week-old) cysticerci. Some proteins and glycoproteins were present on all three of the ages of the parasite examined but there were also distinct age-specific proteins and glycoproteins detected on oncospheres and 4-week-old cysticerci and a marked difference between the protein/glycoprotein profiles of the parasite was apparent at these earlier stages of development and the mature cysticerci. The latter were characterized by the presence of high, 160–200 kDa molecular weight, lysine rich, glycoproteins, whereas small 16 and 18 kDa glycoproteins and a reduction-sensitive 23 kDa glycoprotein were first detected on 4-week-old immature cysticerci. Antigenic characterization of the isotope-labelled proteins and glycoproteins by immunoprecipitation against a panel of clinically defined bovine sera combined with SDS–PAGE analysis indicated that relatively few proteins were precipitated by sera from T. saginata-infected cattle as compared to the glycoproteins. However, both protein and glycoprotein antigens of possible protective and/or diagnostic significance were identified from oncospheres and cysticerci.
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Zhang, Libo, Yanhong Li, Riyao Li, Xiaohong Yang, Zimin Zheng, Jingxin Fu, Hai Yu, and Xi Chen. "Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli." Molecules 28, no. 6 (March 18, 2023): 2753. http://dx.doi.org/10.3390/molecules28062753.
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Protein N-glycosylation is a common post-translational modification that plays significant roles on the structure, property, and function of glycoproteins. Due to N-glycan heterogeneity of naturally occurring glycoproteins, the functions of specific N-glycans on a particular glycoprotein are not always clear. Glycoprotein in vitro N-glycan engineering using purified recombinant enzymes is an attractive strategy to produce glycoproteins with homogeneous N-glycoforms to elucidate the specific functions of N-glycans and develop better glycoprotein therapeutics. Toward this goal, we have successfully expressed in E. coli glycoside hydrolases and glycosyltransferases from bacterial and human origins and developed a robust enzymatic platform for in vitro processing glycoprotein N-glycans from high-mannose-type to α2–6- or α2–3-disialylated biantennary complex type. The recombinant enzymes are highly efficient in step-wise or one-pot reactions. The platform can find broad applications in N-glycan engineering of therapeutic glycoproteins.
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Si, Zhihai, Mark Cayabyab, and Joseph Sodroski. "Envelope Glycoprotein Determinants of Neutralization Resistance in a Simian-Human Immunodeficiency Virus (SHIV-HXBc2P 3.2) Derived by Passage in Monkeys." Journal of Virology 75, no. 9 (May 1, 2001): 4208–18. http://dx.doi.org/10.1128/jvi.75.9.4208-4218.2001.
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ABSTRACT The simian-human immunodeficiency virus SHIV-HXBc2 contains the envelope glycoproteins of a laboratory-adapted, neutralization-sensitive human immunodeficiency virus type 1 variant, HXBc2. Serial in vivo passage of the nonpathogenic SHIV-HXBc2 generated SHIV KU-1, which causes rapid CD4+ T-cell depletion and an AIDS-like illness in monkeys. A molecularly cloned pathogenic SHIV, SHIV-HXBc2P 3.2, was derived from the SHIV KU-1 isolate and differs from the parental SHIV-HXBc2 by only 12 envelope glycoprotein amino acid residues. Relative to SHIV-HXBc2, SHIV-HXBc2P 3.2 was resistant to neutralization by all of the antibodies tested with the exception of the 2G12 antibody. The sequence changes responsible for neutralization resistance were located in variable regions of the gp120 exterior envelope glycoprotein and in the gp41 transmembrane envelope glycoprotein. The 2G12 antibody, which neutralized SHIV-HXBc2 and SHIV-HXBc2P 3.2 equally, bound the HXBc2 and HXBc2P 3.2 envelope glycoproteins on the cell surface comparably. The ability of the other tested antibodies to achieve saturation was less for the HXBc2P 3.2 envelope glycoproteins than for the HXBc2 envelope glycoproteins, even though the affinity of the antibodies for the two envelope glycoproteins was similar. Thus, a highly neutralization-sensitive SHIV, by modifying both gp120 and gp41 glycoproteins, apparently achieves a neutralization-resistant state by decreasing the saturability of its envelope glycoproteins by antibodies.
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Calvete, J. J., J. L. McGregor, G. Rivas, and J. González-Rodríguez. "Identification of a Glycoprotein III a Dimer in Polyacrylamide Gel Separations of Human Platelet Membranes." Thrombosis and Haemostasis 58, no. 02 (1987): 694–97. http://dx.doi.org/10.1055/s-0038-1645957.
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SummaryMembrane glycoproteins IIb and IIIa play a major role in human blood platelet aggregation. The absence or the severe reduction of these two membrane glycoproteins, as observed in platelets of Glanzmann’s thrombasthenic patients, is related to a lack of platelet aggregation. Separation of Glanzmann’s thrombasthenic platelet samples by two-dimensional polyacrylamide O’Farrell gels show the absence of a high and several low molecular mass glycoproteins, in addition to the loss of glycoproteins IIb and IIIa (McGregor J. L. et al. Eur. J. Biochem. 1981; 116: 379-388). The aim of this study was to identify the nature of the high molecular mass component, absent in thrombasthenic platelets. A high molecular mass glycoprotein (200 kDa), present in two-dimensional SDS-polyacrylamide O-Farrell gel separations, was recognized by a monoclonal antibody (MP37) directed against glycoprotein IIIa. Moreover, the tryptic peptide map of this high molecular mass glycoprotein was nearly identical to that of glycoprotein IIIa. These results indicate that this high molecular mass glycoprotein present in SDS-polyacrylamide gels is a dimer of glycoprotein IIIa. This work raises the possibility that the high molecular mass glycoprotein, absent in two-dimensional O’Farrell gel separations of thrombasthenic platelets, is a dimer of glycoprotein IIIa.
