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INTRODUCTION |
Lipid modification of proteins appears to occur in most
eukaryotes. Three general classes of covalent modification have been documented (i.e. N-acylation, isoprenylation, and
thioacylation) (reviewed in Ref. 1). Fatty acids, depending on their
length, are linked to proteins by one of two bonds; long chain fatty
acids (e.g. palmitate) are esterified to cysteines
(i.e. thio-palmitoylation) in varying positions along the
polypeptide (see Refs. 2 and 3 for reviews). (On occasion, long chain
fatty acids occur in amide linkage to lysine (4).) On the contrary,
shorter chain fatty acids (e.g. myristate) have been found
exclusively in amide linkage to N-terminal glycine
(N-acylation) (5).
Trypanosoma brucei, the causative agent of African
trypanosomiasis (sleeping sickness), is a protozoan parasite, which in the bloodstream of a mammal is covered with about 107
molecules of a variant surface glycoprotein
(VSG),1 a GPI-anchored
molecule. A 39-kDa GPI-specific phospholipase C (GPI-PLC) that can
cleave diacylglycerol from VSG and GPI biosynthetic intermediates is
expressed in bloodstream form T. brucei (6-8).
The parasite contains approximately 4 × 104 molecules
of GPI-PLC per cell (6), about one enzyme molecule per 250 molecules of
VSG. With a turnover number (kcat) of 144 min
1 (9), there appears to be sufficient enzyme
intracellularly to cleave all of the VSGs within a few minutes.
However, cleavage of VSG in living T. brucei is not a major
catabolic pathway, although purified GPI-PLC cleaves VSG efficiently
in vitro (9). These observations suggest, among other
possibilities, that (i) GPI-PLC may not have access to VSG in a living
cell and/or (ii) GPI-PLC is not enzymatically active in
vivo. GPI-PLC has been immunolocalized to the cytoplasmic leaflet
of intracellular vesicles (10), apparently sequestered, topologically,
from VSG which is attached to the outer (exoplasmic) leaflet of the
plasma membrane. Such localization studies support the former
hypothesis, although the latter cannot be ruled out completely.
Biosynthesis of GPIs is initiated on the cytoplasmic side of the
endoplasmic reticulum (11), implying that GPI intermediates probably
co-localize with GPI-PLC on the cytoplasmic side of cellular membranes
in T. brucei. This assertion is supported by two studies involving the stable transfection of a cDNA encoding T. brucei GPI-PLC into the related protozoan parasites
Leishmania major and Trypanosoma cruzi. First,
L. major expressing GPI-PLC acquires a GPI-negative
phenotype resulting apparently from cleavage of protein-GPI
intermediates by the enzyme (12). Second, in T. cruzi
GPI-PLC causes a GPI deficiency that is associated with failure of the
parasites to sustain division of the cell nucleus (13). Given these
observations, it is imperative to examine why GPI-PLC in its native
environment (i.e. in T. brucei) does not cause a
GPI deficiency.
As part of efforts aimed at unraveling the mechanisms that regulate
activity of GPI-PLC in T. brucei, we investigated the possibility that the enzyme, itself an integral membrane protein, had a
lipid modification. Herein, we demonstrate that GPI-PLC is covalently
modified with myristic acid in a thioester linkage. The observation is
the first example of acylation of a GPI-PLC in vivo. The
esterified fatty acids modulated activity of this PLC, proving that
phospholipases may be regulated by covalently attached lipids.
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EXPERIMENTAL PROCEDURES |
Cell Types/Strain
T. brucei ILTat 1.3 was harvested from the blood of
infected rats by cardiac puncture. Buffy coats were prepared and
parasites purified by DE52 chromatography (14).
Materials
[9,10-3H]Myristate and Hyperfilm-MP were from
Amersham Pharmacia Biotech. [9,10-3H]Palmitate and
[11,12-3H]laurate were from American Radiolabeled
Chemicals. Hydroxylamine was obtained from Sigma. Cycloheximide was
from Calbiochem (San Diego, CA). Protease inhibitors were obtained from
Boehringer Mannheim. Entensify and EN3HANCE were from
DuPont; MeltiLex was from Amersham Pharmacia Biotech, and Uniplate
RPS-F silica gel (20 × 20 cm) was from Analtech (Newark, DE).
