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130 Cell-Penetrating Peptides: Processes and Applications
61 RNTVNRLLPM LRRKKNEKKN EKIERNNKLK QPPPPPNPND PPPPNPNDPP PPNPNDPPPP
121 NPNDPPPPNA NDPPPPNAND PAPPNANDPA PPNANDPAPP NANDPAPPNA NDPAPPNAND
181 PAPPNANDPP PPNPNDPAPP QGNNNPQPQP RPQPQPQPQP QPQPQPQPQP RPQPQPQPGG
241 NNNNKNNNND DSYIPSAEKI LEFVKQIRDS ITEEWSQCNV TCGSGIRVRK RKGSNKKAED
301 LTLEDIDTEI CKMDKCSSIF NIVSNSLGFV ILLVLVFFN
FIGURE 6.3 Amino acid sequence of P. berghei CS protein. 165 The proline-rich repeat region
is shown in bold.
6.8 PROLINE-RICH PEPTIDES AS TRANSLOCATING PEPTIDES
The translocation activity of a potentially new class of translocating peptides, proline-rich
peptides, has been investigated. Even though each translocating peptide
identified thus far has proven capable of traversing the cell membrane and delivering
biologically active cargo into the cytoplasm and nucleus, several disadvantages are
associated with use of these peptides. For example, even though the basic residues
of cationic peptides seem to aid in interaction with the negatively charged lipid
bilayer and increase translocation, such highly charged peptides are potentially
cytotoxic. In a report published by Singh et al., 159 a loligomer comprising eight
copies of a polylysine sequence had a cytotoxic effect on various eukaryotic cells
at concentrations above 5 µM. Linear polymers of polylysine have also been shown
to possess cytotoxic properties. 160
In a preliminary study we have examined the translocation activity of a peptide
derived from the proline-rich repeat region of the Plasmodium berghei circumsporozoite
surface (CS) protein. The CS protein is the major surface protein of the
Plasmodium sporozoite, 161 the causative agent of malaria. Both the CS protein and
the entire sporozoite possess the ability to translocate from the extracellular environment
to the cytoplasm of hepatocytes. 162-164 Sporozoites have even been shown
to pass back through the membrane and exit cells without fatally disrupting the
membrane. 164 This protein contains a large proline-rich repeat region, the sequence
of the P. berghei CS protein 165 is shown in Figure 6.3. The sequence PPPPNPND-
PPPPNPND was selected for examination of translocation activity; uptake of this
peptide by murine monocytes was found to be greater than that of an RP-rich control
peptide derived from Bac7. 158 The successful uptake of proline-rich peptides has led
to ongoing studies in which the mechanism of uptake is elucidated. These prolinerich
peptides have the desirable properties of being aqueous soluble and not highly
charged and, therefore, more conducive to routine synthesis and delivery.
6.9 FUTURE PERSPECTIVES OF CELL-PERMEANT
PEPTIDE TECHNOLOGY
Peptides should possess several critical properties to be considered translocating vectors:
• Sequence short to simplify synthesis
• Internalization not appreciably affected by coupling to cargo
• Cargo delivered to the desired compartment
• Biologically inactive vector