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128 Cell-Penetrating Peptides: Processes and Applications
TABLE 6.3
Cationic Peptides Tested by Futaki et al. 127
for Translocation Activity
Name Sequence
HIV-1 Rev
FHV coat
HTLV-II Rex
BMV Gag
P22 N
CCMV Gag
λ N
φ21 N
Yeast PRP6
34 TRQARRNRRRRWRERQR 50
35 RRRRNRTRRNRRRVR 49
4 TRRQRTRRARRNR 16
7 KMTRAQRRAAARRNRWTAR 25
14 NAKTRRHERRRKLAIER 30
7 KLTRAQRRAAARKNKRNTR 25
1 MDAQTRRRERRAEKQAQWKAAN 22
12 TAKTRYKARRAELIAERR 29
129 TRRNKRNRIQEQLNRK 144
Source: Futaki, S. et al., J. Biol. Chem., 276, 5836, 2001.
# arginine
residues
on the opposing side of a theoretical helical structure. Based on this observation,
the naturally occurring penetratin peptide sequence has been subsequently modified
to produce a more efficient translocating peptide (Table 6.2) consisting of only
arginine and tryptophan residues. 9,125,126
The translocation activity of several other positively charged peptides has been
investigated by Futaki et al. 127 Peptides representing arginine-rich RNA-binding
regions (Table 6.3) were shown to accumulate in the cytoplasm and nucleus of cells
within 5 min of addition of 1 µM of peptide, adding weight to the premise that those
peptides rich in positively charged residues are efficient translocating peptides.
Peptides containing more than seven arginines exhibited internalization activity
similar to the HIV Tat peptide, whereas those with less arginine residue showed less
extensive internalization. Again, there is a lack of sequence homology between all
the peptides tested; however, all possess several arginine residues, thus implicating
arginine as critical to translocation activity. The ability of arginine oligomers to act
as translocating peptides and also to transport attached cargo such as cyclosporin A
across biological membranes has been reported. 128,129
Based on these observations, polypeptides and peptoids consisting of repeating
units mimicking various characteristics of arginine have been tested as transportants.
130,131 The results generated led to conclusions that the guanidino headgroup is
superior to other cationic subunits and that the length of the alkyl chain between
the headgroup and backbone is also significant.
Possibly the longest translocation peptide, transportan is a 27-residue chimera
(Table 6.2) composed of amino acid sequences derived from two different proteins.
This peptide contains 13 residues from the highly conserved N-terminal region of
galanin and the 14-amino acid-long wasp venom peptide toxin, mastoparan. 10 The most
recent report of use of transportan describes the fusion of transportan to various proteins
ranging in size from the 30-kDa green fluorescent protein to 150-kDa antibodies and
the subsequent delivery of the large protein cargoes into different cell types. 104
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