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PLATELETS AND THROMBOPOIESIS
e-Blood
Proteomic analysis of palmitoylated platelet proteins
*Louisa Dowal,1 *Wei Yang,2-4 Michael R. Freeman,2,3 Hanno Steen,4,5 and Robert Flaumenhaft1
1Division
of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA; 2Urological Diseases Research Center, Department of Urology,
Children’s Hospital Boston, Boston, MA; 3Department of Surgery, Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA;
4Proteomics Center, Children’s Hospital Boston, Boston, MA; and 5Department of Pathology, Harvard Medical School and Children’s Hospital Boston, Boston, MA
Protein palmitoylation is a dynamic process that regulates membrane targeting
of proteins and protein-protein interactions. We have previously demonstrated
a critical role for protein palmitoylation in
platelet activation and have identified palmitoylation machinery in platelets. Using
a novel proteomic approach, Palmitoyl
Protein Identification and Site Characterization, we have begun to characterize
the human platelet palmitoylome. Palmitoylated proteins were enriched from
membranes isolated from resting platelets using acyl-biotinyl exchange chemis-
try, followed by identification using liquid
chromatography-tandem mass spectrometry. This global analysis identified
> 1300 proteins, of which 215 met criteria for significance and represent the
platelet palmitoylome. This collection
includes 51 known palmitoylated proteins, 61 putative palmitoylated proteins identified in other palmitoylationspecific proteomic studies, and 103 new
putative palmitoylated proteins. Of these
candidates, we chose to validate the palmitoylation of triggering receptors expressed on myeloid cell (TREM)–like
transcript-1 (TLT-1) as its expression is
restricted to platelets and megakaryocytes. We determined that TLT-1 is a
palmitoylated protein using metabolic labeling with [3H]palmitate and identified
the site of TLT-1 palmitoylation as cysteine 196. The discovery of new platelet
palmitoyl protein candidates will provide
a resource for subsequent investigations
to validate the palmitoylation of these
proteins and to determine the role palmitoylation plays in their function. (Blood.
2011;118(13):e62-e73)
Introduction
Platelets are key mediators of hemostasis and thrombosis, and their
acute response at sites of vascular injury requires a rapid and
complex series of signaling events. Posttranslational modifications,
such as phosphorylation,1 have been shown to influence plateletsignaling pathways, thus regulating platelet function, and palmitoylation, which is also a reversible modification, has been proposed to
play an analogous regulatory role.2-3 Palmitoylation is the most
common form of S-acylation, which is the covalent attachment of
long chain fatty acids via thioester bonds to cysteine residues.4
Palmitoylation involves the attachment of the 16-carbon saturated
fatty acid palmitate. Although attachment of palmitate enhances the
hydrophobicity of a protein, its function extends beyond that of a
simple membrane anchor as palmitoylation has been shown to
regulate protein trafficking, sorting, stability, and activity.5 Thus,
the addition of this lipid group to a protein has functional
consequences. Palmitoylation is unique among the different types
of lipid modifications in that it is reversible and does not require a
specific sequence motif. The reversible nature of palmitoylation
functions as a regulatory mechanism or molecular switch, directing
protein-lipid and protein-protein interactions6-7 and plays a role in
the regulation of cellular responses to external stimuli.
We have previously shown that platelets possess palmitoylation
machinery and require palmitoylation for activation because chemical inhibition of protein palmitoylation results in abrogation of
platelet aggregation and decreased incorporation of platelets into
thrombi in a murine laser-induced model of vascular injury.8 In
resting platelets, we8 and others9-13 observe incorporation of
[3H]-palmitate into platelet proteins, which indicates that this fatty
acid is being cycled even in the resting state. Although palmitate
accounts for 74% of the fatty acids linked to proteins by a thioester
bond in the platelet,10 protein palmitoylation remains a poorly
understood posttranslational modification in platelets. These observations prompted us to assess the global scope of palmitoylation in
platelets.
Proteomics offers a powerful means to study the biology and
biochemistry of the anucleate platelet.14-15 Recently, palmitoylomes in yeasts,16 rat neurons,17 and human prostate cancer cells18
have been described. In our efforts to define the platelet palmitoylome, we have purified and identified palmitoylated proteins from
membranes of resting platelets using the Palmitoyl Protein Identification and Site Characterization (PalmPISC) method that we
recently developed.18 In this study, we present a comprehensive
identification of palmitoylated platelet proteins. This strategy
identified 215 significantly enriched palmitoylated platelet protein
candidates. Of these, 51 are known palmitoylated proteins and
61 were identified in proteomic studies of other cells with medium
to high confidence, indicating that they are most likely palmitoylated. The remaining 103 palmitoyl protein candidates have not
previously been shown to be palmitoylated. As proof of this
concept, we validated the palmitoylation of triggering receptors
expressed on myeloid cell (TREM)–like transcript-1 (TLT-1), a
platelet- and megakaryocyte-specific protein, and identified its
Submitted May 9, 2011; accepted July 11, 2011. Prepublished online as Blood
First Edition paper, August 2, 2011; DOI 10.1182/blood-2011-05-353078.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*L.D and W.Y. contributed equally to this study.
This article contains a data supplement.
e62
© 2011 by The American Society of Hematology
BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
palmitoylation site. These experiments further our understanding
of the scope of palmitoylation in platelets and will provide a basis
for studying the dynamics and function of palmitoylation in these
putative palmitoyl proteins.
Methods
Chemicals and reagents
Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich.
Complete, EDTA-free protease inhibitor cocktail (11873580001) was
obtained from Roche Diagnostics. [3H]Yohimbine (NET 659250UC), guanosine triphosphate (GTP)[␥-35S] (NEG 030H250UC), and [3H]palmitic acid
(NET043) were obtained from PerkinElmer Life and Analytical Sciences.
Tris(2-carboxyethyl)phosphine (TCEP; 77720) and N-[6-(biotinamido)hexyl]-3⬘(2⬘-pyridyldithio)propionamide (biotin-HPDP; 21341) were obtained from
Thermo Scientific. Coomassie Brilliant Blue and polyvinylidene fluoride (PVDF)
membranes were obtained from Bio-Rad. Iodoacetamide (RPN6302) was
purchased from GE Healthcare. MS-grade trypsin (V5280) was obtained from
Promega. Anti–human TREML1/TLT-1 primary antibody (AF2394) was obtained from R&D Systems, and anti–human Cdc42 (610928) antibody was
obtained from BD Transduction Laboratories. FITC-conjugated secondary
antibodies were obtained from Pierce Biotechnology. Anti-G␤1 primary antibody
(sc-379) and Protein A/G PLUS-agarose (sc-2003) were obtained from Santa
Cruz Biotechnology.
Platelet and platelet membrane preparation
Platelet-rich plasma that had outdated within 24 hours before use was
obtained from the Beth Israel Deaconess Medical Center Blood Bank.
Platelets were washed 3 times and assessed by flow cytometry using a
P-selectin expression assay.8 Only platelet preparations demonstrating
resting P-selectin levels comparable with resting fresh platelets were
further processed. An aliquot of the selected platelets was tested to
confirm normal P-selectin expression in response to 100␮M SFLLRN.