Fatty acid-free bovine serum albumin was from Life Technologies, Inc.,
and DE52 was from Whatman (Hillsboro, OR). p-Nitro blue tetrazolium and 5-bromo-4-chloroindolyl phosphate were purchased from
Bio-Rad. All other reagents were from Sigma.
Metabolic Labeling
A pellet of 2 × 109 purified T. brucei was resuspended in 20 ml of pre-warmed labeling media (RPMI
1640 containing 20 mM HEPES, pH 7.4). Metabolic labeling
was initiated by addition of 400 µCi of
[9,10-3H]myristic acid (51.0 Ci/mmol) or
[9,10-3H]palmitic acid (60 Ci/mmol) or
[11,12-3H]lauric acid (60 Ci/mmol) complexed with fatty
acid-free bovine serum albumin (1 mg) in 40 µl of PBS (140 mM NaCl, 3 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.4). Cells were incubated at
37 °C for 1 h, harvested by centrifugation at 14,000 × g for 10 min, and washed by resuspension in fresh
labeling medium and re-centrifugation.
Pulse-Chase
Parasites (7 × 109) were labeled with
[9,10-3H]myristate (1.4 mCi). The cells were washed and
resuspended in 14 ml of fresh labeling medium and incubated for 60 min
at 37 °C. Two-ml aliquots (109 cells) were harvested
every 10 min, washed with PBS, and frozen at
80 °C until use.
Cycloheximide Treatment
After metabolic labeling of 109 cells with 200 µCi
of [9,10-3H]myristate, the parasites were washed,
resuspended in 5 ml of fresh labeling medium containing 1.4 mM cycloheximide, and incubated for 15 min at 37 °C.
Cells were then harvested, washed with PBS, and frozen at
80 °C
until use. In another set of experiments, 109 cells were
pretreated with 1.4 mM cycloheximide in labeling medium for
15 min at 37 °C prior to addition of 200 µCi of a
[9,10-3H]myristate-bovine serum albumin complex. Labeling
was continued for 1 h. Control cells were labeled with
[9,10-3H]myristate for 1 h, washed, and incubated at
37 °C for 15 min. Parasites were washed with PBS and the resulting
pellets stored at
80 °C until use.
Immunoprecipitation of GPI-PLC
Cells (109) were resuspended in 1 ml of hypotonic
lysis buffer (10 mM sodium phosphate, 1 mM
EDTA, pH 8) containing a protease inhibitor mixture (PIC). (PIC
consisted of leupeptin (2.1 µM), N-tosyl-L-lysine chloromethyl ketone (0.1 mM), antipain dihydrochloride (20 nM),
4-amidinophenylmethylsulfonyl fluoride (20 nM), aprotinin (0.4 units), EDTA (140 µM), and phosphoramidon (20 nM).) Cells were held on ice for 20 min, after which the
lysate was centrifuged at 14,000 × g (4 °C) for 10 min, and the supernatant was discarded. This lysis step was repeated.
A membranous pellet, from the second lysis step (above), was
resuspended in 1 ml of immunoprecipitation dilution buffer (1% (v/v)
Triton X-100, 0.2 M NaCl, 60 mM Tris-HCl, pH
7.5, 6 mM EDTA, 10 units/ml aprotinin, and PIC). The lysate
was incubated on ice for 30 min. One microliter of mc2A6-6 ascites
fluid was added to the detergent-solubilized pellet, which was
incubated (4 °C) with continuous inversion for 2 h. To get rid
of membrane-associated VSG, samples were centrifuged at 14,000 × g for 5 min at 4 °C and the pellet discarded. To the
supernatant, 50 µl of a suspension (1:1) of protein A-Sepharose:water
was added followed by incubation at 4 °C with repeated inversion for
18 h. Samples were centrifuged at 14,000 × g at
4 °C for 10 s, and the supernatants were discarded. Recovered
beads were washed as follows: once with 1 ml of PBS; twice, each time
with 750 µl of buffer KGKOG (3.17 M potassium glutamate,
0.59 M KCl, and 0.1% (w/v) n-octyl glucoside);
twice, 1 ml each, with PBS containing 2% Nonidet P-40 (15). The beads received a final wash in 1 ml of PBS. Protease inhibitors were added to
all buffers before the washes, performed by repeated inversion on a
laboratory rotator at 4 °C for 10 min. Beads were recovered in
between washes by centrifugation at 14,000 × g for 10 s. To help recovery of the protein A-Sepharose beads after washing with KGKOG, 750 µl of deionized water was added, to reduce the viscosity of the solution prior to the centrifugation step.
