Acyl transfer from phosphocholine lipids to melittin

Article abstract of DOI:10.1039/c0cc04677a

Transfer of fatty acyl groups from membrane phospholipids to melittin, a commonly studied membrane-active peptide, has been observed to occur over extended time periods. Transfer can be detected after 1-2 days and selectively targets amino groups at the N-terminal end of the peptide.

Full text of DOI:10.1039/c0cc04677a

Chemical Communications
www.rsc.org/chemcomm
Volume 47 | Number 5 | 7 February 2011 | Pages 1353–1636
ISSN 1359-7345
COMMUNICATION
Catherine J. Pridmore, Jackie A. Mosely, Alison Rodger and John M. Sanderson
Acyl transfer from phosphocholine lipids to melittin

COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Acyl transfer from phosphocholine lipids to melittinw
Catherine J. Pridmore,a Jackie A. Mosely,a Alison Rodgerb and John M. Sanderson*a
Received 29th October 2010, Accepted 24th November 2010
DOI: 10.1039/c0cc04677a
Transfer of fatty acyl groups from membrane phospholipids to
melittin, a commonly studied membrane-active peptide, has been
observed to occur over extended time periods. Transfer can be
detected after 1–2 days and selectively targets amino groups at
the N-terminal end of the peptide.
Chemical modifications to peptides and proteins, most notably
the addition of acyl groups such as myristoyl or palmitoyl, are
known to produce profound changes to the membrane
behaviour of these molecules, with effects including increased
membrane affinity and modified partitioning behaviour.1–3
Acyl groups are usually added enzymatically in vivo (both
translationally and post-translationally).3 In the absence of
enzyme catalysis, the membrane itself is generally seen as an
inert medium. Despite the large number of membrane-active
peptides that are studied for their intrinsic membrane
behaviour, or as models for proteins, the chemical purity and
identity of peptide–lipid systems is seldom examined upon the
Fig. 1 Mass spectrum of melittin following mixing with DOPC (A)
completion of binding experiments. Most biological membranes
have a significant protein content (typically between 20% and
80% by weight)4 and proteins may be in contact with the
membrane for a considerable period of time before recycling,
with half-lifes ranging from minutes to days.5,6 As a con-
sequence, studying the longer term behaviour of peptide– and
protein–lipid systems is of fundamental interest, particularly
with regard to examining the reactivity of these systems towards
reactions such as acyl transfer from the lipids to the protein.
Melittin (H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2)
is a widely studied membrane active peptide, with well-documented
pore-forming properties.7 During the course of experiments to
examine the effects of melittin on lipid stability,8 we undertook
control experiments using synthetic melittin in order to
circumvent the possibility of complications due to any activity
of the enzyme phospholipase A2 (PLA2), which is usually
co-purified with melittin prepared from bee venom.9 Addition
of synthetic melittin to liposomes composed of 1,2-dioleoyl-
sn-glycero-3-phosphocholine (DOPC) in phosphate buffered
saline (PBS) at 37 1C and pH 7 led to the formation of
new species, detectable in the mass spectrum of the sample
(Fig. 1A) after a period of 1–2 days. These new species
are identified as melittin plus the addition of an oleoyl chain,
observed as protonated and sodiated adducts. When incubated
under similar conditions with liposomes prepared using
and POPC liposomes (B) at 37 1C in PBS (8 mM phosphate, 123 mM
NaCl) at pH 7. Melittin and lipid concentrations were 71 mM and
0.35 mM, respectively. Mass spectra were obtained after 14 days (A) or
28 days (B) using matrix-assisted laser desorption ionisation (MALDI)
with a-cyano-4-hydroxycinnamic acid (CHA) as matrix. Ions at m/z
3110.0 and 3132.0 are assigned as [melittin + (oleoyl À H) + H]+ and
[melittin
+
(oleoyl
À
H) + , respectively (calculated
Na]+
monoisotopic m/z 3110.0 and 3132.0); ions at m/z 3084.0 and 3105.9
are assigned as [melittin + (palmitoyl À H) + H]+ and [melittin +
(palmitoyl À H) + Na]+, respectively (calculated monoisotopic m/z
3084.0 and 3106.0).
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), species
were observed after 2 days corresponding to the transfer of
either an oleoyl or a palmitoyl group from the lipid to the
peptide (Fig. 1B), again as protonated and sodiated adducts.
Further analysis by TLC and mass spectrometry revealed
the presence of lyso-phosphatidylcholine (lyso-PC) in the
mixtures, indicating that the reaction to produce the acylated
melittin species was likely to involve attack of a reactive
nucleophile on the peptide with the carbonyl groups of the
lipid esters. In order to monitor the progress of the reaction,
the experiments were repeated and the formation of lyso-PC
was followed by mass spectrometry as an indicator of the
extent of reaction. Experiments were conducted at a molar
peptide : lipid ratio of 1 : 5. Although this ratio is at the upper
end of the range typically used for membrane binding
experiments, at lower peptide concentrations it proved
challenging to detect the formation of small quantities of the
reaction products in the presence of a large excess of lipid.
Even at a molar peptide : lipid ratio of 1 : 5, due to the
excess of lipid it was necessary to remove and concentrate
(by lyophilisation) a significant volume of the sample in order
a Department of Chemistry, Durham University, South Road,
Durham, DH1 3LE, UK. E-mail: j.m.sanderson@durham.ac.uk;
Tel: +44 (0)191 3342107
b University of Warwick, Department of Chemistry, Coventry,
CV4 7AL, UK. E-mail: A.Rodger@warwick.ac.uk;
Tel: +44 (0)24 7652 3234
w Electronic supplementary information (ESI) available: MS/MS data,
TLC data, POPC kinetics, full methods. See DOI: 10.1039/c0cc04677a
c
1422 Chem. Commun., 2011, 47, 1422–1424
This journal is The Royal Society of Chemistry 2011

