Despite this, the modified peptide can still sequester PtdIns4,5P2 into domains, probably because of the presence of a cluster of cationic residues in the peptide.. The intensity of cross
Trang 1peptide and its Tyr mutant
Raquel F Epand1, Brian G Sayer2 and Richard M Epand1,2
1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada
2 Department of Chemistry, McMaster University, Hamilton, Canada
NAP-22 is a 22-kDa protein found in neurons that is
important for neuronal sprouting and plasticity [1]
In addition to the intact 22-kDa protein, significant
amounts of N-terminal myristoylated fragments of this
protein are also found in many tissues [2] A protein
with a high sequence homology to NAP-22 and
prob-ably with very similar properties, cortical
cytoskeleton-associated protein (CAP)-23, was first identified by
Widmer and Caroni [3] Myristoylated proteins are
commonly found in cholesterol-rich domains in
mem-branes [4,5] Full length NAP-22 partitions into the
low density, detergent-insoluble fraction of neuronal membranes [6], suggesting its presence in neuronal rafts Support for this comes from fluorescence micros-copy studies using both intact biological membranes [7,8] as well as model membranes [9] The protein binds to liposomes of phosphatidylcholine only when the bilayer contains high mol fractions of cholesterol [10,11]
Several proteins with cationic clusters, including CAP-23 as well as the MARCKS protein and GAP-43, accumulate in rafts, colocalizing with PtdIns(4,5)P2 [8]
Keywords
cholesterol; domains; differential scanning
calorimetry; MAS ⁄ NMR; phosphatidylinositol
(4,5) diphosphate
Correspondence
R M Epand, Department of Biochemistry
and Biomedical Sciences, McMaster
University, Hamilton, ON Canada L8N 3Z5
Fax: +1 905 521 1397
Tel: 1 905 525 9140, extn 22073
E-mail: epand@mcmaster.ca
(Received 10 December 2004, revised 2
February 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04612.x
The N-terminally myristoylated, 19-amino acid peptide, corresponding to the amino terminus of the neuronal protein NAP-22 (NAP-22 peptide) is a naturally occurring peptide that had been shown by fluorescence to cause the sequestering of a Bodipy-labeled PtdIns(4,5)P2 in a cholesterol-depend-ent manner The prescholesterol-depend-ent work, using differcholesterol-depend-ential scanning calorimetry (DSC), extends the observation that formation of a PtdIns(4,5)P2-rich domain is cholesterol dependent and shows that it also leads to the forma-tion of a cholesterol-depleted domain The PtdIns(4,5)P2 used in the present work is extracted from natural sources and does not contain any label and has the native acyl chain composition Peptide-induced formation of a cho-lesterol-depleted domain is abolished when the sole aromatic amino acid, Tyr11 is replaced with a Leu Despite this, the modified peptide can still sequester PtdIns(4,5)P2 into domains, probably because of the presence of a cluster of cationic residues in the peptide Cholesterol and PtdIns(4,5)P2 also modulate the insertion of the peptide into the bilayer as revealed by 1H NOESY MAS⁄ NMR The intensity of cross peaks between the aromatic protons of the Tyr residue and the protons of the lipid indicate that in the presence of cholesterol there is a change in the nature of the interaction of the peptide with the membrane These results have important implications for the mechanism by which NAP-22 affects actin reorganization in neurons
Abbreviations
DH cal , calorimetric enthalpy; Bodipy-TMR-PI(4,5)P2, BODIPY TMR-X C6-phosphatidylinositol 4,5-diphosphate; CAP-23, cortical cytoskeleton-associated protein (a protein expressed in chicken having a high degree of homology to NAP-22); DP, direct polarization; DSC, differential scanning calorimetry; LUV, large unilamellar vesicle; MAS, magic angle spinning; NAP-22 peptide, the myristoylated amino terminal 19 amino acids of NAP-22 (myristoyl-GGKLSKKKKGYNVNDEKAK-amide); NAP-22, neuronal axonal membrane protein, also referred to as brain acid soluble protein 1 (BASP1 protein), a 22 kDa myristoylated protein; PC, phosphatidylcholine; PO, 1-palmitoyl-2-oleoyl; PtdIns(4,5)P2,
L -a-phosphatidylinositol-4,5-bisphosphate from porcine brain; SO, 1-stearoyl-2-oleoyl; T m , transition temperature.
