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Transportans 65
antibiotin antibodies is strong enough to enable insertion of formed complexes into
plasma membrane of cells and following translocation into the cytosol. Indeed,
antibiotin monoclonal antibodies are detectable in COS-7 cells after 1 to 2 h of
incubation with complexes of biotinyl–transportan with antibiotin monoclonal antibodies
at 37°C (Figure 3.3C).
The respective antibody localizes in the perinuclear and cortical areas of COS-7
cells as well as in the plasma membrane, confirming that transportan has succeeded
to convey noncovalently bound antibodies into a cell’s interior. Seemingly, the
interaction between the transporter peptide and a cargo molecule (protein in this
assay) must not necessarily be extra strong for delivery to occur; complexes of
medium stability can be utilized, too.
The localization of the transduced proteins inside the cells is of primary importance
to understanding whether they retain intactness and activity and if they are
localized or trapped into organelles or accessibly in cytoplasm. Peptide-assisted cell
transduction of the larger proteins has usually led to a granular-reticular pattern of
intracellular distribution. The observed pattern suggests that delivered protein resides
mainly in vesicular structures and that endocytosis could be responsible for most of
the peptide-mediated protein translocation.
Consequently, we incubated COS-7 cells on ice in order to abolish endocytosis
in tissue culture medium containing complexes of biotinyl–transportan with streptavidin–Texas
Red conjugate. Incubation of cells with the respective complexes for 2 h
on ice yielded a marked staining of the plasma membrane (Figure 3.3D) suggesting
that, even at very low temperatures, transportan can target protein cargoes to the
plasma membrane of cells and probably is able to penetrate into the membrane.
Surprisingly, we observed some weak staining of cytosol in the vicinity of plasma
membrane and also in the perinuclear area. Consequently, lowering the temperature
cannot completely abolish internalization of transportan–protein complexes, implying
that endocytosis is not the only mechanism involved in peptide-mediated cellular
translocation of proteins.
Electron microscopy was used in order to get a more detailed view on the cellular
structures where proteins reside after transportan-assisted delivery. Analogously to
the fluorescence microscopy experiment, we applied biotinyl–transportan along with
streptavidin labeled with colloidal gold into the culture medium. Despite the bulkiness
of the label on the protein (the diameter of the gold particle is 10 nm), streptavidin–gold
conjugate is translocated into cells by biotinyl–transportan. Inside the cells, delivered
streptavidin resides mainly in a granulo-vesicular manner in structures, some of which
are surrounded by membrane, but also diffusely in cytoplasm.
Localization of streptavidin–gold conjugates outside membrane-surrounded vesicles
and diffusely in the cytoplasm after only 15 min of incubation suggests that
the transportan-aided uptake of proteins cannot be explained solely by endocytosis.
In principle, a possibility exists that the cell-delivered protein has escaped from
clathrin coated vesicles or early endosomes and been able to localize into cytoplasm.
However, since streptavidin–gold conjugates are diffusely localized in the cytoplasm
already at the initial stages of internalization, this explanation is less plausible.