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58 Cell-Penetrating Peptides: Processes and Applications
fragment (1–12) — the galanin-derived sequence in transportan — more than 100fold.
However, biotinyl-transportan bound to galanin receptors less avidly than its
predecessor, galparan (K D = 6.4). The drop in affinity is probably due to replacement
of galanin’s Pro 13 in galparan to N ε -biotinyl-Lys in transportan. As has been pointed
out, even though transportan can bind to galanin receptors and thereby induce
receptor-mediated endocytosis, this process is not determining the cell entry of this
peptide. 5
Mastoparan, the other component of transportan, is probably responsible for most
of its unexpected properties. A family of 14-amino-acid-long peptides, mastoparans are
one of the best studied amphiphilic peptides. 15 In aqueous solution, mastoparan is
unfolded, but forms an amphiphilic α-helix 16 in the presence of lipids. Furthermore,
mastoparan has been shown to penetrate into cell membranes and into cells.
The representatives of the mastoparan family of peptides show various biological
activities such as degranulation of mast cells, activation of phospholipase A 2 and C,
and release of histamine from mast cells, catecholamines from chromaffin cells, serotonin
from platelets, and insulin from Rin m5F cells (for a review see Soomets et al. 15 ).
Functional responses to the peptide have been proposed to result, at least partly,
from activation of G-proteins 17 and the direct interaction of mastoparan with GTPbinding
proteins has been demonstrated. 18 The activation is exerted through stimulation
of GDP/GTP exchange analogously to G-protein-coupled receptors, indicating
that mastoparans mimic receptors in ligand-bound conformation. 17 Therefore, we
have studied the influence of transportan and all its analogues on GTPase activity.
Contrary to mastoparan that activates GTPases, transportan, as well as its predecessor
galparan, inhibits basal activity of GTPases 19 in Bowes cell membranes. However,
to reveal the inhibitory activity, a rather high concentration of transportan is necessary
(EC 50 21 µM). The inhibitory effect is suggested to be caused by a direct
interaction of transportan with the enzymes or possibly by changes of membrane
properties or structure. 5 It is not clear what relevance this has in cells at the concentration
of transportan used to deliver cargo into the cell.
3.5 STRUCTURE–ACTIVITY RELATIONSHIP OF TRANSPORTAN
To evaluate the significance of either part of the transportan sequence in cellular
penetration and interactions with the signal transduction machinery, we changed the
galanin or mastoparan moieties to different bioactive peptides. 14,20 The C-terminal
half, mastoparan, is known to bind G-proteins, mimicking receptors in active conformation.
In order to decrease this specific side effect, we designed TP 2, in which
the mastoparan part was changed to the inactive analogue, mastoparan 17. 14
In order to elucidate the role of the N-terminal hormone part, the galanin part
was changed to a vasopressin V 1a selective antagonist and coupled to the mastoparan
part through a 6-aminohexanoic linker producing TP 3, even though the predecessor
of transportan 3, M391, was shown to bind to the V 1a vasopressin receptor with high
affinity 21 and also exhibited nonreceptor-mediated biological activity. Finally, since
it was thought that the amphiphilic helix is an important structural motif for membrane
interaction, crabrolin, an amphiphilic peptide of hornet venom, was substituted
for mastoparan in the sequence of TP 4. 22