Trang 2The importance of electrostatic interactions in the
sequestering of PtdIns(4,5)P2 by proteins with a cationic
domain has been demonstrated [12] We have also
dem-onstrated the loss of ability of the NAP-22 peptide to
sequester Bodipy-labeled PtdIns(4,5)P2 in the presence
of high salt concentration [13] In that work we also
demonstrate specificity of the NAP-22-peptide for
labeled PtdIns(4,5)P2 compared with
Bodipy-labeled PtdIns(3,5)P2 [13] In addition, using total
inter-nal reflectance fluorescence microscopy, we have shown
that the sequestering of Bodipy-labeled PtdIns(4,5)P2
into domains can be a cholesterol-dependent
pheno-menon [13] This was demonstrated using a myristoylated
N-terminal peptide of NAP-22, Myristoyl-GGKLSK
KKKGYNVNDEKAK-amide It is known that in vivo,
in addition to the intact NAP-22 protein, a significant
amount of myristoylated N-terminal fragments of this
protein are also present [2], indicating that the
myristo-ylated N-terminal peptide of NAP-22, such as that used
in this work, is also found physiologically In the present
work we demonstrate that not only does cholesterol
affect the ability of the NAP-22-peptide to induce the
formation of PtdIns(4,5)P2 domains, but it also causes
the rearrangement of cholesterol leading to the
forma-tion of cholesterol-depleted domains We also test the
role of the aromatic amino acid residue of the peptide in
these phenomena In addition we show that cholesterol
also affects the arrangement of the peptide in the bilayer
The present study uses PtdIns(4,5)P2 from porcine
brain, a natural form that has long acyl chains
enriched in arachidonic acid, and it also does not
con-tain any fluorescent probes Although PtdIns(4,5)P2
from natural sources is highly enriched in arachidonoyl
groups that should not interact well with liquid
ordered domains of rafts, this lipid nevertheless is
found in raft domains of biological membranes [14]
Results
Differential scanning calorimetry (DSC)
We determined the phase transitions of SOPC and
mixtures of this lipid with one or more of the
fol-lowing components: cholesterol, PtdIns(4,5)P2 and
NAP-22-peptide, using differential scanning
calori-metry (DSC) For each sample, six consecutive DSC
scans were run, three heating scans and three cooling
scans at a scan rate of 2CÆmin)1 Sequential heating
and cooling scans were reproducible In the absence of
cholesterol a prominent transition is observed in the
region 0–10C, corresponding to the chain melting
transition of SOPC This transition is better resolved
in cooling than in heating scans, since in some cases
the heating scans, initiated at 0C, had not reached a steady-state baseline in the temperature range of the transition The transition of POPC would have been even more difficult to measure, although POPC was used for the NMR experiments (see below) because the NMR results could be more directly compared with our earlier observations on other systems and to avoid any artefacts that may result from storing peptide-lipid mixtures that could attain the gel phase Nevertheless,
we would expect that these two lipids, SOPC and POPC, that differ only by two CH2 groups on one of the acyl chains, would interact almost identically with peptides One of the three cooling scans is presented for samples of different compositions (Fig 1A) In the presence of 40 mol% cholesterol, the chain melting transition of SOPC is broadened and the enthalpy lowered (Fig 1B) We also studied the role of the sole aromatic amino acid, Tyr, of the NAP-22-peptide by replacing it with Leu The temperatures and enthalpies for the phospholipid chain melting transition are shown (Table 1) The temperature of the transition is shifted slightly among the different samples and is low-ered by the presence of peptide This is probably a result of the peptide partitioning more favorably into the liquid-crystalline phase than into the gel phase In addition, the enthalpy of this transition in the presence
of cholesterol, PtdIns(4,5)P2 and the NAP-22-peptide
is increased almost threefold This indicates that cho-lesterol has been depleted from a domain of the mem-brane that can now undergo a more cooperative and endothermic transition, more like that of the pure phospholipid Estimates of the transition enthalpy of mixtures containing cholesterol have a higher error because of the low temperature and broadness of the transition In addition to the phospholipid transition, some samples also exhibit a transition corresponding
to the polymorphic transition of anhydrous cholesterol crystals, which appears in the cooling scans at 21C The enthalpy and temperature of this transition was estimated from both cooling and heating scans where this transition occurs at 38C The temperature differ-ence between the heating and cooling curves is charac-teristic of this transition and is caused by the slow rate
of interconversion of two forms of anhydrous choles-terol crystals [15] The polymorphic transition of anhy-drous cholesterol crystals is most clearly seen by DSC
in heating scans We present examples of heating scans
of either SOPC⁄ cholesterol (60 : 40) or SOPC ⁄ choles-terol (50 : 50) containing either 10 or 20 mol% of the NAP-22 peptide or the Y11L NAP-22 (Fig 2) The transition enthalpies of these peaks, obtained from the areas of the peaks, provide an estimate of the amount
of crystalline cholesterol (Table 2) Pure anhydrous
Trang 3Fig 1 DSC cooling scans (A) SOPC alone (curve 1) and SOPC
with 0.2 mol% PtdIns(4,5)P2 added (curve 2); 0.2 mol%
PtdIns(4,5)P2 and 10 mol% NAP-22-peptide added (curve 3);
10 mol% NAP-22-peptide added (curve 4) (B) SOPC ⁄ cholesterol
60 : 40 with 0.2 mol% PtdIns(4,5)P2 and 10 mol% NAP-22-peptide
added (curve 1); SOPC ⁄ cholesterol 60 : 40 with 10 mol%
NAP-22-peptide added (curve 2); SOPC ⁄ cholesterol 60 : 40 with 0.2 mol%
PtdIns(4,5)P2 added (curve 3); SOPC ⁄ cholesterol 60 : 40 (curve 4);
SOPC ⁄ cholesterol 60 : 40 with 10 mol% mutant
Y11L-NAP-22-pep-tide added (curve 5) and SOPC ⁄ cholesterol 60 : 40 with 0.2 mol%
PtdIns(4,5)P2 and 10 mol% mutant Y11L-NAP-22-peptide added
(curve 6); Scan rate 2Æmin)1.