Resting, washed platelets were centrifuged, and platelet pellets were
resuspended in ice-cold lysis buffer (5mM Tris-HCl, pH 7.5, and 5mM
ethylene glyco-bis(b-aminoethyl ester)-N,N,N⬘,N⬘-tetraacetic acid) plus
protease inhibitor cocktail to 4 mL/g wet weight. Platelets were lysed by
successive rounds of sonication; and after removal of intact cells by low
speed centrifugation, the lysates were pooled. Membranes were pelleted
at 50 000g and flash frozen at ⫺80°C. To assess the quality of the
platelet membrane preparation, immunoblotting for the peripheral
membrane protein G␤1 was performed. To further evaluate the integrity
of the membrane preparation, [3H]yohimbine binding and GTP[␥-35S]
binding19 were evaluated. Only preparations in which the receptors
within the membrane retained the ability to bind agonists and stimulate
GTP turnover on stimulation were used for liquid chromatography-mass
spectrometry (LC-MS) studies.
Platelet palmitoyl protein purification, separation, and trypsin
digestion
Proteins were prepared from 100 mg of platelet membranes using our
adaptation of acyl-biotinyl exchange (ABE) chemistry as previously
described.18 Briefly, membrane proteins were denatured with SDS, reduced
with 10mM TCEP, and alkylated with 50mM N-ethylmaleimide (NEM) to
block nonpalmitoylated cysteines. After methanol/chloroform precipitations to remove excess NEM, proteins were treated with 0.75M hydroxylamine (HA) and 1mM biotin-HPDP to replace palmitoyl groups with
biotinyl groups. The in vitro biotinylated (previously palmitoylated)
proteins were enriched by streptavidin affinity purification, specifically
eluted by TCEP, and concentrated by methanol/chloroform precipitation.
Enriched proteins were separated on a 12% SDS-PAGE gel and stained with
Coomassie Brilliant Blue. Each gel lane was cut into 5 slices before
reduction with 10mM dithiothreitol, alkylation with 55mM iodoacetamide,
and in-gel digestion with trypsin.18
PALMITOYLATED PLATELET PROTEINS
e63
Mass spectrometry
Tryptic peptides were analyzed by on-line nanoflow reversed-phase high
performance liquid chromatography (Eksigent nanoLC-2D) connected to
an LTQ Orbitrap mass spectrometer (Thermo Scientific) essentially as
described.18 Samples were loaded onto an in-house packed C18 column
(Magic C18, 5 ␮m, 200 Å, Michrom Bioresources) with 15-cm length and
100-␮m inner diameter, and separated at ⬃ 200 nL/min with 60-minute
linear gradients from 5% to 35% acetonitrile in 0.2% formic acid. Survey
spectra were acquired in the Orbitrap analyzer with the resolution set to a
value of 30 000. Lock mass option was enabled in all measurements, and
decamethylcyclopentasiloxane background ions (at m/z 371.10123) were
used for real-time internal calibration as described previously.20 Up to 5 of
the most intense multiply charged ions per cycle were fragmented and
analyzed in the linear ion trap.
Database searching, spectral counting, and statistical analysis
The Thermo raw files were deposited at Tranche (https://proteomecommons.
org/dataset.jsp?i ⫽ 76 216) and will be made publically accessible on
publication. Raw data were analyzed using MaxQuant Version 1.0.13.13.21
The parameters were set as follows. In the Quant module, Singlets was
selected; oxidation (M), acetyl (protein N-term), carbamidomethyl (C),
and N-ethylmaleimide (C) were set as variable modifications; no fixed
modifications were allowed; concatenated IPI human database Version 3.52
(74 190 forward sequences and 74 190 reverse sequences) downloaded
from www.maxquant.org was used for database searching; all other
parameters used were default values. In the Identify module, all parameters
used were default values, except that maximal peptide posterior error
probability was set as .05. False discovery rates for protein identification
and peptide identification were both set at 1%. The relative protein
abundance changes between the paired HA⫹ and HA⫺ samples were
determined using a label-free spectral counting approach.22 Statistical
analysis was performed as previously described18 with minor modifications.
Briefly, the spectral counts were merged over both biologic replicates, and
all zeros were replaced with 1 in the merged dataset to avoid division by
zero. Given that spectral counting is not accurate for proteins with very
low spectral counts, statistical analysis was only performed on the
proteins with a spectral count of at least 3. The protein-wise log2transformed HA⫹/HA⫺ ratios were calculated and then clustered using a
Bayesian information criterion-based Gaussian mixture model. The resulting 2 Gaussian components represent the log-ratio distributions of protein
sets largely dominated by contaminating proteins or by palmitoyl proteins.
P values were computed based on the distribution of the contaminating
protein-dominant dataset.
Western blot analysis
For immunoblotting, separated proteins were transferred to PVDF membranes and blocked in TBS/0.1% Tween-20/5% milk for 1 hour at room
temperature. Blots were probed with anti–human TREML1/TLT-1 primary
antibody (1:1000) or anti–human Cdc42 (1:500) overnight at 4°C followed
by 1-hour incubation at room temperature with FITC-conjugated secondary
antibodies (1:1000) to detect TLT-1 or Cdc42. Blots were analyzed with an
Amersham Typhoon 9400 molecular imager.
Labeling platelet proteins with [3H]palmitate and
immunoprecipitation of TLT-1
Washed platelets (2 mL at 1 ⫻ 109 platelets/mL) were radiolabeled with
100 ␮Ci/mL [3H]palmitic acid for 1 hour at 37°C, in PIPES/NaCl buffer
with 3.6 mg/mL BSA. Platelets were activated as described in the text,
diluted 5 times with PIPES/NaCl buffer, and pelleted to remove unincorporated [3H]palmitic acid. Platelets were then resuspended in RIPA buffer
(1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158mM NaCl,
10mM Tris-HCl, pH 7.6, 1mM phenylmethylsulfonyl fluoride, and
protease inhibitor cocktail) and allowed to lyse on ice for 5 minutes. The
lysate was centrifuged for 5 minutes at 16 000g, and the supernatant was
used as the RIPA soluble fraction. The platelet lysate was precleared by
adding 20 ␮L of Protein A/G beads and incubating for 30 minutes at
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e64
DOWAL et al
BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
Figure 1. Analysis of ABE-purified palmitoylated proteins from platelet membranes. (A) Electrophoretic analysis. Lane 1 indicates molecular weight marker; lane 2,
experimental samples (HA⫹) with hydroxylamine treatment; and lane 3, control samples (HA⫺) without hydroxylamine treatment. (B) The correlation between proteins identified
in 2 separate rounds of spectral counting analysis. Data were fit with a linear function (R2 ⫽ 0.8645). (C) Distribution of log(HA⫹/HA⫺) ratios. Bayesian Information
Criterion-based Gaussian Mixture modeling suggested that the distribution of log-ratios (solid line) is composed of 2 Guassian components (dashed line). The left Gaussian
represents the log-ratio distribution of most contaminating proteins and some palmitoyl proteins, whereas the right Guassian represents the log-ratio distribution of most
palmitoyl proteins and a small number of contaminants. To distinguish palmitoyl proteins from contaminating proteins, P values were calculated based on the distribution of the
left Gaussian. Proteins with P ⬍ .05 were treated as significant and accepted as palmitoyl protein candidates.
4°C with end-over-end rotation. Beads were pelleted, and TLT-1 was
immunoprecipitated from the supernatant using 2.5 mg/mL human TREML1/
TLT-1 primary antibody overnight at 4°C with end-over-end rotation.