For some studies, an estimate of the total amount of
3H-acylated VSG was needed. For this purpose, a 10-µl
aliquot of the detergent-solubilized pellet (107 cell eq)
was withdrawn (i.e. prior to immunoprecipitation of GPI-PLC)
and added to 10 µl of 2.5× SDS-PAGE sample buffer (25% (v/v)
glycerol, 5% (w/v) SDS, 5% (v/v)
-mercaptoethanol, 0.05% (v/v)
bromphenol blue, and 0.05 M Tris-HCl, pH 6.8). Following a
5-min heating step at 90 °C, a profile of total acylated proteins (mainly VSG) was obtained after SDS-PAGE fluorography (Figs. 2 and
6).
SDS-PAGE and Fluorography
Immunoadsorbed 3H-acylated GPI-PLC (approximately 50 µl) was eluted by heating the protein A-Sepharose-antibody complex in 25 µl of a modified 2.5× SDS-PAGE sample buffer. A SDS-PAGE buffer lacking Tris was used. We found that presence of Tris base in SDS-PAGE
sample buffer deacylated GPI-PLC when heat (90 °C, 5 min) was
applied. Eluted samples were centrifuged for 3 min at 14,000 × g (room temperature). A 20-µl aliquot of the supernatant was analyzed by SDS-PAGE (14% minigel) (Bio-Rad). The gel was soaked
in Entensify (DuPont), dried, and exposed to pre-flashed Hyperfilm-MP
at
80 °C. Alternatively, proteins were transferred to Immobilon P
with a Trans-Blot semi-dry cell (Bio-Rad). It was operated for 2 h
at 20 V at 25 °C and 0.5 A in 48 mM Tris, 39 mM glycine, 20% (v/v) methanol, pH 9.2, containing 1.3 mM SDS. The Immobilon P membrane was coated with molten
MeltiLex wax, air-dried, and exposed to pre-flashed Hyperfilm-MP at
80 °C.
Western Blots
The Immobilon P membrane was first soaked in blocking solution
(1% (v/v) Tween 20, 10% (v/v) newborn calf serum, 13% (w/v) glycerol, 18% (w/v) D-glucose in PBS) for 1 h at room
temperature with shaking. This was followed by 1 h incubation in
blocking solution containing a rabbit polyclonal antibody raised
against GPI-PLC polypeptide (R18B3), at a 1:3000 dilution. The membrane was then washed three times (10 min each) with PBS followed by incubation for another hour with 1:1000 dilution of alkaline
phosphatase-conjugated goat anti-rabbit IgG. The PBS wash was repeated.
The membrane was then washed twice (10 min each) in alkaline
phosphatase reaction buffer (75 mM NaCl, 75 mM
Tris-HCl, pH 9.5, and 3.8 mM MgCl2). Antigens
were visualized in alkaline phosphatase reaction buffer, using
5-bromo-4-chloroindolyl phosphate and p-nitro blue
tetrazolium chloride as substrates.
Deacylation of GPI-PLC
Deacylation on Immobilon P Membrane--
Immunoadsorbed
3H-acylated GPI-PLC or proteins from
[3H]myristate-labeled T. brucei were resolved
by SDS-PAGE and transferred to Immobilon P. The membrane was soaked in
200 ml of one of the following solutions: 1 M
NH2OH (adjusted to pH 7 with 10 N NaOH); 0.2 M KOH; and 1 M Tris-HCl, pH 7. Each solution
was incubated at 30 °C with shaking. These solutions were replaced
three times (every 30 min), after which the membranes were incubated
overnight in 200 ml of the same solution. The membranes were air-dried, coated with MeltiLex wax, and radiolabeled proteins detected by fluorography.