Table 1 DSC Transition of SOPC Transitions observed in cooling scans at 2Æmin)1of SOPC with additional components listed in the first three columns When cholesterol is present it is at a 6 : 4 molar ratio of SOPC:cholesterol PtdIns(4,5)P2 is at 0.2% of total lipids, while NAP-22-peptide is 10 mol% of total lipids when present Additional components
DH (kcalÆmol)1) Cholesterol PtdIns(4,5)P2 Peptide T m (C)
Transition
observed
Fig 2 DSC heating scans (A) NAP-22 peptide (B) Y11L-NAP-22-peptide Curve 1, SOPC ⁄ cholesterol 60 : 40 with 10 mol% peptide; Curve 2, SOPC:cholesterol 60 : 40 with 20 mol% peptide; Curve 3, SOPC ⁄ cholesterol 50 : 50 with 10 mol% peptide; Curve 4, SOPC ⁄ cholesterol 50 : 50 with 20 mol% peptide Scan rate 2Æmin)1.
Trang 4cholesterol crystals have an enthalpy of 910 calÆmol)1
[16] In some cases the height of the transition peak is
not proportional to the area because the peaks differ
in their breadth (cooperativity) At SOPC⁄ cholesterol
(60 : 40) it is clear that the NAP-22 peptide promotes
the formation of a larger amount of cholesterol
crys-tals than does the Y11L mutant peptide However, at
SOPC⁄ cholesterol (50 : 50) the difference between the
two peptides in this regard largely disappears In the
absence of peptide (pure SOPC⁄ cholesterol 60 : 40 or
50 : 50), no peak is observed corresponding to the
formation of cholesterol crystallites (not shown), but
SOPC⁄ cholesterol (50 : 50) is close to the solubility
limit of cholesterol [17]
Fluorescence quenching
Addition of the Y11L-NAP-22-peptide to large
uni-lamellar vesicles (LUVs) containing 0.1 mol%
Bodipy-TMR-PI(4,5)P2 results in quenching of the Bodipy
fluorescence (Fig 3) Self-quenching of the fluorescence
of Bodipy-TMR-PI(4,5)P2 by the MARCKS peptide has
been shown to be caused by sequestering of the labeled
lipid into domains [12] We show that the quenching
of the Bodipy fluorescence by the
Y11L-NAP-22-pep-tide is not significantly affected by cholesterol (Fig 3),
unlike the case of the unmodified NAP-22-peptide
[13] that is shown in this figure for comparison In
addition, in the presence of cholesterol the native
sequence with Tyr is more potent than the
Y11L-NAP-22-peptide in causing quenching of the
Bodipy-TMR-PI(4,5)P2
NMR
The 1H NMR spectra of various lipid mixtures in the
presence of 10 mol% of the NAP-22-peptide show
predominantly the resonances of the protons of
POPC (Fig 4) Because they are well resolved from other peaks, very small peaks arising from the aroma-tic protons of Tyr can also be seen in the region of
7 p.p.m The chemical shifts of these, as well as the major resonances from the phospholipid are summar-ized in Table 3 Each of the aromatic peaks was split into a doublet with a vicinal coupling constant of 7.5 Hz
Table 2 DSC transition of anhydrous cholesterol crystallites
Tran-sitions observed in heating scans at 2Æmin )1 of SOPC with 40 or
50 mol% cholesterol, as well as with added peptide.
%
Cholesterol Peptide
DH (calÆmol cholesterol)1)
Fig 3 Quenching of the fluorescence emission by the NAP-22-pep-tide (dashed lines) or by the Y11L-NAP-22-pepNAP-22-pep-tide (solid lines) of Bodipy-TMR-PI(4,5)P2 LUVs composed of POPC with added NAP-peptide (h) or Y11L-NAP-22-NAP-peptide (d) POPC with 40 mol% cho-lesterol with added NAP-peptide (n) or Y11L-NAP-22-peptide (.) Maximum emission intensity at 571 nm is plotted against the pep-tide to lipid molar ratio (P ⁄ L) LUVs were present in the cuvette at
a concentration of 50 l M and the Bodipy-labelled lipids were pre-sent as 0.1 mol percentage of the total lipid.
Fig 4 1-D 1H MAS ⁄ NMR spectra of several lipid mixture (as indi-cated on the right of each spectrum) and also containing 10 mol% NAP-22-peptide PC, POPC See Table 3 for assignments.