Protein/immune complexes were pulled out by addition of 20 ␮L of Protein
A/G beads and incubation for 60 minutes at 4°C with end-over-end
rotation. Beads were then washed 3 times with 1 mL of RIPA buffer, and
immunoprecipitates were eluted from the beads by the addition of 20 ␮L
SDS sample buffer. [3H]Palmitate-labeled platelet proteins were then
separated by SDS-PAGE and transferred onto a PVDF membrane, which
was exposed to a tritium detection screen for 2 weeks and then analyzed
using an Amersham Typhoon 9400 molecular imager. Tritium blot band
intensities were normalized for the amount of protein loaded.
Identification of TLT-1 palmitoylation site
Enriched palmitoyl proteins were separated by SDS-PAGE. Because the
apparent molecular weight of TLT-1 is ⬃ 37 kDa, a gel slice containing 30to 50-kDa proteins was excised. Proteins were tryptically digested in gel
without reduction and alkylation. Tryptic peptides were extracted and
analyzed by LC tandem MS (LC-MS/MS) as described in “Mass spectrometry.” Free cysteines in the identified peptides are candidate palmitoylation
sites because nonpalmitoylated cysteines were blocked by NEM before
ABE reaction.
Results
Purification and identification of palmitoylated platelet proteins
Palmitoylated proteins were enriched from platelet membranes that
were prepared from resting platelets pooled from 6 aphaeresis
packs, and purification of palmitoylated proteins was carried out
using our adaption of ABE chemistry.18 After extraction of platelet
membrane proteins, all disulfide bonds were reduced with TCEP, a
reducing agent that does not cleave thioester bonds, and free thiols
were blocked with the alkylating agent, NEM. The sample was split
into 2 groups, which were treated with parallel protocols: an
experimental group in which the thioester bonds were specifically
cleaved with neutral HA⫹ and a control group (HA⫺), which was
not treated with hydroxylamine. The HA⫺ group represents background binding to the steptavidin beads, which may be the result of
endogenous biotinylation, nonspecific biotinylation, or nonspecific
binding. After the newly formed thiols were labeled with biotinHPDP, biotinylated proteins were enriched by streptavidin affinity
chromatography and eluted with TCEP. This approach enriched for
a large number of proteins (Figure 1A) that were separated and
visualized by SDS-PAGE followed by in gel digestion and
LC-MS/MS.
LC-MS/MS and MaxQuant analyses of 2 biologic replicates
of enriched palmitoyl proteins resulted in the identification of
⬎ 1300 proteins (supplemental Table 3, see the Supplemental
Materials link at the top of the article), with a false discovery rate of
1% for both proteins and peptides. Plotting both rounds of spectral
counting against each other revealed good agreement between
experimental datasets (R2 ⫽ 0.8645; Figure 1B). Label-free spectral counting quantitation and statistical analysis showed that 215
nonredundant proteins were significantly enriched (P ⬍ .05) in
the HA⫹ sample over that of the HA⫺ control (Figure 1C). To
ensure the accuracy of protein identification based on a single
unique peptide, the MS/MS spectra of all platelet palmitoyl
protein candidates were manually verified (supplemental Figure
1). Proteins that were considered significantly enriched had an
HA⫹/HA⫺ ratio ⬎ 3 (P ⬍ .05); and of the 215 enriched proteins,
103 proteins are not known to be palmitoylated or have not been
described in other palmitoyl proteomic studies (Table 1). The
remaining 112 identified proteins are known to be palmitoylated
or are palmitoyl protein candidates identified in other proteomic
studies (Table 2).
For graphic representation of the proteins identified by LC-MS/
MS, the summed spectral counts for each protein in the HA⫹
sample were plotted against the summed spectral counts for each
protein in the HA⫺ sample (Figure 2A). For the majority of proteins
identified, there was substantial representation in both the HA⫹ and
HA⫺ samples, which resulted in a HA⫹/HA⫺ratio ⬍ 3 (gray dots,
Figure 2A). Clustered around the x-axis, with the known palmitoylated proteins (green circles, Figure 2A), are the newly identified
palmitoyl protein candidates (open black circles, Figure 2A).
Well-established, known palmitoylated platelet proteins identified
in our study are also indicated (blue dots, Figure 2A) and include
the G␣ subunits Gq,11,12 Gi,11,12 and G13,12 platelet glycoprotein 4,25
and CD6326 (Table 2). Several highly abundant platelet proteins
known to be palmitoylated were identified in the list of 1300 purified
proteins but failed to demonstrate a HA⫹/HA⫺ ratio ⱖ 3 (red dots,
Figure 2A). This group includes P-selectin,27 tubulin,28 platelet
glycoprotein Ib,11 PECAM,29 G␣z,11 platelet glycoprotein IX,11 and
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
PALMITOYLATED PLATELET PROTEINS
e65
Table 1. Novel candidates of the platelet palmitoylome
Protein name
Gene name
Ratio
P
Apoptosis
Myc target protein 1
MYCT1
3.10
.039094
Placenta-derived apoptotic factor
PDAF
4.00
.015866
Tetraspanin-33
TSPAN33
6.25
.002368
Claudin-5
CLDN5
3.33
.030650
Dynein heavy chain 1, axonemal
DNAH1
6.00
.002867
Cell growth and/or maintanence
Metalloproteinase inhibitor 1
TIMP1
3.29
.032192
Lysophosphatidylcholine acyltransferase 1
LPCAT1
4.00
.015866
Transmembrane protein 97
TMEM97
3.00
.043487
H2AFX
3.00
.043487
CKLF-like MARVEL transmembrane domain-containing protein 3
CMTM3
11.00
.000116
CKLF-like MARVEL transmembrane domain-containing protein 5
CMTM5
7.43
.001013
Early activation antigen CD69
CD69
5.00
.006457
Minor histocompatibility protein HA-1
HMHA1
3.00
.043487
ORAI1
5.00
.006457
.015866
DNA repair
Histone H2A
Immune response
Ion transport
Calcium release-activated calcium channel protein 1
Metabolism
关3-methyl-2-oxobutanoate dehydrogenase 关lipoamide兴兴 kinase
BCKDK
4.00
␣-amylase 1
AMY1A
3.00
.043487
ATPase family AAA domain-containing protein 3A