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LaBonte, Jason A., Navid Madani, and Joseph Sodroski. "Cytolysis by CCR5-Using Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Is Dependent on Membrane Fusion and Can Be Inhibited by High Levels of CD4 Expression." Journal of Virology 77, no. 12 (June 15, 2003): 6645–59. http://dx.doi.org/10.1128/jvi.77.12.6645-6659.2003.
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ABSTRACT T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4+ CXCR4+ T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5+ target cells. As is the case for CXCR4+ target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4+ CCR5+ cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4+ T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4+ T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.
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Shammala, Farid Abu. "Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery." Brazilian Journal of Biological Sciences 4, no. 7 (2017): 203–15. http://dx.doi.org/10.21472/bjbs.040720.
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Most proteins are glycosylated, glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. With recent advances in proteomics, analytical and computational technologies, glycoproteomics, the global analysis of glycoproteins, is rapidly emerging as a subfield of proteomics with high biological and clinical relevance. Glycoproteomics integrates glycoprotein enrichment and proteomics technologies to support the systematic identification and quantification of glycoproteins in a complex sample. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for mapping glycoprotein glycosylation and detailed glycan structural determination. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.
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Yang, Xinzhen, Juliette Lee, Erin M. Mahony, Peter D. Kwong, Richard Wyatt, and Joseph Sodroski. "Highly Stable Trimers Formed by Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Fused with the Trimeric Motif of T4 Bacteriophage Fibritin." Journal of Virology 76, no. 9 (May 1, 2002): 4634–42. http://dx.doi.org/10.1128/jvi.76.9.4634-4642.2002.
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ABSTRACT The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins. Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X. Yang et al., J. Virol. 74:5716-5725, 2000). To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin. The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct. Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike. Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer. The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins. The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.
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Ji, Xin, Gene G. Olinger, Sheena Aris, Ying Chen, Henry Gewurz, and Gregory T. Spear. "Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization." Journal of General Virology 86, no. 9 (September 1, 2005): 2535–42. http://dx.doi.org/10.1099/vir.0.81199-0.
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Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.
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Ninagawa, Satoshi, Tetsuya Okada, Yoshiki Sumitomo, Satoshi Horimoto, Takehiro Sugimoto, Tokiro Ishikawa, Shunichi Takeda, et al. "Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway." Journal of Cell Biology 211, no. 4 (November 16, 2015): 775–84. http://dx.doi.org/10.1083/jcb.201504109.
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Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.
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Choukhi, Amélie, André Pillez, Hervé Drobecq, Christian Sergheraert, Czeslaw Wychowski, and Jean Dubuisson. "Characterization of aggregates of hepatitis C virus glycoproteins." Journal of General Virology 80, no. 12 (December 1, 1999): 3099–107. http://dx.doi.org/10.1099/0022-1317-80-12-3099.
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Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which assemble in oligomeric structures. Studies of HCV glycoprotein assembly using heterologous expression systems have shown that these glycoproteins can follow two pathways: a productive pathway leading to the formation of a non-covalent heterodimer; and a non-productive pathway leading to the formation of large disulfide-linked aggregates. The non-covalent HCV glycoprotein complex is probably the functional complex which plays an active role in the entry process in host cells. The aggregates are believed to be waste products; however, one can imagine that, in infected cells, they could provide HCV glycoproteins with additional functions. To further understand the potential role played by HCV glycoprotein aggregates in HCV infection, a MAb (H14) was produced which specifically recognizes these aggregates but not the non-covalent E1E2 heterodimer. The H14 epitope was shown to be present on both HCV glycoproteins and was sensitive to deglycosylation. An additional characterization of HCV glycoprotein aggregates, with the help of MAb H14, indicates that they share an epitope with a cellular protein called Mac-2 binding protein. The presence of such an epitope on HCV glycoprotein aggregates could potentially lead to the production of autoantibodies recognizing Mac-2 binding protein in HCV-infected patients.
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Snyder, Aleksandra, Todd W. Wisner, and David C. Johnson. "Herpes Simplex Virus Capsids Are Transported in Neuronal Axons without an Envelope Containing the Viral Glycoproteins." Journal of Virology 80, no. 22 (September 13, 2006): 11165–77. http://dx.doi.org/10.1128/jvi.01107-06.
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ABSTRACT Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.
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Sandrin, Virginie, Delphine Muriaux, Jean-Luc Darlix, and François-Loïc Cosset. "Intracellular Trafficking of Gag and Env Proteins and Their Interactions Modulate Pseudotyping of Retroviruses." Journal of Virology 78, no. 13 (July 1, 2004): 7153–64. http://dx.doi.org/10.1128/jvi.78.13.7153-7164.2004.
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ABSTRACT Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.
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Yang, Xinzhen, Svetla Kurteva, Xinping Ren, Sandra Lee, and Joseph Sodroski. "Subunit Stoichiometry of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers during Virus Entry into Host Cells." Journal of Virology 80, no. 9 (May 1, 2006): 4388–95. http://dx.doi.org/10.1128/jvi.80.9.4388-4395.2006.
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ABSTRACT The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.