Trang 5Static 31P NMR powder patterns demonstrated that
all of the samples used for magic angle spinning
(MAS) studies were in bilayer arrangement (not
shown) Two-dimensional 1H MAS NOESY spectra
were recorded at 25C for four lipid samples, each
with 10 mol% NAP-22-peptide The lipid component
was either POPC; POPC with 0.2 mol% PtdIns(4,5)P2;
POPC⁄ cholesterol (6 : 4); POPC ⁄ cholesterol ⁄ PtdIns(4,
5)P2 (60 : 40 : 0.2) No resonances assignable to
cho-lesterol could be detected either with or without the
peptide, in agreement with earlier observations [18]
The peptide is in relatively low concentration and many
of its resonances would not be well resolved from
those of the lipid, except for the Tyr aromatic protons
We have focused on the relative strength of the NOE
interactions between the Tyr aromatic protons and
other atoms Stronger NOE interactions between two
atoms are a measure of their closer approach These
are observed as peaks in the 2D NOESY spectra
Sli-ces of the NOESY at the resonance position of the
aromatic protons are shown for several lipid mixtures
containing 10 mol% NAP-22-peptide using a delay
time of 50 or 300 ms (Fig 5) The longer delay times can result in larger NOEs by allowing more complete energy transfer through dipolar interactions However, longer delay times can also allow NOE effects to be observed between two groups that are not physically close to each other as a result of spin diffusion It is likely, however, that at least with a 50-ms delay time, spin diffusion does not contribute greatly to the observed dipolar interactions [19] Qualitatively one can conclude that the aromatic residue of the peptide inserts into the bilayer with all of the lipid mixtures, as indicated by the fact that most of the protons of the phospholipid show cross-peaks with the aromatic pro-tons In addition, the presence of cholesterol allows a closer proximity of the Tyr side chain of the peptide with the terminal methyl group of the acyl chain of the lipid as shown by the observation that the intensity
of the cross-peak with the terminal CH3 group (at
1 p.p.m.) relative to that of the CH2 resonances at 1.4 p.p.m is larger in the presence than in the absence
of cholesterol (Fig 5)
In order to specifically assess how PtdIns(4,5)P2 affects the location of the Tyr residue of the NAP-22-peptide in the membrane, we calculated difference spectra by taking a pair of spectra that were identical except for the presence of PtdIns(4,5)P2 Prior to sub-traction the two spectra were adjusted for small dif-ferences in intensity and resonance position so as to visually give the maximal overlap of the two spectra Difference spectra were calculated for pairs of spectra with PtdIns(4,5)P2 minus the spectra for the same lipid mixture without PtdIns(4,5)P2 using a delay time of 50 ms (Fig 6) or 300 ms (Fig 7) Slices from the 2D NOESY spectrum at the two resonance posi-tions for the aromatic residues for pairs of samples with or without cholesterol are shown Peaks of higher intensity, such as the aromatic peaks at 6.9 and 7.2 p.p.m., the HDO peak at 4.8 p.p.m and the quaternary ammonium peak at 3.3 p.p.m show some residual intensity in the difference spectra, that we do not consider significant because the intensity of the
Table 3 Assignment of 1 H NMR resonances.
a Groups correspond to POPC, except for HDO that are the residual
protons of the water and the phenolic CH of the Tyr aromatic
pro-tons from the NAP-22-peptide.
Fig 5 1D slices from the MAS 1 H NOESY spectrum at the chemical shifts of the aro-matic protons using a mixing time of 50 and
300 ms The slice at 7.2 p.p.m corresponds
to the meta CH of Tyr and that at 6.9 p.p.m.
to the phenolic ortho CH.