ATAD3A
3.00
.043487
Catechol-O-methyltransferase
COMT
4.50
.010000
Dihydrolipoamide branched chain transacylase E2
DBT2
8.00
.000690
Dolichyldiphosphatase 1
DOLPP1
3.00
.043487
GPI ethanolamine phosphate transferase 1
PIGN
3.00
.043487
Probable cysteinyl-tRNA synthetase
CARS2
4.00
.015866
Protein disulfide-isomerase TMX3
TMX3
15.00
.000016
Suppressor of Lec15
MPDU1
3.00
.043487
Thioredoxin-related transmembrane protein 1
TMX1
3.45
.027084
Thioredoxin-related transmembrane protein 4
TMX4
6.00
.002867
V-type proton ATPase 116-kDa subunit ␣ isoform 2
ATP6V0A2
3.00
.043487
26S proteasome non-ATPase regulatory subunit 11
PSMD11
6.00
.002867
CAAX prenyl protease 1 homolog
ZMPSTE24
4.00
.015866
Copper chaperone for superoxide dismutase
CCS
4.00
.015866
Protein metabolism
Cystatin-S
CST4
3.00
.043487
E3 ubiquitin-protein ligase MARCH2
MARCH
6.00
.002867
Eukaryotic translation elongation factor 1 ⑀-1
EEF1E1
3.00
.043487
Eukaryotic translation initiation factor 5A-1
EIF5A
6.00
.002867
GrpE protein homolog 1
GRPEL1
3.00
.043487
Leucyl/cystinyl aminopeptidase
LNPEP
4.00
.015866
RING finger protein 11
RNF11
3.50
.025869
Signal transduction
Atlastin-1
ATL1
5.00
.006457
CKLF-like MARVEL transmembrane domain-containing protein 7
CMTM7
4.00
.015866
Endothelial cells scavenger receptor
SCARF1
10.00
.000202
Filaggrin
FLG
3.00
.043487
GTP-binding protein Rheb
RHEB
4.00
.015866
Leucine-rich repeat-containing protein 32
LRRC32
21.00
.000002
Metalloreductase STEAP3
STEAP3
6.00
.002867
Nuclear receptor subfamily 4 group A member 2
NR4A2
3.00
.043487
Platelet-derived endothelial cell growth factor
ECGF1
3.00
.043487
Rho-related GTP-binding protein RhoQ
RHOQ
3.00
.043487
Tescalcin
TESC
18.00
.000005
Tetraspanin-15
TSPAN15
17.00
.000007
Transmembrane protein 11
TMEM11
7.00
.001366
Transmembrane protein 50A
TMEM50A
3.25
.033406
Transmembrane protein 55A
TMEM55A
3.83
.018614
Transcription regulator activity
39S ribosomal protein L12
MRPL12
3.00
.043487
Oxidoreductase HTATIP2
HTATIP2
3.00
.043487
To date, these 103 proteins have not been shown to be palmitoylated and represent putative palmitoylated platelet proteins. Data are the result of 2 independent
experiments, and proteins are listed according to the biologic process they mediate as defined by the HPRD. Also shown is the HA⫹/HA⫺ ratio and corresponding P value.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
DOWAL et al
Table 1. Novel candidates of the platelet palmitoylome (continued)
Protein name
Gene name
Ratio
P
.000202
Transport
BET1 homolog
BET1
10.00
BET1-like protein
BET1L
11.00
.000116
Cholesteryl ester transfer protein
CETP
4.00
.015866
MLN64 N-terminal domain homolog
STARD3NL
4.00
.015866
Nonspecific lipid-transfer protein
SCP2
9.00
.000366
Proteolipid protein 2
PLP2
3.00
.043487
SID1 transmembrane family member 1
SIDT1
4.00
.015866
Sodium/hydrogen exchanger 9
SLC9A9
3.67
.021908
Solute carrier family 43 member 3
SLC43A3
5.00
.006457
Syntaxin-2
STX2
3.00
.043487
Syntaxin-10
STX10
4.00
.015866
Syntaxin-11
STX11
6.18
.002507
Tandem C2 domains nuclear protein
TC2N
42.00
.000000
Vesicle transport protein SFT2C
SFT2D3
6.00
.002867
Vesicle-associated membrane protein 5
VAMP5
15.00
.000016
.025869
Unknown
Catechol-O-methyltransferase domain-containing protein 1
COMTD1
3.50
CD99 antigen-like protein 2
CD99L2
3.00
.043487
Chronic lymphocytic leukemia deletion region gene 6 protein
CLLD6
4.50
.010000
COMM domain-containing protein 9
COMMD9
3.00
.043487
Cyclin-Y
CCNY
9.00
.000366
Exocyst complex component 3-like protein 2
EXOC3L2
4.00
.015866
Leptin receptor gene-related protein
LEPROT
6.00
.002867
Lipoma HMGIC fusion partner
LHFP
8.00
.000690
Lipoma HMGIC fusion partner-like 2 protein
LHFPL2
6.40
.002115
LITAF-like protein
CDIP
6.00
.002867
Major facilitator superfamily domain-containing protein 6
MFSD6
4.00
.015866
Malectin
MLEC
Metallo-␤-lactamase domain-containing protein 2
MBLAC2
4.90
.007035
12.00
.000068
Mps one binder kinase activator-like 3
MOBKL3
5.00
.006457
Oligosaccharyltransferase complex subunit OSTC
OSTC
3.00
.043487
PDZK1-interacting protein 1
PDZK1IP1
8.00
.000690
Prolactin-inducible protein
PIP
4.00
.015866
Protein EFR3 homolog A
EFR3A
9.67
.000245
Protein FAM78A
FAM78A
3.00
.043487
Protein S100-A14
S100A14
3.00
.043487
Putative uncharacterized protein C1orf150
C1orf150
10.00
.000202
Putative uncharacterized protein C1orf95
C1orf95
3.00
.043487
SEC6-like protein C14orf73
C14orf73
24.00
.000001
Small VCP/p97-interacting protein
SVIP
3.00
.043487
Tetraspanin-14
TSPAN14
9.80
.000227
Transmembrane BAX inhibitor motif-containing protein 1
TMBIM1
6.00
.002867
Transmembrane protein 222
TMEM222
10.00
.000202
Transmembrane protein 50B
TMEM50B
8.00
.000690
Transmembrane protein with metallophosphoesterase domain
TMPPE
5.00
.006457
Uncharacterized protein C22orf25
C22orf25
7.00
.001366
Uncharacterized protein KIAA2013
KIAA2013
7.00
.001366
UPF0389 protein FAM162A
FAM162A
8.50
.000500
UPF0598 protein C8orf82
C8orf82
11.00
.003588
UPF0733 protein C2orf88
C2orf88
8.00
.000116
To date, these 103 proteins have not been shown to be palmitoylated and represent putative palmitoylated platelet proteins. Data are the result of 2 independent
experiments, and proteins are listed according to the biologic process they mediate as defined by the HPRD. Also shown is the HA⫹/HA⫺ ratio and corresponding P value.
CD926 (supplemental Table 1). Included among the known palmitoylated proteins is the palmitoylated form of Cdc42, which
previously had been described in neurons17 and identified as a
putative palmitoyl protein in human prostate cancer cells.18 To
confirm that we were observing the palmitoylated form of Cdc42 in
our preparation, samples were stained using antibodies directed
against Cdc42. We found Cdc42 only in the HA palmitoyl-protein
and not the HA⫺ sample, indicating that platelets contain the
palmitoylated form of Cdc42 (Figure 2B).
