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A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway - Adaptation to HIV Envelope Processing.

Stolp ZD, Stotland A, Diaz S, Hilton BJ, Burford W, Wolkowicz R - PLoS ONE (2013)

Bottom Line: Current methodologies fail to link transport and cleavage at the single cell level.Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively.Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, California, United States of America.

ABSTRACT
The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry analysis of cell lines expressing the assay.A. Naïve HEK293T cells or HEK293T clones expressing Env-wt or Env-mut were stained with FLAG-FITC, HA-APC or both antibodies and analyzed by flow cytometry. B. Naïve SupT1 T-cells or SupT1 clones expressing Env-wt or Env-mut were doubled-stained with FLAG-FITC and HA-APC antibodies and analyzed by flow cytometry.
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pone-0068835-g002: Flow cytometry analysis of cell lines expressing the assay.A. Naïve HEK293T cells or HEK293T clones expressing Env-wt or Env-mut were stained with FLAG-FITC, HA-APC or both antibodies and analyzed by flow cytometry. B. Naïve SupT1 T-cells or SupT1 clones expressing Env-wt or Env-mut were doubled-stained with FLAG-FITC and HA-APC antibodies and analyzed by flow cytometry.

Mentions: The Env boundary-bearing scaffold construct was introduced into a Murine Leukemia Virus-based retroviral vector used in consequent experiments for stable expression in mammalian cells [31]. A known non-cleavable recognition sequence containing an Arg-to-Ser mutation was used as control [33]. It is important to mention that this substitution serves as control for the assay and not to prove Furin specificity, which is beyond the goal of the assay. Wild-type and mutant boundaries are referred to as Env-wt and Env-mut, respectively (Figure 1B). Retrovirally transduced adherent HEK293T cells were analyzed by flow cytometry and sorted based on HA cell surface expression, ensuring that proper transport to the cell surface occurred within the purified population. This population was clonally sorted into 96-well plates and amplified to create assay-expressing cell lines. HEK293T clones were then analyzed by flow cytometry following staining with anti-HA, anti-FLAG or both antibodies. A secondary allophycocyanin (APC)-coupled antibody was used to detect HA (HA-APC) and a fluorescein isothiocyanate (FITC)-coupled antibody was used to detect FLAG (FLAG-FITC). The analysis showed a dramatic distinction between Env-wt and Env-mut expressing cells. While HA-APC staining was robust with both cell lines (78-93%), it was FLAG-FITC positive only with Env-mut (81% versus 1%). This result was further corroborated by double staining, with 2% of cells co-expressing FLAG and HA in Env-wt cells in contrast to 75% in Env-mut cells (Figure 2A). Complete abrogation of the Env-mut boundary cleavage nicely corroborates that cleaved and non-cleaved events can be discriminated based on FLAG surface expression.


A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway - Adaptation to HIV Envelope Processing.

Stolp ZD, Stotland A, Diaz S, Hilton BJ, Burford W, Wolkowicz R - PLoS ONE (2013)

Flow cytometry analysis of cell lines expressing the assay.A. Naïve HEK293T cells or HEK293T clones expressing Env-wt or Env-mut were stained with FLAG-FITC, HA-APC or both antibodies and analyzed by flow cytometry. B. Naïve SupT1 T-cells or SupT1 clones expressing Env-wt or Env-mut were doubled-stained with FLAG-FITC and HA-APC antibodies and analyzed by flow cytometry.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686725&req=5

pone-0068835-g002: Flow cytometry analysis of cell lines expressing the assay.A. Naïve HEK293T cells or HEK293T clones expressing Env-wt or Env-mut were stained with FLAG-FITC, HA-APC or both antibodies and analyzed by flow cytometry. B. Naïve SupT1 T-cells or SupT1 clones expressing Env-wt or Env-mut were doubled-stained with FLAG-FITC and HA-APC antibodies and analyzed by flow cytometry.
Mentions: The Env boundary-bearing scaffold construct was introduced into a Murine Leukemia Virus-based retroviral vector used in consequent experiments for stable expression in mammalian cells [31]. A known non-cleavable recognition sequence containing an Arg-to-Ser mutation was used as control [33]. It is important to mention that this substitution serves as control for the assay and not to prove Furin specificity, which is beyond the goal of the assay. Wild-type and mutant boundaries are referred to as Env-wt and Env-mut, respectively (Figure 1B). Retrovirally transduced adherent HEK293T cells were analyzed by flow cytometry and sorted based on HA cell surface expression, ensuring that proper transport to the cell surface occurred within the purified population. This population was clonally sorted into 96-well plates and amplified to create assay-expressing cell lines. HEK293T clones were then analyzed by flow cytometry following staining with anti-HA, anti-FLAG or both antibodies. A secondary allophycocyanin (APC)-coupled antibody was used to detect HA (HA-APC) and a fluorescein isothiocyanate (FITC)-coupled antibody was used to detect FLAG (FLAG-FITC). The analysis showed a dramatic distinction between Env-wt and Env-mut expressing cells. While HA-APC staining was robust with both cell lines (78-93%), it was FLAG-FITC positive only with Env-mut (81% versus 1%). This result was further corroborated by double staining, with 2% of cells co-expressing FLAG and HA in Env-wt cells in contrast to 75% in Env-mut cells (Figure 2A). Complete abrogation of the Env-mut boundary cleavage nicely corroborates that cleaved and non-cleaved events can be discriminated based on FLAG surface expression.

Bottom Line: Current methodologies fail to link transport and cleavage at the single cell level.Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively.Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, California, United States of America.

ABSTRACT
The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.

No MeSH data available.


Related in: MedlinePlus