Trang 6difference spectra peaks represent a small fraction of
the original peak and may arise from imperfect
align-ment of the two spectra In most cases, these
reson-ance positions show closely spaced peaks of positive
and negative sign, indicating a small difference in
chemical shift between the two spectra However,
with cholesterol, the difference spectra using a 50-ms
delay time clearly shows several positive peaks in the
region 1–2 p.p.m (Fig 6, left) This indicates that in
the presence of cholesterol, PtdIns(4,5)P2 allows a
closer proximity of the Tyr side chain of the peptide
with the methylene groups of the acyl chains of the
lipid This phenomenon is not observed in the
absence of cholesterol (Fig 6, right) However, for
the samples without cholesterol, for the slice at
6.9 p.p.m., the difference spectra shows a decrease of
intensity at the resonance position of the CH2 groups,
compared to the samples with cholesterol, and an
increase of the peak intensity at the resonance
posi-tion of the terminal methyl group at 1 p.p.m This is
particularly clear from the spectra using a 300-ms
delay time (Fig 7, lower right spectrum) This indi-cates the ortho protons of the Tyr side chain gain closer approach to the terminal methyl groups of the acyl chains, on a millisecond time scale, in the pres-ence of PtdIns(4,5)P2, but not cholesterol It should
be pointed out, however, that there could be contri-butions to the weaker signals in the difference spectra from cross-peaks between the aromatic protons and aliphatic protons of the peptide that are not well resolved in the 1D spectrum Even if there was such
a contribution, the results would still indicate that PtdIns(4,5)P2 affects the geometrical relationship between the peptide and lipid
Peptide-induced changes in the chemical shift of the carbon atoms as measured by 13C direct polarization (DP)⁄ MAS indicate that the peptide affects the chem-ical shift at many positions in the lipid molecule Such shifts are usually interpreted in terms of ring-current effects caused by the aromatic group of the peptide However, it is unlikely that similar ring-current effects could occur at both the glycerol C3 and terminal methyl group of the acyl chain in the absence of cholesterol or at the glycerol C2 and the cholesterol C18 in the presence of cholesterol (Table 4) We suggest that in addition to ring-current effects there are peptide-induced changes in lipid packing and interaction with water It is known that dehydration will cause an upfield chemical shift of
13C resonances [20]
Fig 6 Calculated differences of spectra shown in Fig 4 using
50 ms delay time Difference of spectra with PtdIns(4,5)P2 minus
the spectra of the same mixture without PtdIns(4,5)P2 Pairs of
spectra are either from samples with cholesterol (+ cholesterol) or
without cholesterol (– cholesterol) Resonance position of the slice
is indicated on the graph.
Fig 7 Same as Fig 6 but for data with 300 ms delay time.
Trang 7In addition to electrostatic interactions with PtdIns(4,
5)P2, the NAP-22-peptide has two features that can
contribute to its interaction with membranes These
features include membrane interactions of the
N-ter-minal myristoyl group and the phenolic side chain of
the Tyr residue, both of which are hydrophobic
moiet-ies known to partition into membranes [21,22]
With regard to myristoylation of NAP-22, this
post-translational modification has been found to be
required for the interaction of this protein with
mem-branes [23] In addition, the protein has no
hydropho-bic segment and its free energy of partitioning into
membranes can be accounted for by the insertion of its
myristoyl group [11] Myristoylated proteins are often
found to sequester to cholesterol-rich domains in
bio-logical membranes We suggest that this group
contri-butes to the cholesterol modulation of the membrane
interaction of the NAP-22-peptide
We have directly tested the role of the Tyr residue in
the membrane interactions of the NAP-22-peptide by
comparing it with a myristoylated peptide in which the
sole Tyr residue was substituted with Leu The
NAP-22-peptide is more effective in sequestering cholesterol
than is the Y11L mutant From the DSC results, this
is indicated by fact that the NAP-22-peptide is able
to promote the formation of a greater
cholesterol-depleted domain as shown by the higher enthalpy of the SOPC transition in the presence of this peptide compared with the Y11L-NAP-22-peptide, both in the presence and absence of PtdIns(4,5)P2 (Table 1) In addition, in mixtures of SOPC⁄ cholesterol (60 : 40) the NAP-22-peptide induces the formation of more anhy-drous cholesterol crystals than the Y11L mutant (Table 2) We suggest that these crystals form because cholesterol surpasses its solubility limit in the mem-brane in cholesterol-rich domains whose formation is promoted by the peptides It should also be pointed out that any cholesterol that is directly bound to a peptide would be less likely to form crystals However, the amount of cholesterol is much larger than the amount of peptide, so that most of the cholesterol in these domains will not be binding directly to the pep-tide The Y11L-NAP-22-peptide is slightly less effective than the NAP-22-peptide in sequestering Bodipy-TMR-PI(4,5)P2 in the presence of cholesterol (Fig 3), but more dramatic is that the cholesterol dependence
of Bodipy-TMR-PI(4,5)P2 sequestering is almost com-pletely eliminated Tyr is an essential element in the CRAC motif, suggested to be responsible for choles-terol recognition [24] Although the NAP-peptide does not have other elements required for a CRAC motif, the sole presence of an aromatic residue may be a con-tributing factor for cholesterol interaction We have previously shown that the aromatic side chains of the
Table 4 Peptide-induced 13 C chemical shift differences of lipid resonances Data show the chemical shift differences in p.p.m for the indi-cated lipid mixture between the pure lipid and lipid with 10 mol% NAP-22 peptide Cholesterol present in equimolar ratio with POPC and PtdIns(4,5)P2 as 0.2 mol% ND, Not determined because of poor resolution of the peak.