In Gel Deacylation of GPI-PLC--
Immunopurified
[3H]GPI-PLC was electrophoresed on an SDS-PAGE. Gel
slices containing the radiolabeled polypeptide were excised, sliced,
and placed in a 1.5-ml microcentrifuge tube (approximate volume of gel
was 400 µl). After rinsing with 1 ml of deionized water,
[3H]GPI-PLC was deacylated in situ with 1 ml
of neutral hydroxylamine by incubation at 30 °C. At 20-h intervals,
the gel suspension was centrifuged at 14,000 × g for 1 min, and the supernatant was withdrawn. The incubation and
centrifugation steps were repeated five more times, and the
supernatants were pooled and dried in vacuo. Released
3H-fatty acids were identified as described below.
Deacylation of Native 3H-Acylated GPI-PLC and
3H-Myristoylated mfVSG--
A complex of
3H-acylated GPI-PLC (from 4 × 109 cells)
adsorbed to monoclonal antibody 2A6-6, which was in turn bound to
protein A-Sepharose (in 100 µl), was resuspended in 250 µl of 1 M NH2OH, pH 7.0. The suspension was incubated
at 30 °C for 20 min with shaking, followed by centrifugation at
14,000 × g for 1 min, and withdrawal of the
supernatant. Two more hydroxylamine treatments were performed. Eluates
were pooled, and 54 µl of concentrated HCl added, to acidify the
solution. [3H]mfVSG was deacylated to provide
cell-derived [3H]myristate as a control in the TLC
analysis. Approximately 300,000 dpm of purified protein was incubated
at 30 °C for 1 h with 900 µl of 0.2 M KOH, and
the solution was acidified with 20 µl of concentrated HCl.
3H-Fatty acids released from both [3H]GPI-PLC
and [3H]mfVSG were recovered into hexane by three
extractions, each with 500 µl of the solvent. The hexane phases for
each sample were pooled, back-extracted twice with 700 µl of 10 mM HCl, and dried under nitrogen gas. Methyl ester
derivatives of the fatty acids were generated by resuspending the dried
fatty acids in 200 µl of BF3-methanol (Supelco,
Bellefonte, PA) and heating for 2 min at 100 °C. Two hundred µl of
5 M NaCl, 0.5 M acetic acid was added, and the
fatty acid methyl esters (FAMES) extracted three times, each time with 200 µl of toluene. Toluene extracts were combined, dried under nitrogen gas, and resuspended in 10 µl of the solvent. Then,
radioactivity in a 2-µl portion was quantitated in a liquid
scintillation counter and the remaining 8 µl analyzed by reverse
phase-high performance thin layer chromatography (RP-HPTLC) (see
below). Radiolabeled FAMES were detected by fluorography.
Identification of Fatty Acids by Reverse-phase High Performance
Thin Layer Chromatography
Uniplate RPS-F silica gel (20 × 20 cm) were used with
chloroform:methanol:water (15:45:3, v/v/v) as the mobile phase (16). The developed HPTLC plate was air-dried, sprayed with
EN3HANCE, and FAMES detected by fluorography. Standard
FAMES of [3H]laurate, [3H]myristate,
[3H]palmitate, and [3H]stearate were
prepared from the corresponding free fatty acids as described above.
Deacylation of GPI-PLC and Determination of Phospholipase C
Activity
GPI-PLC (from 2 × 1010 cells of a hypotonic
lysate of T. brucei) was adsorbed to monoclonal antibody
2A6-6 (mc2A6-6) and washed as described above. The immunoadsorbent was
incubated in 5 ml of 1 M neutral hydroxylamine at 30 °C
for 40 min with shaking. One-ml aliquots (approximately 2 µg of
GPI-PLC) were withdrawn after 0, 10, 20, 30, and 40 min and centrifuged
at 14,000 × g for 1 min. Recovered beads were washed
three times in 3 ml of PBS. GPI-PLC was eluted from the column with 100 µl of 50 mM Tris-HCl, pH 12, containing 0.1% Nonidet
P-40, into an equal volume of chilled (0 °C) 1 M
Tris-HCl, pH 6.0, containing 1.0% Nonidet P-40 to neutralize the
elution buffer.
Enzyme activity in the eluate was assayed as follows. A reaction
mixture containing 2 µg of [3H]myristate-labeled VSG in
25 µl of AB (50 mM Tris-HCl, 5 mM EDTA, 1%
Nonidet P-40) was assembled on ice (in a 1.5-ml microcentrifuge tube).
