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Transportans 59
In the penetration studies performed with transportan and its analogues, transportan
and TP 2 (galanin (1–12)-Lys-mastoparan 17) showed comparable uptake.
TP 3 was found to be localized mainly to the plasma membrane rather than inside
the cells, probably due to structural differences between galanin and vasopressin
antagonist moieties. The TP 4 did not insert into the plasma membrane or enter the
cells in a detectable amount; hence the mastoparan part with its amphipathic properties
seems to contribute vastly to the ability of these peptides to traverse the cellular
membranes. 14
Penetratin and Tat-peptide are shorter than transportan, but still possess ability
to penetrate into cells. In order to find the shortest transportan analogue with no or
low biological activity that has similar cell-penetrating properties as the original
peptide, we synthesized different deletion analogues of transportan (Table 3.1). 23
Deletion of three or six residues from the N terminus of transportan (TP 7, TP
10) or the first two residues of the mastoparan part (TP 9) did not change the cellular
penetration ability of the peptides. All three peptides could be detected in the
cytoplasm and nucleus of Bowes cells. This lends further support for a nonendocytotic
uptake mechanism for the transportans, since Trp 2 and Asn 5 , which have been
shown to be necessary for high affinity binding of this N-terminal fragment to the
galanin receptor, 8 are not present in TP 10 molecule.
However, truncation of nine amino acid residues from the N terminus (TP 8)
diminished the cellular penetration and deletion of amino acids 7 to 12 (TP 11)
drastically decreased the uptake, possibly hinting at significance of the tyrosine in
position 9.
It has been shown that mastoparan, the C-terminal part of transportan, penetrates
into cell membranes and can be internalized to some small degree. 15 Therefore, it
could be expected that the mastoparan part of transportan would be important in the
uptake of this peptide. TP 9, where two amino acids in the N terminus of the
mastoparan part are deleted, showed similar uptake as transportan in Bowes cells.
Building on this, we deleted three (TP 12) or six (TP 14) amino acids from the N
terminus of TP 9, respectively. The uptake of both peptides was significant, but
somewhat lower than for transportan 9. Transportans 13 and 15, where a Lys residue
has been deleted from the mastoparan part, did not show any detectable cellular
uptake, pointing to the importance of positive charges for cell penetration. 23
3.6 TRANSPORTAN AS DELIVERY VECTOR
3.6.1 DELIVERY OF PEPTIDES
In order to measure the delivery kinetics of transportan, constructs based on energy
transfer quenching of the 2-amino-benzoic acid fluorophore were used. 24 The fluorophore
is compatible with t-Boc chemistry, is resistant to photo-bleaching, and has
a well-characterized amino acid-like quencher. The fluorophore model peptide cargo
and the quencher CPP were connected by a disulfide bond that should be rapidly
reduced in the cellular interior. After cleavage of the construct, the fluorescence
intensity increased at least tenfold. This allowed us to follow the uptake of the
peptide–cargo construct in real time without time-consuming cell isolation steps.