Assignment
Chemical shift
POPC + PtdIns(4,5)P2
POPC ⁄ cholesterol (1 : 1)
POPC ⁄ cholesterol (1 : 1) + PtdIns(4,5)P2
Trang 8peptide N-acetyl-LWYIK-amide can interact with the
A ring of cholesterol [25] The Y11L-NAP-22-peptide
is also less potent in inducing the formation of
choles-terol clusters than is the NAP-22-peptide This is
indi-cated by the observation that no cholesterol crystallites
are observed with SOPC and 40 mol% cholesterol in
the presence of Y11L-NAP-22-peptide, while they do
form in the presence of the NAP-22 peptide In
addi-tion, there is no evidence for the formation of a
choles-terol-depleted phase with the Y11L-NAP-22-peptide,
which would result in a more cooperative chain
melt-ing transition of SOPC with higher enthalpy (Table 1
and Fig 1)
Peptides with cationic clusters, even simple
oligo-mers of Lysine, will sequester the polyanionic
PtdIns(4,5)P2 [12,26–28] The unique feature of the
NAP-22 peptide is that this clustering of PtdIns(4,5)P2
is strongly dependent on the presence of cholesterol
[13] A well studied peptide that does not require
cho-lesterol for sequestering PtdIns(4,5)P2 is the MARCKS
peptide [12] There are several differences between
the MARCKS peptide and the NAP-22-peptide The
MARCKS peptide has 13 positive charges compared
to only seven cationic residues for the NAP-22-peptide
As a consequence, electrostatic interactions alone will
provide a stronger driving force for the MARCKS
peptide to sequester PtdIns(4,5)P2, compared with the
NAP-22-peptide Although the MARCKS protein, like
NAP-22, is N-terminally myristoylated, the longest
cluster of five Lys residues in MARCKS begins at
resi-due 86, far removed from the amino-terminal
myris-toyl group Also the model MARCKS peptide is not
myristoylated, unlike the peptides used in the present
work With regard to aromatic residues, the MARCKS
peptide has five Phe residues while the NAP-22-peptide
has only one Tyr In the case of MARCKS peptide,
the major cross-peak between the aromatic resonance
of the peptide and the lipid protons is with the
methy-lene peak [19], while in the case of the NAP-22-peptide
there is a more intense cross-peak with the terminal
methyl group of the acyl chain, particularly when
cho-lesterol is present (Fig 5) The depth of insertion is
not greatly altered when all but two of the Phe
resi-dues of the MARCKS peptide are replaced with Ala
[19] However, when all five Phe residues are replaced
with Ala, spin label studies indicate less penetration of
the peptide into the membrane [29] Nevertheless, this
Ala substituted peptide has only somewhat diminished
ability to sequester PtdIns(4,5)P2 This is not that
dif-ferent from the effects of removal of the Tyr residue
from the NAP-22 peptide when studied in membranes
containing cholesterol However for membranes devoid
of cholesterol, the Y11L-NAP-22-peptide has greater
activity in sequestering PtdIns(4,5)P2 than the unmodi-fied NAP-22 peptide We suggest that the ability of peptides to form domains of PtdIns(4,5)P2 is a conse-quence of the combined interactions of the cationic cluster of amino acid residues and the insertion of hydrophobic amino acids into the membrane In some cases, the insertion of groups that promote the forma-tion of cholesterol-rich domains will result in the pref-erential sequestering of PtdIns(4,5)P2 into one of the domains This would be a mechanism additional to the direct electrostatic interaction between the peptide and PtdIns(4,5)P2
When electrostatic interactions predominate, there is sequestering of PtdIns(4,5)P2, independently of the nat-ure of the surrounding lipid However, when the elec-trostatic interactions are reduced, as it is in NAP-22 compared with the MARCKS peptide, then sequester-ing of PtdIns(4,5)P2 is also affected by the insertion of hydrophobic moieties into the membrane that change the depth of burial of the peptide, the orientation of the peptide with respect to the membrane, and the lat-eral distribution of lipids into domains through hydro-phobic interactions These hydrohydro-phobic interactions alone are insufficient in the case of the Y11L-NAP-22-peptide to modulate the sequestering of cholesterol In the case of NAP-22, the combined interactions of the myristoyl group, the Tyr side chain and the cationic cluster in the peptide, result in a cholesterol-dependent sequestering of PtnIns(4,5)P2 into domains
There is also a structural aspect that makes NAP-22 unusual Many proteins are N-terminally myristoylated [30] but only a few have in addition, clusters of cationic residues comprised of four or more Lys or Arg residues
in sequence One of the few examples we have found is the membrane fusion protein, p15, of baboon reovirus that is both myristoylated and has a cluster of four cationic residues [31] Two other examples we have discsussed earlier are MARCKS and NAP-22 The structural difference between these two proteins is that the cationic cluster of NAP-22 is close to the myristoyl group at the amino terminus This is not the case for MARCKS Since myristoylation is a factor that causes proteins to sequester into raft domains, it would seem
a priori more likely that sequestering of PtnIns(4,5)P2 would be coupled to translocation to a cholesterol-rich domain for NAP-22 than for MARCKS, as is found The rearrangement of PtdIns(4,5)P2 and cholesterol