Using the Human Protein Reference Database (HPRD),30 we
classified all the known and putative palmitoylated proteins by
biologic process (Figure 3). We find that the largest percentage of
palmitoylated platelet protein candidates (31.3%) is involved in
signal transduction processes. A substantial number of proteins
(18.3%) involved in transport processes, which includes the
movement of vesicles and small molecules and ions within or out
of the cell, were found to be palmitoylated as well. Although close
to half of these transport proteins are known to be palmitoylated or
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
PALMITOYLATED PLATELET PROTEINS
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Table 2. Known palmitoylated proteins of the platelet palmitoylome
Protein name
Gene name
Ratio
P
Source
16.50
.000009
Proteomic studies18
2.90
.048440
Proteomic studies17
Cell growth and/or maintenance
Claudin-3
CLDN3
Desmoplakin
DSP
Flotillin-1
FLOT1
14.80
.000018
Manual
55-kDa erythrocyte membrane protein
MPP1
3.00
.043487
Uniprot
Proteomic studies17
Immune response
Complement component 1 Q subcomponent-binding protein
C1QBP
3.00
.043487
HLA class I histocompatibility antigen, B-27 ␣-chain
HLA-B
9.00
.000366
Manual
Interferon-induced transmembrane protein 3
IFITM3
3.00
.043487
Uniprot
Linker for activation of T-cell family member 1
LAT
7.33
.001082
Uniprot
CANX
2.88
.049774
Proteomic studies18
3-mercaptopyruvate sulfur transferase
MPST
3.33
.030650
Proteomic studies17
4-aminobutyrate aminotransferase
ABAT
4.00
.015866
Proteomic studies17
ADP-ribosyl cyclase 1
CD38
5.00
.006457
Proteomic studies17
Acid ceramidase
ASAH1
5.25
.005232
Proteomic studies18
Protein folding
Calnexin
Metabolism
ATP synthase subunit ␥
ATP5L
4.00
.015866
Proteomic studies17
CTD small phosphatase-like protein
CTDSPL
8.25
.000586
Proteomic studies18
Cytochrome b-c1 complex subunit Rieske
UQCRFS1
3.00
.043487
Proteomic studies17
Glutaminase kidney isoform
GLS
3.00
.043487
Proteomic studies17
NAD(P) transhydrogenase
NNT
7.92
.000726
Proteomic studies17
Probable phospholipid-transporting ATPase IF
ATP11B
11.00
.000116
Proteomic studies18
Sn1-specific diacylglycerol lipase-␤
DAGLB
29.00
1.29E-07
Proteomic studies18
Protein metabolism
Cation-dependent mannose-6-phosphate receptor
M6PR
7.00
.001366
Manual
DnaJ homolog subfamily C member 5
DNAJC5
27.00
2.29E-07
Uniprot
Endothelin-converting enzyme 1
ECE1
35.00
2.72E-08
Manual
F-box/LRR-repeat protein 20
FBXL20
6.00
.002867
Proteomic studies18
Adenylate cyclase type 6
ADCY6
3.40
.028628
Proteomic studies17
ADP-ribosylation factor 5
ARF5
3.00
.043487
Proteomic studies17
ADP-ribosylation factor-like protein 15
ARL15
5.25
.005232
Proteomic studies18
Casein kinase I ␥1 isoform
CSNK1G1
3.00
.043487
Proteomic studies17
Casein kinase I isoform ␥3
CSNK1G3
8.00
.000690
Proteomic studies17
CD151 antigen
CD151
3.00
.043487
Uniprot
CD63 antigen
CD63
3.67
.021908
Manual
CD82 antigen
CD82
4.00
.015866
Manual
CDC42 small effector protein 1
CDC42SE1
3.00
.043487
Uniprot
CDC42 small effector protein 2
CDC42SE2
9.00
.000366
Uniprot
Cell division control protein 42 homolog
CDC42
3.00
.043487
Manual
Choline transporter-like protein 2
CTL2
27.00
2.29E-07
Disks large-associated protein 4
DLGAP4
6.00
.002867
Proteomic studies17
Disheveled-associated activator of morphogenesis 1
DAAM1
9.00
.000366
Proteomic studies17
Signal transduction
Proteomic studies18
Dystroglycan
DAG1
4.00
.015866
Proteomic studies17
Erbb2-interacting protein
ERBB2IP
8.88
.000395
Manual
Flotillin-2
FLOT2
10.55
.000149
Manual
G protein-coupled receptor kinase 6
GRK6
8.00
.000690
Probable
G(i) ␣-1
GNAI1
3.80
.019226
HPRD
G(i) ␣-2
GNAI2
3.25
.033301
Manual
G(i) ␣-3
GNAI3
4.86
.007300
HPRD
G(q) ␣
GNAQ
5.17
.005609
HPRD
G(s) ␣
GNAS
5.80
.003352
Manual
G␣-11
GNA11
6.00
.002867
HPRD
G␣-13
GNA13
7.45
.000998
Uniprot
G␣-15
GNA15
4.00
.015866
Manual
GTPase Hras
HRAS
7.00
.001366
Uniprot
GTPase Nras
NRAS
5.25
.005232
Uniprot
Junctional adhesion molecule C
JAM3
5.14
.005722
Proteomic studies17
Kalirin
KALRN
7.50
.000965
Proteomic studies17
Linker for activation of T-cell family member 2
LAT2
7.00
.001366
Probable
Mitochondrial import inner membrane translocase subunit TIM50
TIMM50
4.00
.015866
Proteomic studies24
Known palmitoylated proteins as catalogued by the Uniprot or HPRD databases or by review of published palmitoylation-related research articles (manual). Also included
in this list are proteins characterized as being palmitoylated by similarity, probably palmitoylated, or potentially palmitoylated as defined by the Uniprot database. A total of 61 of
the identified proteins were discovered as palmitoyl protein candidates in other proteomic studies.17,18,24 Data are the result of 2 independent experiments, and proteins are
grouped according to the biologic process they mediate as defined by the HPRD. Also shown is the HA⫹/HA⫺ ratio and corresponding P value.
