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288 Cell-Penetrating Peptides: Processes and Applications
to elucidate the receptor dependency of the internalization. In TP4 mastoparan part
was replaced by crabrolin, a peptide from hornet venom. TP5 and TP6 were obtained
by introduction of Pro into TP sequence as a replacement of amino acids at positions
6 or 6 and 7, respectively.
The order of cellular uptake estimated by immunofluorescence was TP, TP2 >
TP3, TP5, TP6 > TP4. This speaks for the great importance of mastoparan part and
its amphipathic properties for the ability of these types of peptides to translocate
cellular membranes, while modifications in galanin part have much less dramatic
effect. In immunofluorescence experiments, longer incubation times were used;
therefore, results might reflect the yield and not the rate of cellular uptake.
For this reason, it was important to compare data on immunofluorescence with
time courses of cellular uptake of [ 125 I]-labeled peptides. Iodination on Tyr was
successfully carried out with TP, TP2, and TP3 — peptides with highest demonstrated
cellular localization (cf. above). All listed [ 125 I]–TPs were internalized into
Bowes melanoma cells, even at 10 nM concentration, and time courses of cell
penetration of the peptides were comparable. The efficiency and time course of
uptake were in accordance with earlier results shown for TP, 8 but the biphasic kinetic
behavior, characterized by slow decrease of cellular concentration of TP after reaching
maximal value (see above), was not observed with TP2 and TP3. In the case of
TP3, this might be because of lack of a cleavage point between Gly 12 and Lys 13 ,
while in the case of TP2 the reason is not clear (see degradation steps in Figure 13.1).
Thus, analysis according to Scheme 13.2 (see above) could not be applied for
comparison of internalization kinetics of TP, TP2, and TP3. Therefore, a simplified
approach was used in which experimental data for the time courses of cellular uptake
for all three peptides in question were approximated to the first-order scheme and
Equation 13.1 was used for fitting time course curves to the experimental points.
Results confirmed that the immunofluorescence method mainly detects yield of
uptake and does not necessarily reflect rate of penetration. The maximum uptake of
labeled TP and TP2 were nearly identical, 9.8 and 10.7% of total peptide, respectively,
while the same value for TP3 was significantly lower: 6% of total peptide
added. On the other hand, the rate of cell penetration was nearly the same for all
three peptides and was characterized by half-life (t 0.5) of uptake of 14 to 17 min.
The second class of TP analogues (TP7–TP15) was obtained by truncation of
amino acids in the original TP sequence with the aim to minimize the undesired
biological activities of TP (the affinity for galanin receptors and the interaction with
G-proteins), but to retain the cell-penetration efficiency of the peptide. 9 In the first
subgroup of the TP analogues, amino acids were deleted from the N terminus of
TP, yielding three, six, or nine amino acid residues shorter peptides (TP7, TP10,
and TP8), respectively. In the second subgroup, the peptides were truncated in the
middle of the sequence (TP9 and TP11). The third subgroup represents peptides
truncated at the N terminus and in the middle of the sequence (TP12–TP14). Finally,
in TP15, the amino acids were deleted at the N and C termini as well as in the
middle of the TP sequence.
Most of the short TP analogues have retained the ability to penetrate Bowes
cells. As estimated from immunofluorescence experiments, the deletion of three or
six residues from the N terminus of TP (TP7 and TP10, respectively) or two residues