conditions, enabled direct comparisons to be made between
them. MS/MS of peptides generally induces cleavage along the
backbone. Products of such cleavages are labelled as a-, b- or
c-type should the fragment retain the N-terminus, and x-, y- or
z-type should it retain the C-terminus.10 In our case, a key
requirement for confirming acylation at a particular site was
the ability to identify unique product ions that only matched
modification at that site and were therefore absent from the
spectrum of the unmodified peptide. Furthermore, for reaction
at a melittin amino group leading to amide formation, it was to
be expected that cleavage of the acyl group would occur under
MS/MS conditions, resulting in the formation of ions with
similar or identical masses to the unmodified peptide. This was
confirmed in the product ion spectra of both modified forms of
melittin (m/z 3083 and 3110), in which an ion corresponding to
loss of the acyl group was observed at m/z 2845. As a
consequence, ladders of unmodified ions at the N-terminus
or C-terminus of the peptide were unreliable as a means of
identifying sites of modification.
Fig.
2 The formation of lyso-PC monitored by MALDI-MS
following mixing of melittin (71 mM) with DOPC liposomes
(0.35 mM) in PBS at 37 1C and pH 7. MALDI-MS was performed
using 2,5-dihydroxybenzoic acid as matrix. Normalised peak
intensities for lyso-PC are calculated as the sum of the intensities of
the protonated and sodiated lyso-PC peaks divided by the sum of the
intensities of the protonated and sodiated lyso-PC and DOPC peaks.
Errors are estimated from repeat experiments.
In the product ion spectrum of oleoyl–melittin (m/z 3110), a
ladder of b-type product ions can be identified that
corresponds to N-terminal modification (Fig. 3a), although
the first ion in this series (corresponding to the oleoylated
N-terminal Gly) is absent. Nevertheless, the ions with m/z
435.3, 492.2 and 563.2 are unique to this spectrum and can be
assigned as oleoylated fragments with confidence. These
N-terminal fragment ions are consistent with the addition of
an oleoyl group to the N-terminus of the peptide. A weak
fragment with m/z 758.4 is also unique to this spectrum and
matches a b-type fragment that has lost ammonia (oleoyl-
GIGAVL, b-17-type). An additional b-type fragment is found
at m/z 1000.3 (oleoyl-GIGAVLKV) and a fragment assigned
as an a-18-type at m/z 1069.5 (oleoyl-GIGAVLKVL). These
latter two product ions are again consistent with N-terminal
modification, but may also arise through modification of K7.
Interestingly, the high-intensity ion with m/z 492.2 is also a
match for an internal fragment with K7 oleoylation (oleoyl-
KV, formed via b/y-type cleavage). A corresponding ion at
m/z 464.1 matches both an a-type fragment (oleoyl-GIG) and
an internal fragment (oleoyl-KV, formed via a/y-type
cleavage). Weak ions at m/z 888.7 and 808.3 match internal
fragments that include only K7 (oleoyl-AVLKVL and oleoyl-
KVLTT), although the latter of these is also present in the
spectrum of the unmodified melittin (Fig. 3c). None of the
higher mass fragments enabled the position of modification to