To this mixture, several dilutions (in AB) of the eluate from the
mc2A6-6·protein A-Sepharose column was added (the objective being to
obtain values within the linear range of the assay (9)). This reaction
mixture was incubated at 37 °C for 30 min and terminated by vortex
mixing with 500 µl of water-saturated n-butanol (at room
temperature). Phases were separated by centrifugation (12,000 × g, 1 min, 25 °C), and enzyme activity was quantified by
measuring the amount of [3H]dimyristoylglycerol released
into the upper butanol phase (9).
Kinetic Analysis of Acylated and Deacylated GPI-PLC
To obtain GPI-PLC for kinetic analysis, the immunoadsorption
protocol was modified to reduce contamination of the purified enzyme
with antibody leaching from the monoclonal antibody column. GPI-PLC
(from 2.5 × 109 parasites) was adsorbed to a 100-µl
suspension of mc2A6-6 that had been chemically cross-linked to protein
A-Sepharose (9). The complex was treated with 1 M neutral
hydroxylamine or PBS (see above, "Deacylation of Native
3H-Acylated GPI-PLC and 3H-Myristoylated
mfVSG") for 40 min. GPI-PLC was eluted from the immune complex as
described above. Amount of GPI-PLC that cleaved approximately 20%
of [3H]myristate-labeled VSG was determined
empirically and used in the kinetic analysis.
Reaction mixtures were assembled on ice as described above with the
exception that the AB buffer contained 0.1% Nonidet P-40. VSG cleavage
was allowed to proceed for 15 min. Lineweaver-Burk plots were used to
determine the Michaelis constant (Km) (Table I). VSG
was assumed to be a dimer in the calculation of substrate
concentration. For purposes of determining the turnover number
(kcat), protein concentration of eluates from
the mc2A6-6 column was determined by quantitative Western blots using
known amounts of purified recombinant GPI-PLC as standards.
 |
RESULTS |
GPI-PLC Is Esterified with Myristic and Palmitic
Acids--
Metabolic labeling of T. brucei with
[3H]myristate (C14),
[3H]palmitate (C16), or
[3H]laurate (C12) led to incorporation of
radiolabel into GPI-PLC detected after SDS-PAGE (Fig.
1). This observation suggested that a
fatty acid might be covalently bound to GPI-PLC. Lipids are covalently
attached to proteins by one of three general mechanisms as follows: (i)
through an amide bond (e.g. N-acylation), (ii) by
esterification, as in S-palmitoylation; or (iii) in an ether linkage (e.g. S-isoprenylation). To define the
link between the acyl group and GPI-PLC, attempts were made at
deacylation after electrotransfer of purified 3H-acylated
GPI-PLC to Immobilon P membrane. Reagents tested were 0.2 M
KOH (pH ~13), which cleaves both oxyesters and thioesters, 1 M NH2OH, pH 7 (neutral hydroxylamine), which
can specifically cleave thioesters (17), and 1 M Tris-HCl,
pH 7, a control. Cleavage (by de-O-myristoylation) of a GPI
anchor (on VSG) was studied as a control for the effectiveness and
specificity of these reagents.

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Fig. 1.
[3H]Laurate,
[3H]myristate, and [3H]palmitate
metabolically label GPI-PLC. Bloodstream form trypanosomes
(109) were metabolically labeled either with
[3H]laurate (lane 1),
[3H]myristate (lane 2), or
[3H]palmitate (lane 3). GPI-PLC was
immunoprecipitated from a lysate of 5 × 108 cell eq
and analyzed by SDS-PAGE (14% minigel)/fluorography. Dried gels were
exposed to HyperfilmTM (Amersham Pharmacia Biotech) for 7 days (lanes 2 and 3) or 12 days
(lane 1).
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Neutral Tris-HCl failed to deacylate either VSG (Fig.