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
DOWAL et al
Table 2. Known palmitoylated proteins of the platelet palmitoylome (continued)
Protein name
Phosphatidylinositol 4-kinase type 2-␣
Gene name
Ratio
P
PI4K2A
10.00
.000202
Source
Manual
Phosphatidylinositol 4-kinase type 2-␤
PI4K2B
3.00
.043487
Manual
Phospholipid scramblase 1
PLSCR1
20.50
.000002
Potential
Platelet glycoprotein 4
CD36
6.14
.002569
Uniprot
Prostacyclin receptor
PTGIR
10.00
.000202
Uniprot
Proto-oncogene tyrosine-protein kinase Fyn
FYN
4.91
.006980
HPRD
Proto-oncogene tyrosine-protein kinase Yes
YES1
10.00
.000202
Proteomic studies18
Raftlin
RFTN1
5.00
.006457
Probable
Ras-related protein Rap-2a
RAP2A
28.00
.000000
by similarity
Ras-related protein Rap-2b
RAP2B
3.93
.017042
by similarity
Ras-related protein Rap-2c
RAP2C
11.50
.000089
by similarity
Ras-related protein R-Ras
RRAS
7.44
.001002
Manual
Regulator of G-protein signaling 19
RGS19
15.00
.000016
Uniprot
Semaphorin-4D
SEMA4D
6.00
.002867
Proteomic studies17
Sortilin
SORT1
3.00
.043487
Proteomic studies17
Stomatin
STOM
3.29
.031966
Uniprot
Tetraspanin-9
TSPAN9
6.67
.001737
Proteomic studies18
Thromboxane A2 receptor
TBXA2R
4.00
.015866
Manual
3.50
.025869
Proteomic studies17
10.67
.000139
Potential
LYN
5.00
.006457
by similarity
HIST1H2BN
6.00
.002867
Proteomic studies17
Transmembrane protein 55B
TMEM55B
Type I inositol-1,4,5-trisphosphate 5-phosphatase
INPP5A
Tyrosine-protein kinase Lyn
Transcription regulator activity
Histone H2B type 1-N
Transport
AFG3-like protein 2
AFG3L2
4.00
.015866
Proteomic studies17
ATP-binding cassette subfamily B member 6
ABCB6
5.00
.006457
Proteomic studies18
Choline transporter-like protein 1
CTL1
14.20
.000024
Proteomic studies18
Cytochrome b5 type B
CYB5B
3.67
.021908
Proteomic studies17
Golgin subfamily A member 7
GOLGA7
17.00
.000007
Uniprot
Multidrug resistance-associated protein 4
ABCC4
3.00
.043487
Proteomic studies18
Phospholipid scramblase 3
PLSCR3
13.00
.000042
Probable
Phospholipid scramblase 4
PLSCR4
7.00
.001366
Probable
Pituitary tumor-transforming gene 1 protein-interacting protein
PTTG1IP
19.50
.000003
Proteomic studies18
PRA1 family protein 2
PRAF2
14.00
.000026
Proteomic studies17
Protein tweety homolog 3
TTYH3
11.00
.000116
Proteomic studies17
Secretory carrier-associated membrane protein 1
SCAMP1
10.25
.000175
Proteomic studies17
Secretory carrier-associated membrane protein 2
SCAMP2
3.95
.016681
Proteomic studies17
Secretory carrier-associated membrane protein 3
SCAMP3
16.00
.000011
Proteomic studies17
Secretory carrier-associated membrane protein 4
SCAMP4
3.00
.043487
Proteomic studies17
Sodium/potassium-transporting ATPase subunit ␣-1
ATP1A1
3.00
.043487
Proteomic studies17
SNAP-23
SNAP23
3.74
.020385
HPRD
Manual
Syntaxin-8
STX8
11.00
.000116
Syntaxin-12
STX12
4.00
.015866
Proteomic studies17
Trafficking protein particle complex subunit 3
TRAPPC3
8.67
.000450
by similarity
Transferrin receptor protein 1
TFRC
3.00
.043487
Uniprot
Vesicle-associated membrane protein 3
VAMP3
8.43
.000523
Proteomic studies18
Vesicle-associated membrane protein 4
VAMP4
10.00
.000202
Proteomic studies17
Vesicle-associated membrane protein 7
VAMP7
3.69
.021361
Proteomic studies18
Unknown
3-oxoacyl-关acyl-carrier-protein兴 synthase
OXSM
6.00
.002867
Proteomic studies17
Abhydrolase domain-containing protein FAM108B1
FAM108B1
8.33
.000556
Proteomic studies18
Coiled-coil domain-containing protein 109A
CCDC109A
4.50
.010000
Proteomic studies17
Endoplasmic reticulum-Golgi intermediate compartment protein 3
ERGIC3
3.00
.043487
Proteomic studies17
Abhydrolase domain-containing protein FAM108A1
FAM108A1
7.50
.000965
Proteomic studies23
Protein FAM49B
FAM49B
21.00
.000002
Proteomic studies18
Protein LYRIC gene-1 protein
LYRIC
18.00
.000005
Proteomic studies17
Transmembrane protein 63A
TMEM63A
7.29
.001118
Proteomic studies18
Transmembrane protein 63B
TMEM63B
3.00
.043487
Proteomic studies18
UPF0404 protein C11orf59
C11orf59
5.71
.003588
Proteomic studies18
Known palmitoylated proteins as catalogued by the Uniprot or HPRD databases or by review of published palmitoylation-related research articles (manual). Also included
in this list are proteins characterized as being palmitoylated by similarity, probably palmitoylated, or potentially palmitoylated as defined by the Uniprot database. A total of 61 of
the identified proteins were discovered as palmitoyl protein candidates in other proteomic studies.17,18,24 Data are the result of 2 independent experiments, and proteins are
grouped according to the biologic process they mediate as defined by the HPRD. Also shown is the HA⫹/HA⫺ ratio and corresponding P value.
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
PALMITOYLATED PLATELET PROTEINS
e69
Figure 2. Global analysis of the platelet palmitoylome. (A) Graphical depiction of the 1300 proteins identified in resting platelet membranes. Gray dots represent
identified proteins not meeting the criteria for significance. Candidate palmitoyl proteins (E) cluster around
the x-axis with known palmitoylated proteins and putative
palmitoyl proteins identified in other palmitoylationspecific proteomic studies (green dots). Also shown are
well-established palmitoylated platelet proteins identified
(blue dots) and not identified (red dots) as being palmitoylated in this study. Inset: Expanded view of the graph for
proteins with ⬍ 50 spectral counts. Diagonal line in each
graph indicates the HA⫹/HA⫺ cut-off. (B) Western blotting
analysis of ABE-purified proteins from platelet membranes, as prepared for proteomic analysis, in the presence and absence of HA using antibodies directed
against Cdc42. Also shown is the total protein input for
the HA⫹ and HA⫺ samples.
have been described in other proteomic studies, the rest are newly
identified palmitoyl candidates. This group includes syntaxins-2,
-10, and -11 and VAMP-5. These results indicate that signaling and
transport proteins represent approximately half of all palmitoylated
platelet proteins identified in this study.
TLT-1 is a palmitoyl protein candidate
Because the expression of TLT-1 is restricted to megakaryocytes
and platelets, we chose it for further analysis. TLT-1 demonstrated
an HA⫹/HA⫺ ratio of 2.5 (P ⫽ .076). The full-length form of
Figure 3. The biologic processes mediated by proteins of the platelet palmitoylome. Analysis of the platelet palmitoylome indicates that many of the identified platelet proteins are
involved in signal transduction pathways and transport processes. Each protein was assigned a biologic process as defined by the HPRD, which is Gene Ontology compliant.
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DOWAL et al
BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
Figure 4. TLT-1 is enriched in HAⴙ samples in resting
and activated platelets. (A) Domain organization of
full-length TLT-1 and the TLT-1 splice variant depicting an
Ig-like V-type domain (blue) with 4 cysteine residues
followed by a single-pass transmembrane (TM) domain
(green). The TLT-1 splice variant has a truncated cytoplasmic tail and does not contain the 3 intracellular cysteines
or ITIM region.32 (B) Western blot analysis of TLT-1 using
ABE-purified palmitoylated proteins from resting and
thrombin-activated platelets. Arrow indicates full-length
TLT-1; and arrowhead, a 25 kDa TLT-1 splice variant.
Bands seen directly below full-length TLT-1 and bands
seen below the TLT-1 splice variant are degradation
products.33 Also shown are 20 ␮g of the sample inputs
that represent the protein sample added to the streptavidin agarose beads.
TLT-1 has a 126-amino acid cytosolic C-terminal tail, which
includes an immunoreceptor tyrosine-based inhibitory motif (ITIM)
region and a proline-rich region that may mediate protein-protein
interactions.31 The cytosolic tail also contains 3 cysteine residues
(Figure 4A), and analysis of the sequence using the palmitoylation
site predicting software, CSS-Palm 2.0, indicated that TLT-1 was
probably palmitoylated only on cysteine 196 (Cys196).32 TLT-1
exists as 2 species: a full-length form with a molecular mass of
35 kDa and a splice variant with a molecular mass of 25 kDa. The
splice variant differs from full-length TLT-1 in that it has a
truncated cytosolic region, which encodes a short, 14-amino acid
tail (Figure 4A).31 In addition, amino acids 190 to 199 of the splice
variant differ from the full-length sequence. Instead of GNRLGVCGRF, the splice variant encodes for ESLLSGPPRQ, which
does not contain the predicted palmitoylation site at Cys196
(Uniprot database).