be identified with certainty. Taken together, it is clear that the
N-terminal amino group is a significant site of modification,
with likely additional modification of the side chain of K7. No
evidence could be found for the addition of oleoyl groups to
the remaining two lysines (K21 and K23).
to detect the lyso-PC by TLC. With DOPC, in the initial stages
(Fig. 2) the reaction was slow, with the first statistically
significant detection of lyso-PC after 2 days. The intensity of
the lyso-PC peaks increased steadily over the next 14 days and
then increased slowly thereafter. No formation of lyso-PC
was observed in control experiments without melittin and oleic
acid was not detected in any of the samples (with or
without melittin), indicating that the level of background
lipid hydrolysis was negligible. Due to absence of either
competing hydrolysis or multiple acylation products, the
formation of lyso-PC was therefore taken to be a reliable
indicator of the extent of melittin acylation.
After 16 days, the relative intensity of the lyso-PC peaks was
0.3 Æ 0.05. By comparison with a 1-oleoyl-sn-glycero-3-
phosphocholine/DOPC calibration curve, this corresponded
to a mole fraction of lyso-PC in the sample of 0.1 Æ 0.02 and a
corresponding extent of conversion of the melittin to acylated
product of 50 Æ 10%. This compared favourably with estima-
tion of the extent of lyso-PC formation by TLC, which also
indicated an extent of conversion of 50 Æ 10% after 17 days
(see the ESI1). In experiments with POPC, similar kinetic
profiles were obtained, with relative lyso-PC peak intensities
of 0.15 for palmitoyl-sn-glycero-3-phosphocholine (corre-
sponding to oleoyl transfer) and 0.07 for oleoyl-sn-glycero-3-
phosphocholine (corresponding to palmitoyl transfer) after
14 days, and corresponding values of 0.18 and 0.10 after
28 days (see the ESIw). These data indicated that acyl transfer
from POPC was slower than from DOPC, and transfer of the
oleoyl group was favoured over the palmitoyl group.
Fragmentation of the palmitoylated melittin ion at m/z 3083
yields an N-terminal ladder of b-type ions at m/z 296.1, 409.2,
466.2, 537.2 and 636.3 (Fig. 3b). Of these, the ions at m/z 409.2,
466.2 and 537.2 are unique to this spectrum. As with the
product ion spectrum of oleoylated melittin, the peak at
466.2 can also be assigned as a palmitoylated internal
fragment (palmitoyl-KV in this case). An additional
fragment unique to this spectrum at m/z 782.1 is assigned as
an internal fragment involving modification of the side chain of
The most favourable positions of melittin for nucleophilic
attack could reasonably be proposed as the N-terminal amino
group and the e-amino groups of the three lysines (K7, K21
and K23). Tandem mass spectrometry (MS/MS) was therefore
performed on unmodified melittin and acylated melittin
precursor ions in order to generate fragments that would
permit the site of acylation to be localised. Performing all of
these MS/MS analyses on the same sample, under identical
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 1422–1424 1423