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Rosenberg, Arielle R., Lélia Delamarre, Anna Preira, and Marie-Christine Dokhélar. "Analysis of Functional Conservation in the Surface and Transmembrane Glycoprotein Subunits of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2." Journal of Virology 72, no. 9 (September 1, 1998): 7609–14. http://dx.doi.org/10.1128/jvi.72.9.7609-7614.1998.
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ABSTRACT Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are closely related retroviruses with nucleotide sequences that are 65% identical. To determine whether their envelope glycoproteins function similarly and to define the molecular determinants of HTLV-2 envelope-mediated functions, we have used pseudotyped viruses and have introduced mutations into regions of the HTLV-2 glycoproteins homologous to those known to be important for HTLV-1 glycoprotein functions. The envelopes of the two viruses could be exchanged with no loss of infectivity, suggesting that the glycoproteins function in broadly similar ways. However, comparative analysis of the HTLV-1 and HTLV-2 glycoproteins showed subtle differences in the structure-function relationships of the two surface glycoprotein (SU) subunits, even though they recognize the same receptor. Indeed, mutations introduced at equivalent positions in the two SU glycoproteins resulted in different phenotypes in the two viruses. The scenario is the opposite for the transmembrane glycoprotein (TM) subunits, in which the functional domains of the two viruses are strictly conserved, confirming the involvement of the TM ectodomain in postfusion events required for full infectivity of the HTLVs. Thus, although they recognize the same receptor, the HTLV-1 and HTLV-2 SU subunits have slightly different ways of transducing the conformational information that primes a common fusion mechanism effected by similar TM subunits.
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Karger, Axel, Ulrike Schmidt, and Ursula J. Buchholz. "Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin." Journal of General Virology 82, no. 3 (March 1, 2001): 631–40. http://dx.doi.org/10.1099/0022-1317-82-3-631.
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Bovine respiratory syncytial virus (BRSV) encodes three transmembrane envelope glycoproteins, namely the small hydrophobic (SH) protein, the attachment glycoprotein (G) and the fusion glycoprotein (F). The BRSV reverse genetics system has been used to generate viable recombinant BRSV lacking either the G gene or the SH gene or both genes. The deletion mutants were fully competent for multicycle growth in cell culture, proving that, of the BRSV glycoprotein genes, the SH and G genes are non-essential. Virus morphogenesis was not impaired by either of the deletions. The deletion mutants were used to study the role of the F glycoprotein and the contributions of SH and G with respect to virus attachment. Attachment mediated by the F protein alone could be blocked by soluble heparin, but not by chondroitin sulphate. Heparin affinity chromatography revealed that both the BRSV G and F glycoproteins have heparin-binding activity, with the affinity of the F glycoprotein being significantly lower than that of G. Therefore, the roles of the BRSV glycoproteins in virus attachment and receptor binding have to be reconsidered.
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Børsum, Tone. "Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells." Thrombosis and Haemostasis 63, no. 02 (1990): 303–11. http://dx.doi.org/10.1055/s-0038-1645214.
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SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.
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Bloodgood, R. A., and N. L. Salomonsky. "The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein." Journal of Cell Biology 127, no. 3 (November 1, 1994): 803–11. http://dx.doi.org/10.1083/jcb.127.3.803.
Full textAbstract:
Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60-kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.
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Weaver, T. E., J. A. Whitsett, W. M. Hull, and G. Ross. "Identification of canine pulmonary surfactant-associated glycoprotein A precursors." Journal of Applied Physiology 58, no. 6 (June 1, 1985): 2091–95. http://dx.doi.org/10.1152/jappl.1985.58.6.2091.
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Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000–28,000 daltons; glycoprotein A2, 32,000–34,000 daltons; and glycoprotein A3, 37,000–38,000 daltons; pH at isoelectric point (pI) 4.5–5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8–5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000–34,000 daltons, pI 4.8–5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000–28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.
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Batonick, Melissa, and Gail W. Wertz. "Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells." Advances in Virology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/343408.
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Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.
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Bieńkowska-Szewczyk, K., and B. Szewczyk. "Expression of genes coding for animal virus glycoproteins in heterologous systems." Acta Biochimica Polonica 46, no. 2 (June 30, 1999): 325–39. http://dx.doi.org/10.18388/abp.1999_4166.
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The outermost layers of animal viruses are usually composed of glycoproteins. They are responsible not only for the entrance of viruses into, and release from host cells but also for the initial interaction of a viral particle with immunological defense of the host. It is therefore not surprising that many laboratories devote a lot of effort to study viral glycoproteins at the molecular level. Very often such studies are possible only after the introduction of a glycoprotein gene into a heterologous system. Expression of glycoprotein genes is usually obtained in mammalian or insect cells. Expression in mammalian cells yields viral glycoproteins with glycan chains indistinguishable from the original counterparts in virion particles but the level of synthesis of glycoproteins is very low. Vaccinia virus is the most common vector for expression in mammalian cells. It is easy to grow, the introduction of foreign genes is relatively simple and, due to the size of the vaccinia genome, it can accept large pieces of foreign DNA. Glycosylation in insect cells is not as complex as in mammalian cells and usually glycoproteins produced in insect cells are of slightly lower molecular mass than those produced in mammalian cells. The most common vector for expression of glycoproteins in insect cells is a baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The great advantage of this system is a very high level of expression of foreign genes.
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Kukushkin, Nikolay V., Dominic S. Alonzi, Raymond A. Dwek, and Terry D. Butters. "Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase." Biochemical Journal 438, no. 1 (July 27, 2011): 133–42. http://dx.doi.org/10.1042/bj20110186.
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During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase.