2, lane 1) or GPI-PLC (Fig. 2,
lane 2). Hydroxylamine, on the other hand, deacylated
GPI-PLC (Fig. 2, lane 6) but did not deacylate VSG (Fig. 2,
lane 5). These observations indicate that the acyl moiety on
GPI-PLC is present in a thioester link. Consistent with the specificity
of hydroxylamine the oxy-esterified [3H]myristate on VSG
was left intact, apparently. KOH deacylated GPI-PLC (Fig. 2, lane
4) confirming that the fatty acid was indeed esterified to
GPI-PLC. VSG was deacylated with KOH (Fig. 2, lane 3). In
another control experiment, neutral hydroxylamine did not release
[3H]myristate from purified
[3H]myristate-labeled VSG in aqueous solution (data not
presented).

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Fig. 2.
GPI-PLC is thioacylated. GPI-PLC
immunoprecipitated from [3H]myristate-labeled cells
(5 × 108) and total acylated membrane proteins
(107 cell eq) from T. brucei were resolved by
SDS-PAGE (14% minigel). The proteins were electro-transferred to
Immobilon P. The membranes were treated with 1 M Tris-HCl,
pH 7 (lanes 1 and 2), 0.2 M KOH (lanes 3 and 4), or
1 M NH2OH, pH 7 (lanes 5 and 6). Lanes 1, 3, and 5 contain total acylated membrane proteins, and lanes 2, 4, and 6 contain GPI-PLC immunoprecipitates.
HyperfilmTM (Amersham Pharmacia Biotech) was developed
after gels were exposed for either 18 h (lanes
1 and 5) or 7 days (lanes 2-4, and
6).
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Metabolic labeling of GPI-PLC with three fatty acids (i.e.
[9,10-3H]palmitate (C16),
[9,10-3H]myristate (C14), and
[11,12-3H]laurate (C12)) (Fig. 1) raised the
possibility that the enzyme was heterogeneously acylated. To address
this issue, the fatty acid on GPI-PLC was characterized. Cells were
metabolically labeled with [9,10-3H]myristate; GPI-PLC
was immunoadsorbed to protein A-Sepharose and deacylated with neutral
hydroxylamine. Methyl esters of the released fatty acids were generated
and analyzed by reversed phase-high performance thin layer
chromatography (RP-HPTLC)/fluorography (Fig.
3, lane 2). Using fatty acid
methyl ester standards (Fig. 3, lane 1), myristic and
palmitic acids were detected (Fig. 3, lane 2). The ratio of
the myristate:palmitate was 3:1, as determined by laser scanning
densitometry of the fluorographs. When the parasites were metabolically
labeled with [3H]palmitate, myristate and palmitate were
detected on GPI-PLC, although the efficiency of labeling the enzyme was
reduced significantly (data not presented). Together these data suggest
that T. brucei metabolizes fatty acids before their
incorporation into GPI-PLC.

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Fig. 3.
GPI-PLC is modified by both myristate and
palmitate. GPI-PLC was immunopurified from
[3H]myristate-labeled 4 × 109 T. brucei and deacylated with neutral hydroxylamine (see
"Deacylation of Native 3H-Acylated GPI-PLC and
3H-Myristoylated mfVSG" under "Experimental
Procedures"). Purified [3H]mfVSG was deacylated with
KOH. Lane 1, fatty acid methyl ester standards; lane
2, FAMES of fatty acids released from [3H]GPI-PLC;
lane 3, from [3H]mfVSG.
HyperfilmTM (Amersham Pharmacia Biotech) was developed
after plate was exposed for 2 weeks. The experiment described in
lane 2 has been repeated 15 times.
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Because this report is the first in the literature of myristate in a
thioester bond to an enzyme, a second approach was adopted to check
whether the assignment of one of the fatty acids on GPI-PLC as myristic
acid was in error. For this purpose, purified VSG was deacylated with
KOH, and the released fatty acid was converted to the methyl ester and
compared with the FAMES from lipids of GPI-PLC. The VSG fatty acids
were correctly identified as myristic acid (Fig. 3, lane 3)
(18). More importantly, the VSG fatty acid comigrated with the
myristate from GPI-PLC (Fig. 3, lane 2). Thus, the
assignment of the faster migrating fatty acid species in the GPI-PLC
sample as myristate is not an artifact caused by spurious migration of
a longer chain fatty acid on the HPTLC plate.