in a membrane caused by the presence of NAP-22 pro-vides a mechanism by which this protein can affect the actin cytoskeleton PtdIns(4,5)P2 plays an important role in the attachment of the cytoskeleton to the plasma membrane as well as affecting actin dynamics [32] Since NAP-22 causes the sequestering of both
Trang 9cholesterol and PtnIns(4,5)P2 into domains, we suggest
that the protein recruits more PtnIns(4,5)P2 into
raft-like domains This will result in an increase in the
interactions between the cytoskeleton and plasma
membrane occurring at rafts and hence the
rearrange-ment of the spatial distribution of the cytoskeleton In
neurons, several proteins including NAP-22, GAP-43
and MARCKS, affect the efficiency of raft dependent
signaling [33] Both the kinase that catalyses the
syn-thesis of PtdIns(4,5)P2 [34] as well as the phosphatase
that degrades it [35], affect cytoskeletal organization
NAP-22 together with related proteins, function to
enhance the accumulation and assembly of PtdIns(4,
5)P2-rich raft domains [36] During neuronal
develop-ment, axonal elongation and branching are regulated
by the activity of PI(4)P5 kinase [37], an enzyme that
synthesizes PtdIns(4,5)P2 Thus, the amount and
distri-bution of PtdIns(4,5)P2 will regulate cytoskeletal
dynamics, which in turn will affect neuronal growth
and development CAP-23 accumulates in the neuronal
growth cone and has a marked effect on the
rearrange-ment of the actin cytoskeleton [38] An early
conse-quence of CAP-23 accumulation is an increase in
dynamic actin structures and the loss of more stable
actin filaments such as stress fibers
We can use this simplified system to identify certain
molecular interactions that we can suggest form the
basis for events that are observed at the cellular level
In this work we use a 19 amino acid lipopeptide
cor-responding to the amino terminus of NAP-22 With
this peptide, the consequences of the rearrangements
of PtdIns(4,5)P2 we observe by fluorescence or by
DSC are significantly greater than we observe with the
intact protein It is known that there are N-terminal
fragments of NAP-22 present in cells [2] Furthermore,
a construct composed of the N-terminal segment of
CAP-23 and containing 40 amino acids arranges in a
punctate pattern on the cell surface and is associated
with the cytoskeleton Like the full length protein, this
short construct produces marked changes in cell
mor-phology but unlike the full length protein, it does not
produce blebbing [38] It has been estimated that
PtnIns(4,5)P2 comprises 0.3–1.5% of the phospholipid
of the plasma membrane of mammalian cells [12] If
dissolved in the total cell volume, this amount of
PtnIns(4,5)P2 would have a concentration in the range
2–30 lm, although the PtnIns(4,5)P2 varies
consider-ably among cell types and is particularly low in
some cells [39] Nevertheless, our use of 0.2 mol%
PtnIns(4,5)P2 in the model membranes is within the
physiological range In comparison, in the developing
brain NAP-22 comprises 0.4–0.8% of the total protein,
corresponding to a concentration of 20–40 lm [40]
Thus, there are comparable amounts of PtnIns(4,5)P2 and NAP-22 in the cell and the ratio is within the range used in our work Since NAP-22 binds to PtnIns(4,5)P2 by nonspecific electrostatic interactions, one molecule of NAP-22 can promote the formation
of a domain of many molecules of PtnIns(4,5)P2 [27] Thus not all of the NAP-22 has to be bound to PtnIns(4,5)P2 in order for a major fraction of this lipid to be sequestered into a domain This is different from proteins with specific folded domains that bind PtnIns(4,5)P2 in a stoichiometric fashion [41] The greater potency of the N-terminal peptide in forming domains would suggest that membrane lipid domain formation may be facilitated by proteolytic processing
of NAP-22 Myristoylated proteins interacting with membranes through both electrostatic interactions as well as insertion of a myristoyl group, can be dissoci-ated from the membrane by proteolytic cleavage [42]
It is possible that this is an example of the opposite, i.e proteolytic cleavage would cause increased seques-tration to the membrane by removing the anionic por-tion of the protein that would repel anionic lipids The
pI of rat NAP-22 is only 4.5 Another indication of the importance of the amino terminal fragment of NAP-22 is that the first 21 amino acids are invariant among NAP-22 of several mammalian species and this segment differs by only one residue with chicken NAP-22 (CAP-23)
Thus both cholesterol and PtdIns(4,5)P2 affect the location of the NAP-22-peptide in a bilayer The lipo-peptide has little capability of inducing phase separ-ation in mixtures of SOPC and cholesterol, but with addition of PtdIns(4,5)P2 there is a cholesterol-dependent separation into a cholesterol enriched and a cholesterol-depleted domain This segregation is repre-sented in the drawing in Fig 8 (not drawn to scale) These results demonstrate how sensitive the interaction
Fig 8 Schematic representation of the domain enrichment caused
by the peptide (red) in the presence of cholesterol (blue) and PtdIns(4,5)P2 (green) The other lipid headgroups are presented in grey and the acyl chains in orange The clustering of charges in the peptide permits interaction with the negative charges on the head-group of PtdIns(4,5)P2 concomitantly resulting in the redistribution
of cholesterol.