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Lisanti, M. P., A. Le Bivic, M. Sargiacomo, and E. Rodriguez-Boulan. "Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery." Journal of Cell Biology 109, no. 5 (November 1, 1989): 2117–27. http://dx.doi.org/10.1083/jcb.109.5.2117.
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We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.
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Wang, Qiong, and Michael J. Betenbaugh. "Metabolic engineering of CHO cells to prepare glycoproteins." Emerging Topics in Life Sciences 2, no. 3 (October 18, 2018): 433–42. http://dx.doi.org/10.1042/etls20180056.
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As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.
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Bloodgood, R. A., and N. L. Salomonsky. "Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane." Journal of Cell Science 96, no. 1 (May 1, 1990): 27–33. http://dx.doi.org/10.1242/jcs.96.1.27.
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The Chlamydomonas flagellar surface exhibits a number of dynamic membrane phenomena associated with whole-cell gliding locomotion and the early events in fertilization. Crosslinking of a specific population of flagellar surface-exposed glycoproteins with the lectin concanavalin A or an anti-carbohydrate mouse monoclonal antibody, designated FMG-1, results in a characteristic pattern of glycoprotein redistribution within the plane of the flagellar membrane. Recent evidence suggests that flagellar membrane glycoprotein movements are associated with both whole-cell gliding motility and the early events in mating. It is of interest to determine the transmembrane signaling pathway whereby crosslinking of the external domains of flagellar glycoproteins activates the intraflagellar machinery responsible for translocation of flagellar membrane glycoproteins. The redistribution of flagellar membrane glycoproteins requires micromolar levels of free calcium in the medium; lowering the free calcium concentration to 10(−7) M results in complete but reversible inhibition of redistribution. Redistribution is maximal in the presence of 20 microM free calcium in the medium. Redistribution is inhibited in the presence of 20 microM free calcium by the calmodulin antagonists trifluoperazine, W-7 and calmidazolium, the calcium channel blockers diltiazem, methoxyverapamil (D-600) and barium chloride, and the local anesthetics, lidocaine and procaine. The actions of all of these agents can be interpreted in terms of a requirement for calcium in the signaling mechanism associated with flagellar glycoprotein redistribution. In particular, the requirement for micromolar calcium in the external medium and the effects of specific calcium channel blockers suggest that flagellar membrane glycoprotein crosslinking may induce an increase in calcium influx, which may be the initial trigger for activating the flagellar machinery responsible for active movement of flagellar membrane glycoproteins.
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Hulswit, Ruben J. G., Guido C. Paesen, Thomas A. Bowden, and Xiaohong Shi. "Recent Advances in Bunyavirus Glycoprotein Research: Precursor Processing, Receptor Binding and Structure." Viruses 13, no. 2 (February 23, 2021): 353. http://dx.doi.org/10.3390/v13020353.
Full textAbstract:
The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.
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Srivastav, Archana, Balvir Singh, Abhishek Chandra, Farrukh Jamal, Mohammad Y. Khan, and Sunil R. Chowdhury. "Partial characterization, sperm association and significance of N- and O-linked glycoproteins in epididymal fluid of rhesus monkeys (Macaca mulatta)." Reproduction 127, no. 3 (March 2004): 343–57. http://dx.doi.org/10.1530/rep.1.00119.
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The present study investigated regional modifications of glycosylation status, sperm association and functional significance of N- and O-linked glycoproteins in epididymal luminal fluid of the rhesus monkey (Macaca mulatta). The predominant glycoproteins of the epididymal luminal fluid that increase in the extent of glycosylation or unmasking of exposed epitopes in a region-specific, maturation-dependent manner, included those of 150, 116, 68, 64, 58 (N- and O-linked) and 170 kDa (O-linked). The higher expression of 40 (N-linked), 38 (N- and O-linked) and 60, 56 and 33 kDa (O-linked) glycoproteins in the proximal caput epididymal fluid was followed by alteration or reorganization of 60, 38 and 33 kDa (O-linked) glycoproteins in the distal segments of the epididymis. The association of epididymal fluid glycoproteins with maturing spermatozoa was identified by generating polyclonal antiserum against monkey caudal sperm membrane in female albino rabbits. The antiserum crossreacted strongly with 58 and 33 kDa epididymal fluid glycoproteins of monkeys and also reacted with 116, 68, 58, 56 and 33 kDa glycoproteins from Triton X-100 extracts of human spermatozoa, indicating the presence of antigenically related components in both species. The functional significance of epididymal fluid glycoproteins in sperm functions was investigated by raising antiserum against a heavily glycosylated 58 kDa glycoprotein (MEF1) of caudal epididymal fluid, which crossreacted with the Triton X-100 extracts of epididymal spermatozoa of monkey and ejaculated human spermatozoa on immunoblots. In an in vitro micro-sperm agglutination assay, anti-MEF1 serum agglutinated both rat caudal epididymal spermatozoa and human spermatozoa. MEF1 seemed to be involved in fertilization as demonstrated by inhibition of fertility (100%) in female albino rabbits and rats immunized with this protein. A sperm-agglutinating 58 kDa glycoprotein of rhesus monkey epididymis with functional significance in fertility was identified, thus indicating that it is a potential candidate for contraceptive vaccine development.
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Horvat, B., H. A. Multhaupt, and I. Damjanov. "Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity." Journal of Histochemistry & Cytochemistry 41, no. 9 (September 1993): 1351–57. http://dx.doi.org/10.1177/41.9.8354876.
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We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had a unique binding pattern different from the patterns revealed by other lectins. However, several estrous cycle phase-specific glycoproteins reacted with more than one lectin. The most prominent of these glycoproteins (M(r) 92-95 KD) was weakly expressed in late diestrus and fully expressed only in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle.
