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Hydrophobic Membrane Translocating Sequence Peptides 123
expression of α-2,8-polysialic acid on cell surface neuronal cell adhesion molecules
(NCAM) is required. 91 Uptake by T lymphocytes indicates that the mechanism
involved does not depend on membrane caveolae because T lymphocytes do not
express these structures. 37 The addition of inhibitors of the endosomal pathway to
cell culture does not prevent the intracellular localization of MTS peptides; therefore,
uptake does not rely on endocytosis. 33
Furthermore, in a study using electron microscopy co-staining techniques,
MTS-cargo peptides have been observed to translocate across the cell membrane by
free penetration and without specific association with any membrane-bound vesicles,
such as endosomes. 92 These observations, together with results suggesting that
import of MTS peptides is ATP-independent and temperature-dependent, and that
intact architecture of the plasma membrane is required, 33 lead to conclusions that
translocation occurs directly through the lipid bilayer and that membrane fluidity
and lateral mobility of membrane proteins influence this import process.
The secondary conformation adopted by MTS peptides is potentially responsible
for membrane interaction. The residues located in the h-regions of signal sequences
are often those with a propensity to form α-helices; data from circular dichroism
(CD) studies show that various signal peptides can adopt an α-helical conformation
in membrane-mimetic environments. 31,93-98 Similarly, the correlation of the stability
of the helix–turn–helix conformation of h-regions with in vivo function is observed
with the prokaryotic LamB protein. 99 Furthermore, it has been suggested that a
helical hairpin structure makes hydrophobic peptides prone to interaction with membrane
lipid 93-95,100 and that this interaction unloops the peptide, resulting in a transmembrane
conformation. 101
Du and co-workers performed a detailed study aimed at defining the conformational
requirements of cell-permeant hydrophobic MTS peptides and the correlation
between translocation activity and sequence topology. 102 For this, SA peptide with
sequence AAVALLPAVLLALLAPAAANYKKPKL 34 and several variants were synthesized
and analyzed for secondary conformation and translocating activity. The
16 N-terminal residues of SA represent the h-region of kFGF, which is separated
from the NLS of FGF-1 by a trialanine spacer region. Results confirm that the MTS
induces α-helix formation in membrane-mimetic environments and the authors concluded
that a tendency to adopt α-helical conformation in a heterogeneous
amphiphilic environment suggests a possible interaction with hydrophilic headgroups
of plasma membrane lipids.
These findings suggest that signal sequence-based MTS peptides can be modified
to retain or improve their import activity as long as the secondary conformation
remains unchanged. In fact, Rojas et al. have designed a 12-residue MTS derived
from the 16-residue h-region of kFGF mentioned above. They have shown that this
shortened MTS peptide is also capable of efficiently delivering peptides and proteins
into cells. 36 This 12-residue MTS may be the shortest hydrophobic translocating
peptide reported thus far.
Du et al. also addressed the effect of the location of the MTS on peptide import
function. It was found that the peptide still adopted an α-helical structure in the
membrane-mimetic environment and was able to penetrate across the cell membrane
when the MTS was attached at the C terminus of the NLS. 102 Thus, the site of