To confirm that TLT-1 is palmitoylated and to determine which
TLT-1 species is palmitoylated, we directly assessed samples
purified by ABE chemistry for TLT-1. Platelets from outdated
aphaeresis packs were washed and divided into 2 equal aliquots.
One was left resting and the other was stimulated with 1 U/mL
␣-thrombin for 5 minutes at room temperature. Prostaglandin E1
was added to both samples, and the platelets were immediately
pelleted and dissolved in lysis buffer. Palmitoylated proteins were
subsequently purified using the ABE method from membranes
prepared from resting and thrombin-activated platelets, and were
separated by gel electrophoresis and transferred to a PVDF
membrane (Figure 4B). Immunoblotting of ABE-purified TLT-1
demonstrated 3 major bands corresponding to full-length TLT-1, a
previously described degradation product,33 and the splice variant.
Full-length TLT-1 and its degradation product were palmitoylated
because the HA⫹ samples were enriched for full-length TLT-1 in
resting and activated platelets compared with the HA⫺ samples
(Figure 4B). In contrast, the TLT-1 splice variant was not enriched.
Bands seen for TLT-1 in the HA⫺ sample represent background
binding to the streptavidin beads, which may be the result of
improper biotinylation or nonspecific binding. Western blot analysis of the ABE-purified samples in the resting and activated HA⫹
lanes did not demonstrate a change in the palmitoylation state of
TLT-1 on platelet activation under the conditions in which this
assay was performed (Figure 4B). These results suggest that
full-length TLT-1, but not the TLT-1 splice variant, is palmitoylated
in platelets.
TLT-1 is a novel palmitoylated platelet protein
To determine whether TLT-1 is a bona fide palmitoylated protein,
we metabolically labeled platelets with [3H]palmitic acid and
assayed for incorporation of [3H]palmitate. Resting platelets were
labeled with [3H]palmitic acid for 1 hour at 37°C and then divided
into 2 equal portions. One portion remained resting, and the other
was activated with 1 U/mL ␣-thrombin. Samples were lysed and
TLT-1 was immunoprecipitated (Figure 5A). The minor band seen
at 33 kDa represents a TLT-1 degradation product.33 Subsequent
audioradiography revealed that full-length TLT-1 incorporated
[3H]palmitate in both resting and activated platelets (Figure 5B).
The 25-kDa splice variant, which was immunoprecipitated with
full-length TLT-1 (Figure 5A lanes 3 and 4), did not incorporate
[3H]palmitate (Figure 5B), as predicted by the absence of the
cytosolic cysteine residues and studies of TLT-1 using ABE
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BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
Figure 5. [3H]Palmitate incorporation confirms that TLT-1 is a palmitoylated
protein. Platelets were metabolically labeled with [3H]palmitic acid and separated
into resting (lane 1) and thrombin-activated (lane 2) samples and lysed. TLT-1 was
then immunoprecipitated from resting (lane 3) and thrombin-activated platelet lysates
(lane 4). Samples were subjected to Western blot analysis (A) or audioradiography to
assess [3H] palmitate incorporation (B). Full-length TLT-1 is the band corresponding
to 37 kDa. This blot is representative of 3 independent experiments.
chemistry (Figure 4). These studies confirm that TLT-1 is a platelet
palmitoyl protein.
To further characterize the palmitoylation of TLT-1, we used
PalmPISC to identify the candidate palmitoylation site. In this
approach, palmitoylated cysteines can be readily distinguished
from nonpalmitoylated cysteines because nonpalmitoylated cysteines are irreversibly blocked by NEM, whereas palmitoylated
cysteines become free cysteines after the ABE reaction and TCEP
elution. The resulting spectrum of a peptide derived from TLT-1,
LGVCGR, shows a free cysteine residue based on the m/z
difference of 103 between the y2 and y3 fragment ion (Figure 6).
The fact that this cysteine (Cys196) is free and not NEM-modified
indicates that Cys196 is a putative palmitoylation site. This result is
in agreement with the CSS-Palm prediction that Cys196 is a
candidate palmitoylation site of TLT-1.
Discussion
Our analysis of the platelet palmitoylome resulted in the identification of ⬎ 1300 proteins. We found that 215 of these proteins were
significantly enriched in the HA⫹ sample, suggesting that they are
palmitoyl proteins. However, there are several reasons to think that
even more palmitoylated proteins exist in platelets. First, we did
not observe some of the known palmitoylated proteins as expected
in the platelet palmitoylome. These proteins include members of
the acyl-transferase family, known as DHHC proteins, which have
been observed in other proteomic studies.18 The reason for this
could lie in the low abundance of these proteins. Second, to avoid
falsely identifying palmitoyl protein candidates, we used strict
cut-off values to define the HA⫹ enriched dataset and excluded
actual palmitoyl proteins. For example, TLT-1, which was not
significantly enriched in the HA⫹ sample, was confirmed to be a
palmitoylated platelet protein (Figure 5). Similarly, we did not
identify all of the platelet proteins that have previously been found
to be subject to palmitoylation. P-selectin, tubulin, platelet glycoproteins IX and Ib, PECAM, G␣z, and CD9 were identified (Figure
1B red dots; supplemental Table 2), but their HA⫹/HA⫺ ratios did
not meet the stringent threshold (ratio ⱖ 3, P ⬍ .05) that was used
to identify candidate palmitoyl proteins. It is possible that, although
these proteins are highly abundant in platelets, their palmitatemodified, membrane-associated forms are not. Another potential
PALMITOYLATED PLATELET PROTEINS
e71
source of error is the copurification of contaminating proteins, a
common issue for affinity purification-based methods. Nonspecific
copurification of nonpalmitoylated proteins in the HA⫺ sample will
lower the HA⫹/HA⫺ ratio and is a limitation of the PalmPISC
method. In addition, the widely used spectral counting quantitation
approach is only semiquantitative. Development of better methods
to reduce the background contribution of contaminating proteins is
currently ongoing and will increase the sensitivity and specificity
for identification of palmitoyl proteins in platelets. Yield of
palmitoyl proteins could also be improved with better solubilization of the membranes to release more integral membrane proteins.
Studies are currently underway to test an alternative method using
pressure cycling technology34 to extract more proteins from platelet
membranes.
Approximately 50% of identified palmitoyl proteins mediate
signal transduction and transport processes. Because palmitoylation is a dynamic lipid modification, the palmitoylation state of
platelet proteins may present another layer of regulation governing
the localization and protein-protein interactions necessary to carry
out these processes. Included in our list of signaling proteins is the
palmitoylated form of Cdc42. This result was intriguing because
Cdc42 exists as 2 isoforms: a prenylated form that has ubiquitous
expression and a palmitoylated form that has been described
previously in the rat neuronal palmitoyl proteome.17 Immunoblotting of the ABE-purified palmitoylated proteins demonstrated the
HA dependence of Cdc42 (Figure 2C) in our preparations and
indicates that platelets possess this palmitoylated isoform. Unlike
metabolic labeling with [3H]palmitate, the ABE method provides a
snapshot of all the palmitoylated proteins in a cell at a particular
moment in time. Although ABE methodology cannot give information about the rate of palmitate turnover, when combined with a
global proteomic approach, it can provide the identity of proteins
for further targeted study.