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Zimmer, Gert, Klaus-Peter Zimmer, Ina Trotz, and Georg Herrler. "Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells." Journal of Virology 76, no. 8 (April 15, 2002): 4103–7. http://dx.doi.org/10.1128/jvi.76.8.4103-4107.2002.
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ABSTRACT In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.
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Vollenweider, Florence, Felix Kappeler, Christian Itin, and Hans-Peter Hauri. "Mistargeting of the Lectin ERGIC-53 to the Endoplasmic Reticulum of HeLa Cells Impairs the Secretion of a Lysosomal Enzyme." Journal of Cell Biology 142, no. 2 (July 27, 1998): 377–89. http://dx.doi.org/10.1083/jcb.142.2.377.
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ERGIC-53, a homo-oligomeric recycling protein associated with the ER–Golgi intermediate compartment (ERGIC), has properties of a mannose-selective lectin in vitro, suggesting that it may function as a transport receptor for glycoproteins in the early secretory pathway. To investigate if ERGIC-53 is involved in glycoprotein secretion, a mutant form of this protein was generated that is incapable of leaving the ER. If expressed in HeLa cells in a tetracycline-inducible manner, this mutant accumulated in the ER and retained the endogenous ERGIC-53 in this compartment, thus preventing its recycling. Mistargeting of ERGIC-53 to the ER did not alter the gross morphology of the early secretory pathway, including the distribution of β′-COP. However, it impaired the secretion of one major glycoprotein, identified as the precursor of the lysosomal enzyme cathepsin C, while overexpression of wild-type ERGIC-53 had no effect on glycoprotein secretion. Transport of two other lysosomal enzymes and three post-Golgi membrane glycoproteins was unaffected by inactivating the recycling of ERGIC-53. The results suggest that the recycling of ERGIC-53 is required for efficient intracellular transport of a small subset of glycoproteins, but it does not appear to be essential for the majority of glycoproteins.
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Rosenberg, Arielle R., Lélia Delamarre, Claudine Pique, Isabelle Le Blanc, Graziella Griffith, and Marie-Christine Dokhélar. "Early Assembly Step of a Retroviral Envelope Glycoprotein: Analysis Using a Dominant Negative Assay." Journal of Cell Biology 145, no. 1 (April 5, 1999): 57–68. http://dx.doi.org/10.1083/jcb.145.1.57.
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As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.
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Culp, D. J., L. R. Latchney, M. W. Frampton, M. R. Jahnke, P. E. Morrow, and M. J. Utell. "Composition of human airway mucins and effects after inhalation of acid aerosol." American Journal of Physiology-Lung Cellular and Molecular Physiology 269, no. 3 (September 1, 1995): L358—L370. http://dx.doi.org/10.1152/ajplung.1995.269.3.l358.
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Characterization of normal airway mucus is required to elucidate mechanisms protecting the airways and to understand changes associated with disease and environmental insult. Toward this goal, we collected bronchial washes (10 ml saline) from healthy human subjects to 1) evaluate the yield of high-density material (delta > or = 1.35 g/ml), and 2) characterize glycoconjugates associated with collected secretions. Samples were lipid extracted followed by CsCl density gradient centrifugation. The yield of high-density material from individual subjects was variable but sufficient to demonstrate that mucin glycoproteins are a major constituent of mucus from healthy airways and that proteoglycans are absent. Next, we investigated whether inhalation of H2SO4 aerosol (1,000 microgram/m3), an environmental insult associated with alterations in mucociliary clearance, changes the composition of high-density glycoproteins in airway secretions. In a paired, double-blinded study, high-density fractions of bronchial secretions from 12 subjects were collected 18 h after exposures of 2 h to aerosolized NaCl and H2SO4. In all cases the high-density material displayed characteristics of mucin glycoproteins. In addition, a unique 150-kDa glycoprotein was detected in most but not all samples and may represent a small mucin glycoprotein differentially expressed in humans. No differences were noted between the two exposure conditions in the profiles of the glycoproteins or proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Statistically, large changes with acid exposure in the composition of carbohydrates and amino acids were absent. Thus no substantial systematic changes in airway mucin glycoproteins or closely associated proteins and glycoproteins were correlated with H2SO4 exposure. Alternatively, statistical analysis of the differences between exposures in glycoprotein constituents among subjects denoted greater variability in carbohydrates compared with amino acids with repeated sampling, suggesting normal daily variations in the mucin composition of individual airway mucus.
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Wu, R., C. G. Plopper, and P. W. Cheng. "Mucin-like glycoprotein secreted by cultured hamster tracheal epithelial cells. Biochemical and immunological characterization." Biochemical Journal 277, no. 3 (August 1, 1991): 713–18. http://dx.doi.org/10.1042/bj2770713.