Trang 10of even small peptides with membranes is to the lipid
composition of the membrane
Experimental procedures
Materials
The synthetic lipopeptide with the sequence:
myristoyl-GGKLSKKKKGYNVNDEKAK-amide, corresponding to
the 19 amino terminal residues of NAP-22, as well as a
variant of this lipopeptide, Y11L were purchased from
Bio-Source International (Hopkinton, MA, USA)
Phospho-lipids and cholesterol were purchased from Avanti Polar
Lipids (Alabaster, AL, USA) PtdIns(4,5)P2 was purified
from porcine brain Bodipy-TMR-PI(4,5)P2 was purchased
from Molecular Probes (Eugene, OR, USA)
Preparation of samples for DSC and NMR
experiments
Lipid components were codissolved in chloroform⁄
meth-anol (2 : 1, v⁄ v) For samples containing peptide, an
ali-quot of a solution of the peptide in methanol was added to
the lipid solution in chloroform⁄ methanol The amount of
peptide used was monitored by the absorbance at 280 nm
using an extinction coefficient calculated from the amino
acid composition [43] The solvent was rapidly evaporated
at 30C under a stream of nitrogen with constant rotation
of a test tube to avoid separation of lipid components [12]
and to deposit a uniform film of lipid over the bottom
third of the tube Last traces of solvent were removed by
placing the tube under high vacuum for at least 2 h The
lipid film was then hydrated with 20 mm Pipes, 1 mm
EDTA, 150 mm NaCl with 0.002% NaN3, pH 7.40 and
suspended by intermittent vortexing and heating to 50C
over a period of 2 min under argon Samples used for
NMR analysis were hydrated with the same buffer made in
2
H2O and adjusted to a pH meter reading of 7.0 (pD¼
7.4) and incubated at least 24 h at 4C to allow conversion
of any anhydrous cholesterol crystals to the monohydrate
form For the NMR measurements, the samples were first
spun in an Eppendorf centrifuge at room temperature The
resulting hydrated pellet was transferred to a 4 mm
zirco-nia rotor with the 12-lL Kel-F insert, attempting to pack
the maximal amount of lipid into the rotor while keeping it
wet
DSC
Measurements were made using a Nano Differential
Scan-ning Calorimeter (Calorimetry Sciences Corporation,
American Fork, UT, USA) The scan rate was 2CÆmin)1
and there was a delay of 5 min between sequential scans in
a series to allow for thermal equilibration The features of
the design of this instrument have been described [44] DSC curves were analyzed by using the fitting program, DA-2, provided by Microcal Inc (Northampton, MA, USA) and plotted with origin, version 5.0
Preparation of LUV for fluorescence spectroscopy
A solution of POPC and 0.1 mol% Bodipy-TMR-PI(4,5)P2 with or without 40 mol% cholesterol was prepared in chlo-roform⁄ methanol (2 : 1) and the lipid deposited on the walls of a glass test tube by solvent evaporation with a stream of nitrogen gas Last traces of solvent were then removed by evaporation for 2 h under vacuum Films were hydrated with a 10 mm Hepes buffer pH 7.4 containing
1 mm EDTA and 140 mm NaCl The lipid suspensions were further processed by five cycles of freezing and thawing, fol-lowed by 10 passes through two stacked 0.1 lm polycar-bonate filters, using a Lipex extruder [45], to convert the lipid suspension to LUVs The content of lipid phospho-rous was determined by the method of Ames [46]
Fluorescence quenching
Fluorescence measurements were made in silanized glass cuvettes containing 2 mL of the appropriate buffer, at
25C, under constant stirring with Teflon magnets An amount of LUVs were added to the cuvette and then titra-ted with successive additions of small aliquots of peptide solution, using silanized Eppendorf tips Peptide solutions were made in the appropriate buffer and the peptide con-centration was quantified by absorbance at 275 nm Peptide solutions were kept in silanized containers at 4C until used
The excitation and emission monochromators were set at
542 nm and 571 nm, respectively, with a 500-nm cut-off fil-ter in the emission path The excitation and emission band-pass slits were set at 4 nm Cuvettes were maintained in the dark with the shutters closed between additions of peptide; the shutter was toggled only at the beginning of the record-ing of each emission scan, to prevent photobleachrecord-ing of the probe Two independent determinations were performed with each batch of LUVs The corresponding set of titra-tion curves with buffer not containing peptide were subtrac-ted from the titration with peptide
1H NOESY MAS/NMR
High resolution MAS spectra were acquired using a spin-ning speed of 5.5 kHz in a Bruker AV 500 NMR spectro-meter Probe temperature was 24 ± 1C The 2D NOESY spectra were obtained using delay times of 50 and 300 ms Resonances were assigned based on reports of phosphat-idylcholine [18], cholesterol [47] and amino acid residues [48]