Fig. 3 A section of the MALDI-MS/MS spectra of (a) oleoyl–melittin (m/z 3110, offset on the y-axis by 50%), (b) palmitoyl–melittin (m/z 3083,
offset on the y-axis by 25%) and (c) melittin (m/z 2845) from a sample of melittin incubated with POPC in PBS for 28 days at 37 1C, pH 7. Ladders
corresponding to modified b-type fragments are indicated by dashed vertical lines. Key ions corresponding to modified (or potentially modified)
fragments are indicated by diamonds. Spectra were obtained using CHA as matrix and are base peak normalised. See the ESIw for full spectra and
assignments.
K7 (palmitoyl-KVLTT formed via b/y-type cleavage). A
number of product ions in this spectrum correspond to
oleoylated products (e.g. the ion at m/z 492.2), which
indicates that isolation of the parent ion was not complete.
Nevertheless, these ions do not prohibit the identification
of unique palmitoylated fragments. It may therefore be
concluded that palmitoyl modification occurs at the
N-terminal amino group, with the side chain of K7 likely to
be an additional site of modification. No acyl melittin could be
detected when peptide : lipid ratios were lowered to 1 : 50,
although this may be more a consequence of a lack of relative
sensitivity of the analytical methods than a lack of reaction. A
number of other experiments were conducted in order to
examine the effects of salts and metal ions on the acylation
process. Repeating the experiments with DOPC in the presence
of zinc or calcium salts gave data comparable to the PBS/NaCl
data. Conducting the experiments with DOPC in phosphate
buffer alone (i.e. without NaCl or any other metal salts) yielded
no detectable oleoylated products. This latter result may reflect
either a change in reactivity, or salt-related effects on melittin
conformation.11
found for proteins in biological membranes, and as a
consequence raises the possibility that similar processes may
occur in vivo. It should be noted that it is unclear whether this
reactivity of melittin is typical of all membrane peptides (and
proteins), or the result of a particular chemical or structural
feature of the peptide that favours reaction. It is perfectly
feasible that this reaction will be promoted by the presence of
particular amino acids near the sites of acylation and the scope
of this reaction therefore merits further scrutiny.
Notes and references
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M. J. Nadolski and M. E. Linder, FEBS J., 2007, 274, 5202.
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3 A. N. Hannoush and J. Sun, Nat. Chem. Biol., 2010, 6, 498.
4 P. J. Quinn, in The Lipid Handbook, ed. F. D. Gunstone,
J. L. Harwood and A. J. Dijkstra, CRC Press, Boca Raton, 2007,
3rd edn, p. 512.
5 Y. Ohsumi, IUBMB Life, 2006, 58, 363.
6 J. Hare and R. Hodges, J. Biol. Chem., 1982, 257, 3575;
S. M. Russell, J. S. Amenta and R. J. Mayer, Biochem. J., 1984,
220, 489.
7 H. Raghuraman and A. Chattopadhyay, Biosci. Rep., 2007, 27,
189.
8 A. Damianoglou, A. Rodger, C. Pridmore, T. R. Dafforn,
J. A. Mosely, J. M. Sanderson and M. R. Hicks, Protein Pept.
Lett., 2010, 17, 1351.
9 C. Code, Y. A. Domanov, J. A. Killian and P. K. J. Kinnunen,
Biochim. Biophys. Acta, Biomembr., 2009, 1788, 1064; L. Vernon
and J. Bell, Pharmacol. Ther., 1992, 54, 269.
Overall, it is apparent that the amino groups of a
prototypical membrane-active peptide are able to react with
the ester groups of phosphocholines to yield product amides
with an acyl group appended to the peptide. In the case of
melittin at least, this process is selective for amino groups near
the N-terminus of the peptide. The rate of acylation, although
slow, is nevertheless relevant for membrane proteins that
exhibit low rates of turnover in biological membranes. The
ratio of peptide to lipid used, although higher than normal for
many peptide binding experiments, is still within the range
10 P. Roepstorff and J. Fohlman, Biol. Mass Spectrom., 1984, 11, 601.
11 H. Raghuraman, S. Ganguly and A. Chattopadhyay, Biophys.
Chem., 2006, 124, 115.
c
1424 Chem. Commun., 2011, 47, 1422–1424
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