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We isolated mucin-like glycoproteins from the conditioned medium of primary hamster tracheal epithelial (HTE) cell culture and characterized them biochemically and immunologically. These glycoproteins were purified on Sepharose CL-4B after Streptomyces hyaluronidase treatment and then by CsCl-density-gradient centrifugation in the presence of 4 M-guanidinium chloride. The purified glycoproteins were resistant to digestion by chondroitin AC lyase, heparinase, heparitinase and endo-N-acetylglucosaminidases A, D and H, but susceptible to endo-beta-galactosidase and keratanase. SDS/PAGE demonstrated no contamination by low-molecular-mass proteins. The purified glycoproteins showed a peak buoyant density of 1.56 g/ml in CsCl-density-gradient centrifugation, and contained 10% peptide and 90% carbohydrate by weight. Carbohydrates in these glycoproteins contained N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, sialic acid and a trace amount of mannose, but no uronic acid. Serine and threonine together accounted for 27% of the total amino acid residues. In addition, the mucin-like glycoproteins exhibited blood-group A and B activities, and very strong inhibitory activity for influenza A virus haemagglutination. With the use of the purified glycoprotein as an antigen, six monoclonal antibodies that stained mucus granules in hamster tracheal epithelium were obtained. We characterized the antibody produced by one of the clones, HM D46. We conclude that HTE cells cultured in the serum-free medium secrete a glycoprotein with physicochemical properties similar to those known in various airways mucins.
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Garry, Courtney E., and Robert F. Garry. "Proteomics Computational Analyses Suggest that the Antennavirus Glycoprotein Complex Includes a Class I Viral Fusion Protein (α-Penetrene) with an Internal Zinc-Binding Domain and a Stable Signal Peptide." Viruses 11, no. 8 (August 14, 2019): 750. http://dx.doi.org/10.3390/v11080750.
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A metatranscriptomic study of RNA viruses in cold-blooded vertebrates identified two related viruses from frogfish (Antennarius striatus) that represent a new genus Antennavirus in the family Arenaviridae (Order: Bunyavirales). Computational analyses were used to identify features common to class I viral fusion proteins (VFPs) in antennavirus glycoproteins, including an N-terminal fusion peptide, two extended alpha-helices, an intrahelical loop, and a carboxyl terminal transmembrane domain. Like mammarenavirus and hartmanivirus glycoproteins, the antennavirus glycoproteins have an intracellular zinc-binding domain and a long virion-associated stable signal peptide (SSP). The glycoproteins of reptarenaviruses are also class I VFPs, but do not contain zinc-binding domains nor do they encode SSPs. Divergent evolution from a common progenitor potentially explains similarities of antennavirus, mammarenavirus, and hartmanivirus glycoproteins, with an ancient recombination event resulting in a divergent reptarenavirus glycoprotein.
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Hortin, G., E. D. Green, J. U. Baenziger, and A. W. Strauss. "Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on α2HS-glycoprotein." Biochemical Journal 235, no. 2 (April 15, 1986): 407–14. http://dx.doi.org/10.1042/bj2350407.
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Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides.
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Kassa, Aemro, Andrés Finzi, Marie Pancera, Joel R. Courter, Amos B. Smith, and Joseph Sodroski. "Identification of a Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Variant Resistant to Cold Inactivation." Journal of Virology 83, no. 9 (February 11, 2009): 4476–88. http://dx.doi.org/10.1128/jvi.02110-08.
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ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.
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Snyder, Aleksandra, Katarina Polcicova, and David C. Johnson. "Herpes Simplex Virus gE/gI and US9 Proteins Promote Transport of both Capsids and Virion Glycoproteins in Neuronal Axons." Journal of Virology 82, no. 21 (August 27, 2008): 10613–24. http://dx.doi.org/10.1128/jvi.01241-08.
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ABSTRACT Following reactivation from latency, alphaherpesviruses replicate in sensory neurons and assemble capsids that are transported in the anterograde direction toward axon termini for spread to epithelial tissues. Two models currently describe this transport. The Separate model suggests that capsids are transported in axons independently from viral envelope glycoproteins. The Married model holds that fully assembled enveloped virions are transported in axons. The herpes simplex virus (HSV) membrane glycoprotein heterodimer gE/gI and the US9 protein are important for virus anterograde spread in the nervous systems of animal models. It was not clear whether gE/gI and US9 contribute to the axonal transport of HSV capsids, the transport of membrane proteins, or both. Here, we report that the efficient axonal transport of HSV requires both gE/gI and US9. The transport of both capsids and glycoproteins was dramatically reduced, especially in more distal regions of axons, with gE−, gI−, and US9-null mutants. An HSV mutant lacking just the gE cytoplasmic (CT) domain displayed an intermediate reduction in capsid and glycoprotein transport. We concluded that HSV gE/gI and US9 promote the separate transport of both capsids and glycoproteins. gE/gI was transported in association with other HSV glycoproteins, gB and gD, but not with capsids. In contrast, US9 colocalized with capsids and not with membrane glycoproteins. Our observations suggest that gE/gI and US9 function in the neuron cell body to promote the loading of capsids and glycoprotein-containing vesicles onto microtubule motors that ferry HSV structural components toward axon tips.
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Lamers, Susanna L., Ruchi M. Newman, Oliver Laeyendecker, Aaron A. R. Tobian, Robert C. Colgrove, Stuart C. Ray, David M. Koelle, Jeffrey Cohen, David M. Knipe, and Thomas C. Quinn. "Global Diversity within and between Human Herpesvirus 1 and 2 Glycoproteins." Journal of Virology 89, no. 16 (May 27, 2015): 8206–18. http://dx.doi.org/10.1128/jvi.01302-15.
