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Two complementary approaches for intracellular delivery of exogenous enzymes.

Rust A, Hassan HH, Sedelnikova S, Niranjan D, Hautbergue G, Abbas SA, Partridge L, Rice D, Binz T, Davletov B - Sci Rep (2015)

Bottom Line: Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research.Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions.We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes.

View Article: PubMed Central - PubMed

Affiliation: The University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT
Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes.

No MeSH data available.


The macrophage J774.2 cell line is sensitive to the botulinum enzyme type D.(A) Immunoblot showing cleavage of VAMP3 in J774.2, but not in N2a cells, by botulinum enzyme type D at the indicated concentrations after 72 hrs incubation. (B) The J774.2 cells exhibit splayed morphology after 72 hrs incubation in the presence of botulinum enzyme type D (100 nM).
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f2: The macrophage J774.2 cell line is sensitive to the botulinum enzyme type D.(A) Immunoblot showing cleavage of VAMP3 in J774.2, but not in N2a cells, by botulinum enzyme type D at the indicated concentrations after 72 hrs incubation. (B) The J774.2 cells exhibit splayed morphology after 72 hrs incubation in the presence of botulinum enzyme type D (100 nM).

Mentions: To assess whether J774.2 macrophages are penetrable to structurally/functionally distinct proteins, we analysed biological effects of the enzyme derived from botulinum neurotoxin type D. This protease cleaves the vesicle-associated membrane proteins (VAMPs) involved in membrane trafficking including VAMP3, also known as cellubrevin, which is ubiquitously present in all cells10. When the botulinum protease was incubated with J774.2 cells and neuroblastoma N2a cells for 72 hrs, we detected efficient cleavage of VAMP3 by Western immunoblotting only in the J774.2 cells (Fig. 2A). Interestingly, cleavage of VAMP3 resulted in a dramatic change in cell morphology with the macrophage-derived cells adopting a stellate phenotype (Fig. 2B). A similar phenomenon has been observed in previous studies where VAMP3 expression was knocked down using a siRNA approach implicating VAMP3 in cell spreading, adhesion and migration of macrophages11. Thus the J774.2 cell line may present a convenient model to study effects of externally applied enzymes on intracellular substrates.


Two complementary approaches for intracellular delivery of exogenous enzymes.

Rust A, Hassan HH, Sedelnikova S, Niranjan D, Hautbergue G, Abbas SA, Partridge L, Rice D, Binz T, Davletov B - Sci Rep (2015)

The macrophage J774.2 cell line is sensitive to the botulinum enzyme type D.(A) Immunoblot showing cleavage of VAMP3 in J774.2, but not in N2a cells, by botulinum enzyme type D at the indicated concentrations after 72 hrs incubation. (B) The J774.2 cells exhibit splayed morphology after 72 hrs incubation in the presence of botulinum enzyme type D (100 nM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4513551&req=5

f2: The macrophage J774.2 cell line is sensitive to the botulinum enzyme type D.(A) Immunoblot showing cleavage of VAMP3 in J774.2, but not in N2a cells, by botulinum enzyme type D at the indicated concentrations after 72 hrs incubation. (B) The J774.2 cells exhibit splayed morphology after 72 hrs incubation in the presence of botulinum enzyme type D (100 nM).
Mentions: To assess whether J774.2 macrophages are penetrable to structurally/functionally distinct proteins, we analysed biological effects of the enzyme derived from botulinum neurotoxin type D. This protease cleaves the vesicle-associated membrane proteins (VAMPs) involved in membrane trafficking including VAMP3, also known as cellubrevin, which is ubiquitously present in all cells10. When the botulinum protease was incubated with J774.2 cells and neuroblastoma N2a cells for 72 hrs, we detected efficient cleavage of VAMP3 by Western immunoblotting only in the J774.2 cells (Fig. 2A). Interestingly, cleavage of VAMP3 resulted in a dramatic change in cell morphology with the macrophage-derived cells adopting a stellate phenotype (Fig. 2B). A similar phenomenon has been observed in previous studies where VAMP3 expression was knocked down using a siRNA approach implicating VAMP3 in cell spreading, adhesion and migration of macrophages11. Thus the J774.2 cell line may present a convenient model to study effects of externally applied enzymes on intracellular substrates.

Bottom Line: Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research.Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions.We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes.

View Article: PubMed Central - PubMed

Affiliation: The University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT
Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes.

No MeSH data available.