We chose TLT-1 as a palmitoyl protein candidate for further
study because its palmitoylation has not previously been described
and its expression is restricted to megakaryocytes and platelets.35
The limited expression pattern of TLT-1 suggests that it may play a
specific role in platelet function and makes it a potential target for
the modulation of hemostasis and thrombosis because antibodies
directed against TLT-1 are able to block thrombin-mediated platelet
aggregation.33 Furthermore, TLT-1 knockout mice demonstrate
Figure 6. Representative tandem mass spectrum of a candidate palmitoyl
peptide derived from TLT-1 protein. Enriched palmitoyl proteins were separated by
SDS-PAGE and a gel slice containing 30- to 50-kDa proteins excised. Proteins were
digested in gel, followed by the extraction of tryptic peptides, which were analyzed by
LC-MS/MS. Free cysteines in the purified peptides are candidate palmitoylation sites.
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DOWAL et al
BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
Figure 7. Venn diagrams representing the overlap
between the platelet and rat neuronal palmitoylomes.
Number of overlapping proteins identified in the platelet (solid line) and rat neuronal (dashed line) palmitoylomes (A). Number of overlapping known palmitoylated
proteins identified in the platelet and rat neuronal
palmitoylomes (B).
extended tail bleeding times and are predisposed to hemorrhage in
an inflammatory model. Platelets from these mice are defective in
aggregation in response to adenosine diphosphate and U46619
stimulation.36 TLT-1 is part of a family of receptors termed
triggering receptors expressed on myeloid cells (TREMs)37; and
although TLT-1 is homologous to TREMs in its V-type immunoglobulin-like extracellular domain, TLT-1 has a longer cytoplasmic
tail with a proline-rich region and contains an ITIM instead of an
immunoreceptor tyrosine-based activation motif (Figure 3). It has
been shown in vitro that the ITIM region of TLT-1 is capable of
being phosphorylated and can recruit SH2-domain–containing
protein tyrosine phosphatases 1 and 2.31,38
We present evidence here that TLT-1 is a novel palmitoylated
protein and is palmitoylated on Cys196. Full-length TLT-1 has
7 cysteine residues.39 Four of these reside in the extracellular
domain and form disulfide bonds.40 Of the 3 intracellular cysteine
residues, Cys196 occurs just after the transmembrane domain. Our
identification of Cys196 as a putative palmitoylation site is in
agreement with the observation that the likelihood of a cysteine
being palmitoylated increases after stretches of hydrophobic amino
acids, such as a transmembrane domains. As expected, the truncated 25-kDa TLT-1 splice variant did not incorporate [3H]palmitate (Figure 5B), consistent with the lack of intracellular cysteines.
The role that palmitoylation serves in TLT-1 function is unknown.
Many of the palmitoyl proteins that we found in the platelet had
previously been identified in our analysis of palmitoyl proteins in
lipid rafts (supplemental Table 2). We have found that a fraction of
TLT-1 incorporates into lipid rafts (supplemental Figure 2). TLT-1
palmitoylation could affect its incorporation into rafts, as has been
demonstrated for the immunoreceptor tyrosine-based activation
motif-containing protein PECAM-1.29 Alternatively, palmitoylation may influence phosphorylation of TLT-1, as observed in
tissue factor41 and linker for activation of T cells (LAT).42 Our
methodologies and experimental conditions did not demonstrate
activation-induced palmitoylation of TLT-1. However, activation
could affect palmitate cycling, and presently we cannot separate
palmitoylation and depalmitoylation activities. Therefore, we cannot conclude whether or not activation-induced TLT-1 palmitoylation occurs. We are currently developing methods to study
activation-dependent platelet palmitoylation and determine the
significance of TLT-1 palmitoylation in platelets.
We compared the platelet palmitoylome with other palmitoylation-specific proteome studies16-18,23,24,43 and identified 103 new
palmitoyl protein candidates. In our analysis of the platelet
palmitoylome, we determined the overlap between it and the rat
neuronal palmitoylome.17 We chose the neuronal palmitoylome for
comparison because megakaryocytes/platelets and neurons share
structural and functional characteristics, and there are numerous
studies in which platelets have been proposed as a model for
neuronal function.44 Of the 215 proteins that compose the platelet
palmitoylome, 75 proteins overlap between our study and the rat
neuronal palmitoylome (Figure 7). We also find that, of the
51 known palmitoylated proteins identified in the platelet palmitoylome, 37 overlap with the 68 known palmitoylated proteins identified in
the neuronal palmitoylome (Figure 7).17 Of particular interest was our
identification of the palmitoylated form of Cdc42. Although Cdc42 null
platelets display a complex phenotype,45 Cdc42 is implicated in platelet
cytoskeletal remodeling46,47 and filopodia formation.48 In neurons, the
palmitoylated form of Cdc42 induces the formation of dendritic
spines.17 It is tempting to speculate that the palmitoylated form of Cdc42
may play a similar role in megakaryocytes or platelets contributing to
proplatelet formation or morphologic changes seen on platelet activation. Studies are currently underway to assess the implications of this
finding.
This study is the first comprehensive description of the platelet
palmitoylome and expands our understanding of the scope of
palmitoylation in platelets. However, given the importance of
palmitoylation in regulating protein function, our findings extend
beyond platelet biology as our global characterization of the
platelet palmitoylome resulted in the identification of 103 novel
palmitoyl protein candidates. The validation of the palmitoylation
of these candidate proteins will provide opportunities for future
studies aimed at increasing our understanding of the role palmitoylation plays in platelet function. The platelet palmitoylome will
establish a platform, providing an essential first step to the
determination of how palmitoylation alters the function of the
novel palmitoyl protein candidates.
Acknowledgments
The authors thank Valance Washington (University of Puerto Rico–
Mayaguez) for helpful discussions and the Beth Israel Deaconess
Medical Center Blood Bank for providing a source of platelets.
This work was supported by the National Institutes of Health
(grant HL87203, R.F.) and the United States Army (grant PC093459,
M.R.F. and H.S.). L.D. was supported by the National Institutes of
Health (grant T32 HL07917). R.F. is a recipient of an Established
Investigator Award from the American Heart Association.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 29 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 13
Authorship
Contribution: L.D. designed and performed research, analyzed
data, and wrote the paper; W.Y. designed and performed research,
analyzed data, and contributed to writing the paper; M.R.F.
provided reagents and contributed to writing the paper; H.S.
facilitated the mass spectrometric analysis and contributed to
PALMITOYLATED PLATELET PROTEINS
e73
writing the paper; and R.F. conceived of study, analyzed data, and
contributed to writing the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Robert Flaumenhaft, Division of Hemostasis
and Thrombosis, Department of Medicine, Beth Israel Deaconess
Medical Center, 330 Brookline Ave, Boston, MA 02215; e-mail:
[email protected].
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2011 118: e62-e73
doi:10.1182/blood-2011-05-353078 originally published
online August 2, 2011
Proteomic analysis of palmitoylated platelet proteins
Louisa Dowal, Wei Yang, Michael R. Freeman, Hanno Steen and Robert Flaumenhaft
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