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ABSTRACTHuman herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large-genome DNA viruses that establish a persistent infection in sensory neurons and commonly manifest with recurring oral or genital erosions that transmit virus. HSV encodes 12 predicted glycoproteins that serve various functions, including cellular attachment, entry, and egress. Glycoprotein G is currently the target of an antibody test to differentiate HSV-1 from HSV-2; however, this test has shown reduced capacity to differentiate HSV strains in East Africa. Until the recent availability of 26 full-length HSV-1 and 36 full-length HSV-2 sequences, minimal comparative information was available for these viruses. In this study, we use a variety of sequence analysis methods to compare all available sequence data for HSV-1 and HSV-2 glycoproteins, using viruses isolated in Europe, Asia, North America, the Republic of South Africa, and East Africa. We found numerous differences in diversity, nonsynonymous/synonymous substitution rates, and recombination rates between HSV-1 glycoproteins and their HSV-2 counterparts. Phylogenetic analysis revealed that while most global HSV-2 glycoprotein G sequences did not form clusters within or between continents, one clade (supported at 60.5%) contained 37% of the African sequences analyzed. Accordingly, sequences from this African subset contained unique amino acid signatures, not only in glycoprotein G, but also in glycoproteins I and E, which may account for the failure of sensitive antibody tests to distinguish HSV-1 from HSV-2 in some African individuals. Consensus sequences generated in the study can be used to improve diagnostic assays that differentiate HSV-1 from HSV-2 in global populations.IMPORTANCEHuman herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large DNA viruses associated with recurring oral or genital erosions that transmit virus. Up to 12 HSV-1 and HSV-2 glycoproteins are involved in HSV cell entry or are required for viral spread in animals, albeit some are dispensable for replicationin vitro. The recent availability of comparable numbers of full-length HSV-1 and HSV-2 sequences enabled comparative analysis of gene diversity of glycoproteins within and between HSV types. Overall, we found less glycoprotein sequence diversity within HSV-2 than within the HSV-1 strains studied, while at the same time, several HSV-2 glycoproteins were evolving under less selective pressure. Because HSV glycoproteins are the focus of antibody tests to detect and differentiate between infections with the two strains and are constituents of vaccines in clinical-stage development, these findings will aid in refining the targets for diagnostic tests and vaccines.
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Stein, W. D. "Kinetics of the multidrug transporter (P-glycoprotein) and its reversal." Physiological Reviews 77, no. 2 (April 1, 1997): 545–90. http://dx.doi.org/10.1152/physrev.1997.77.2.545.
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Most cancer deaths result from the cancer's either being intrinsically resistant to chemotherapeutic drugs or becoming resistant after being initially sensitive. Often, in cells grown in cell culture, drug resistance correlates with the presence of one or more of the so-called P-glycoproteins or multidrug resistance proteins, products of the mdr family of genes. This review is largely concerned with the transport kinetics of the P-glycoproteins. We first present a brief overview of the P-glycoproteins, their properties, and their clinical significance. Later sections of the review expand on this material with special emphasis on the substrates of P-glycoprotein and how they cross the cell membrane, on the transport kinetics of the P-glycoprotein, on reversers of its action, and on its activity as an ATPase. In a final section, we consider the mechanism of action of P-glycoprotein as an actively transporting membrane pump. The characteristic of P-glycoprotein considered the most difficult to explain is its very broad specificity (or lack of specificity), but there are precedents for this property in well-known proteins such as serum albumin, which binds a range of molecular types, including substrates and reversers of P-glycoprotein, seemingly as broad as does P-glycoprotein. Pointing out this analogy does not provide a molecular explanation for the substrate-binding properties of P-glycoprotein but does make those properties more assimilable.
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Lin, Borong, Xue Qing, Jinling Liao, and Kan Zhuo. "Role of Protein Glycosylation in Host-Pathogen Interaction." Cells 9, no. 4 (April 20, 2020): 1022. http://dx.doi.org/10.3390/cells9041022.
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Host-pathogen interactions are fundamental to our understanding of infectious diseases. Protein glycosylation is one kind of common post-translational modification, forming glycoproteins and modulating numerous important biological processes. It also occurs in host-pathogen interaction, affecting host resistance or pathogen virulence often because glycans regulate protein conformation, activity, and stability, etc. This review summarizes various roles of different glycoproteins during the interaction, which include: host glycoproteins prevent pathogens as barriers; pathogen glycoproteins promote pathogens to attack host proteins as weapons; pathogens glycosylate proteins of the host to enhance virulence; and hosts sense pathogen glycoproteins to induce resistance. In addition, this review also intends to summarize the roles of lectin (a class of protein entangled with glycoprotein) in host-pathogen interactions, including bacterial adhesins, viral lectins or host lectins. Although these studies show the importance of protein glycosylation in host-pathogen interaction, much remains to be discovered about the interaction mechanism.
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Bowden, Thomas A., Max Crispin, Stephen C. Graham, David J. Harvey, Jonathan M. Grimes, E. Yvonne Jones, and David I. Stuart. "Unusual Molecular Architecture of the Machupo Virus Attachment Glycoprotein." Journal of Virology 83, no. 16 (June 3, 2009): 8259–65. http://dx.doi.org/10.1128/jvi.00761-09.
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ABSTRACT New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel α/β fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
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Jiang, Lingdong, Rui Lu, and Lei Ye. "Towards Detection of Glycoproteins Using Molecularly Imprinted Nanoparticles and Boronic Acid-Modified Fluorescent Probe." Polymers 11, no. 1 (January 18, 2019): 173. http://dx.doi.org/10.3390/polym11010173.
