scholarly journals Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

1988 ◽  
Vol 8 (4) ◽  
pp. 1709-1714 ◽  
Author(s):  
S Vijaya ◽  
N Elango ◽  
F Zavala ◽  
B Moss
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Stop Codon ◽  
Integral Membrane Protein ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Recombinant Vaccinia ◽  
Hybrid Proteins ◽  
Syncytial Virus

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein

1988 ◽  
Vol 8 (4) ◽  
pp. 1709-1714
Author(s):  
S Vijaya ◽  
N Elango ◽  
F Zavala ◽  
B Moss
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Stop Codon ◽  
Integral Membrane Protein ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Recombinant Vaccinia ◽  
Hybrid Proteins ◽  
Syncytial Virus

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.

scholarly journals Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus

2004 ◽  
Vol 85 (11) ◽  
pp. 3229-3238 ◽  
Author(s):  
Carolina Johnstone ◽  
Patricia de León ◽  
Francisco Medina ◽  
José A. Melero ◽  
Blanca García-Barreno ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Vaccinia Virus ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Recombinant Vaccinia Virus ◽  
Recombinant Vaccinia ◽  
F Protein ◽  
Syncytial Virus ◽  
Lymphocyte Responses

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85–93 and F92–106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85–93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249–258, which is presented by Kd as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous Kd ligand. The CD8+ T-lymphocyte responses to epitopes F85–93 and F249–258 present in the F protein of RSV Long were found to be strongly skewed to F85–93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8+ T-lymphocyte responses to F85–93 and F249–258 epitopes was observed in vivo during a primary response.

scholarly journals Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine

2001 ◽  
Vol 82 (9) ◽  
pp. 2107-2116 ◽  
Author(s):  
Teresa R. Johnson ◽  
Julie E. Fischer ◽  
Barney S. Graham
Keyword(s):  
Respiratory Syncytial Virus ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Antigen Expression ◽  
Virus Protein ◽  
Recombinant Vaccinia ◽  
Vaccinia Viruses ◽  
Ifn Γ ◽  
Syncytial Virus ◽  
The Impact

Recombinant vaccinia viruses are well-characterized tools that can be used to define novel approaches to vaccine formulation and delivery. While vector co-expression of immune mediators has enormous potential for optimizing the composition of vaccine-induced immune responses, the impact on antigen expression and vector antigenicity must also be considered. Co-expression of IL-4 increased vaccinia virus vector titres, while IFN-γ co-expression reduced vaccinia virus replication in BALB/c mice and in C57BL/6 mice infected with some recombinant viruses. Protection against respiratory syncytial virus (RSV) challenge was similar in mice immunized with vaccinia virus expressing RSV G glycoprotein and IFN-γ, even though the replication efficiency of the vector was diminished. These data demonstrate the ability of vector-expressed cytokine to influence the virulence of the vector and to direct the development of selected immune responses. This suggests that the co-expression of cytokines and other immunomodulators has the potential to improve the safety of vaccine vectors while improving the immunogenicity of vaccine antigens.

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

2005 ◽  
Vol 58 (2) ◽  
pp. 426-440 ◽  
Author(s):  
Berit Sondén ◽  
Dana Kocíncová ◽  
Caroline Deshayes ◽  
Daniel Euphrasie ◽  
Lamya Rhayat ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Integral Membrane Protein

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

scholarly journals Isolation and partial characterization of antibody- and globin-enriched complexes from membranes of dense human erythrocytes

1991 ◽  
Vol 278 (1) ◽  
pp. 57-62 ◽  
Author(s):  
R Kannan ◽  
J Yuan ◽  
P S Low
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Integral Membrane Protein ◽  
Protein Band ◽  
Reticuloendothelial System ◽  
Band 3 ◽  
Isolation And Characterization ◽  
Protein Clusters ◽  
Associated Proteins

In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or ‘non-self’ by the immune system and aggressively opsonized with IgG.

P-selectin binds to the D′-D3 domains of von Willebrand factor in Weibel-Palade bodies

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3922-3924 ◽  
Author(s):  
Grégoire Michaux ◽  
Timothy J. Pullen ◽  
Sandra L. Haberichter ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Integral Membrane Protein ◽  
Hek293 Cells ◽  
Binding Partner ◽  
Lumenal Domain ◽  
Von Willebrand ◽  
Willebrand Factor

It has recently been shown that the ultralarge platelet–recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade body–like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D′-D3 domains of VWF.

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