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Transportans 61
A 21-mer antisense PNA complementary to type 1 galanin receptor mRNA,
delivered into Bowes melanoma cells either by transportan or penetratin, led to
decrease of the level of the protein by more than 90% after 36 h at 3 µM concentration
of the construct. Moreover, de novo synthesis of GalR1 protein was blocked or
reduced below the level of detection upon treatment of Bowes cells with the cellpenetrating
PNA constructs. Antisense PNA cell-penetrating constructs are applicable
in cells growing in culture and also in vivo. Administration of PNA constructs
targeting the analogous region of the rat GalR1 mRNA into the lumbar spinal cord
in rat leads to a profound down-regulation of galanin-binding sites. The loss of
galanin-binding sites is accompanied by a reduction of the inhibition of galanin on
the facilitation of the flexor reflex, suggesting that galanin receptors of type 1 mediate
this physiological effect of galanin. 29
3.6.3 DELIVERY OF PROTEINS
In addition to small- or medium-sized hydrophilic biomolecules like peptides and
oligonucleotides, proteins can also be transduced into a cell’s interior by cellpenetrating
peptides (for review, see Swartze et al. 31 ).
We have studied whether transportan is able to convey proteins, ranging in
molecular weight from 30 to 150 kDa, into cells by coupling transportan to the
proteins with different coupling strategies. The connection of transportan to peptides
or PNA oligomers via a disulfide bond enabled the transduction of the molecules
into cells with subsequent liberation in the cytosol. 29 Hence, the disulfide bond was
used to couple transportan with the green fluorescent protein (GFP) (Figure 3.2A). 32
Incubation of COS-7 cells for 10 min with 0.5 µM construct suffices for GFP to
insert into the membrane and translocate into cytosol. In 1 h most of the GFP
fluorescence located to cytoplasmic membranes and, to a smaller extent, to the
plasma membrane as judged by the presence of reticular structures detected by
fluorescence microscopy and confocal laser scanning microscopy.
Treatment of GFP-S-S-TP adduct with dithiothreitol (DTT) before applying to
the cells completely abolished GFP internalization, thus suggesting that the coupling
of GFP to transportan is indeed necessary for translocation into the cells. However,
when DTT was added to the medium after incubation with constructs, no decrease
in fluorescence intensity was detected in either the cytosol or plasma membrane.
This shows that transportan does not simply function as a membrane anchor for the
protein.
The fluorescence of GFP is detectable at all stages of its cell translocation: in
the solution surrounding the cells, in the plasma membrane, and inside the cells.
The staining was seen both diffusely and in granular or vesicular structures. Since
native GFP has a very tight barrel-shaped structure which must be preserved to be
able to fluoresce, we suggest that GFP is not unfolded upon the passage across the
membrane or in the cell interior. We may conclude that, even if unfolding of proteins
facilitates its transduction into the cells, as was suggested previously, 33 it is not
obligatory.
The 30-kDa green fluorescent protein is a rather small protein, but its efficient
transportan-assisted translocation into cells inspired us to continue with bigger