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Glycoproteins represent a group of important biomarkers for cancer and other life-threatening diseases. Selective detection of specific glycoproteins is an important step for early diagnosis. Traditional glycoprotein assays are mostly based on lectins, antibodies, and enzymes, biochemical reagents that are costly and require special cold chain storage and distribution. To address the shortcomings of the existing glycoprotein assays, we propose a new approach using protein-imprinted nanoparticles to replace the traditional lectins and antibodies. Protein-imprinted binding sites were created on the surface of silica nanoparticles by copolymerization of dopamine and aminophenylboronic acid. The imprinted nanoparticles were systematically characterized by dynamic light scattering, scanning and transmission electron microscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy, and elemental analysis. A boronic acid-modified fluorescent probe was used to detect the target glycoprotein captured by the imprinted nanoparticles. Using horseradish peroxidase as a model glycoprotein, we demonstrated that the proposed method can be applied to detect target protein containing multiple glycosylation sites. Because of their outstanding stability and low cost, imprinted nanoparticles and synthetic probes are attractive replacements of traditional biochemical reagents to develop simpler, faster, and more cost-effective analytical methods for glycoproteins.
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KONRAD, Zvia, and Jerry EICHLER. "Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation." Biochemical Journal 366, no. 3 (September 15, 2002): 959–64. http://dx.doi.org/10.1042/bj20020757.
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Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg2+-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [3H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane.
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Batonick, Melissa, Antonius G. P. Oomens, and Gail W. Wertz. "Human Respiratory Syncytial Virus Glycoproteins Are Not Required for Apical Targeting and Release from Polarized Epithelial Cells." Journal of Virology 82, no. 17 (June 18, 2008): 8664–72. http://dx.doi.org/10.1128/jvi.00827-08.
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ABSTRACT Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.
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Santos, Joy Ramielle L., Weijie Sun, Tarana A. Mangukia, Eduardo Reyes-Serratos, and Marcelo Marcet-Palacios. "Challenging the Existing Model of the Hexameric HIV-1 Gag Lattice and MA Shell Superstructure: Implications for Viral Entry." Viruses 13, no. 8 (July 31, 2021): 1515. http://dx.doi.org/10.3390/v13081515.
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Despite type 1 human immunodeficiency virus (HIV-1) being discovered in the early 1980s, significant knowledge gaps remain in our understanding of the superstructure of the HIV-1 matrix (MA) shell. Current viral assembly models assume that the MA shell originates via recruitment of group-specific antigen (Gag) polyproteins into a hexagonal lattice but fails to resolve and explain lattice overlapping that occurs when the membrane is folded into a spherical/ellipsoidal shape. It further fails to address how the shell recruits, interacts with and encompasses the viral spike envelope (Env) glycoproteins. These Env glycoproteins are crucial as they facilitate viral entry by interacting with receptors and coreceptors located on T-cells. In our previous publication, we proposed a six-lune hosohedral structure, snowflake-like model for the MA shell of HIV-1. In this article, we improve upon the six-lune hosohedral structure by incorporating into our algorithm the recruitment of complete Env glycoproteins. We generated the Env glycoprotein assembly using a combination of predetermined Env glycoprotein domains from X-ray crystallography, nuclear magnetic resonance (NMR), cryoelectron tomography, and three-dimensional prediction tools. Our novel MA shell model comprises 1028 MA trimers and 14 Env glycoproteins. Our model demonstrates the movement of Env glycoproteins in the interlunar spaces, with effective clustering at the fusion hub, where multiple Env complexes bind to T-cell receptors during the process of viral entry. Elucidating the HIV-1 MA shell structure and its interaction with the Env glycoproteins is a key step toward understanding the mechanism of HIV-1 entry.
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Wahl-Jensen, Victoria, Sabine K. Kurz, Paul R. Hazelton, Hans-Joachim Schnittler, Ute Ströher, Dennis R. Burton, and Heinz Feldmann. "Role of Ebola Virus Secreted Glycoproteins and Virus-Like Particles in Activation of Human Macrophages." Journal of Virology 79, no. 4 (February 15, 2005): 2413–19. http://dx.doi.org/10.1128/jvi.79.4.2413-2419.2005.
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ABSTRACT Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Δ-peptide), GP1, and GP1,2Δ. While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP1,2 in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.
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Roberts, G. P. R., and J. Brunt. "Differentiation-related changes in glycoprotein synthesis by human keratinocytes." Biochemical Journal 237, no. 2 (July 15, 1986): 519–25. http://dx.doi.org/10.1042/bj2370519.
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Human keratinocytes were cultured in media in which the Ca2+ concentration controlled the stage of differentiation. In media containing less than 0.1 mM-Ca2+ keratinocytes grew as a monolayer, but in the presence of 2mM-Ca2+ the cells differentiated and formed stratified colonies. Glycoproteins of both stratified and unstratified cells were radiolabelled by metabolic incorporation of radioactive precursors and by cell-surface labelling using galactose oxidase/NaB3H4. The radiolabelled keratinocytes were extracted with 0.5% Triton X-100, and the glycoproteins in both the Triton X-100-soluble and Triton X-100-insoluble fractions were analysed by polyacrylamide-gel electrophoresis in the presence of SDS. Two Triton X-100-soluble glycoproteins with high Mr values (greater than 200,000) were major glycoproteins in stratified keratinocytes, but were present in only trace amounts in unstratified keratinocytes. Characterization of these glycoproteins by examination of the effect of tunicamycin on their synthesis and the effect of neuraminidase on their migration characteristics showed that they were cell-surface sialoglycoproteins containing O-glycosidically linked oligosaccharides. Analysis of the adherent cytoskeletons left after Triton X-100 extraction of stratified and unstratified keratinocytes revealed that a glycoprotein of Mr 184,000 was decreased in stratified keratinocytes. Incubation of unstratified keratinocytes in high-Ca2+ medium resulted in a rapid modification of the glycoprotein of Mr 184,000, and it is suggested that this event may be related to desmosome formation and stratification.