Co- and Post-translational Acylation of GPI-PLC--
To
investigate the temporal order of GPI-PLC acylation with regard to
protein synthesis, cycloheximide (CHX), an inhibitor of T. brucei protein synthesis, was used in experiments in which metabolic labeling with [3H]myristate was attempted
before or after addition of CHX. When the trypanosomes were labeled
with [3H]myristate prior to CHX treatment (CHX-II), the
inhibitor appeared to have no effect on acylation of GPI-PLC within
that time frame studied (Fig.
4A, compare lane
1 to lane 3). However, if the
parasites were treated with cycloheximide (CHX-I) prior to addition of
[3H]myristate, acylation of GPI-PLC was inhibited
partially (Fig. 4A, lane 2), compared with the control (Fig.
4A, lane 1). Approximately 50% of the lipid modification on
GPI-PLC (as estimated by laser scanning densitometry of fluorographs)
occurs in the absence of new protein synthesis. We infer that 50% of
the acylation occurred on nascent GPI-PLC, possibly on ribosomes. These
observations indicate that acylation of GPI-PLC occurs both
co-translationally and post-translationally.

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Fig. 4.
Acylation of GPI-PLC occurs co- and
post-translationally. Cells were preincubated with cycloheximide
(CHX-I) (lane 2) for 15 min prior to addition of
[3H]myristate (A and C) or
[35S]methionine (B). The parasites were
metabolically labeled for 1 h. Alternatively, cells were
metabolically labeled for 1 h, followed by addition of CHX
(CHX-II) (lane 3) for 15 min (37 °C). Control
cells (lane 1) were labeled for 1 h, washed, and
incubated in fresh labeling medium for 15 min (37 °C). Each lane
represents GPI-PLC immunoadsorbed from a lysate of 5 × 108 T. brucei. Dried gels were exposed to
HyperfilmTM (Amersham Pharmacia Biotech) for either 5 h (B) or 5 days (A). C, GPI-PLC was
detected by Western blotting with R18B3.
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In control experiments, the effect of CHX on the GPI-PLC polypeptide
backbone was studied in [35S]methionine-labeled T. brucei (Fig. 4B). Here, CHX blocked synthesis of the
enzyme if pre-incubated with the cells (Fig. 4B, lane 2), but no effect on GPI-PLC was apparent when added after metabolic labeling (Fig. 4B, lane 3). These observations confirm our
previous conclusion that a significant proportion of the
S-acylation of GPI-PLC occurs post-translationally. When a
gel identical to that depicted in Fig. 4A was analyzed by
Western blotting with an anti-GPI-PLC antibody, similar amounts of
GPI-PLC were detected in all three lanes (Fig. 4C). We
conclude that loss of 3H-lipid (Fig. 4A, lane 2)
was due to a decrease in fatty acid addition instead of protein degradation.
To investigate whether myristate or palmitate was preferentially added
to GPI-PLC post-translationally, trypanosomes were pretreated with CHX
(CHX-I) prior to metabolic labeling with [3H]myristate.
[3H]Acyl-GPI-PLC was immunoadsorbed and deacylated (see
"Experimental Procedures"). The released fatty acid(s) were
analyzed by RP-HPTLC/fluorography (see "Experimental Procedures")
(Fig. 5). Both myristate and palmitate were added to GPI-PLC post-translationally, but myristate addition dropped about 50% (Fig. 5, compare lanes 2 and
3). The ratio of myristate:palmitate (recovered from
GPI-PLC) decreased to 2:1 (i.e. from 3:1). As compared with
the control lane (Fig. 5, lane 2), the amount of palmitate
added post-translationally to GPI-PLC did not change. Apparently, most
of the post-translational acylation of GPI-PLC is due to myristate
incorporation. These experiments also ruled out the possibility that
the palmitate and myristate detected on GPI-PLC when the enzyme was
adsorbed to a mc2A6-6·protein A-Sepharose column (Fig. 3) originated
from contaminating phospholipid or VSG. Since immunopurified
[3H]GPI-PLC isolated from polyacrylamide gel slices
contained [3H]palmitate and
[3H]myristate (Fig. 5, lane 2), it is
clear that both fatty acids are covalently attached to GPI-PLC. The
ratio of [3H]myristate:[3H]palmitate on
GPI-PLC was again 3:1, as determined by laser scanning densitometry of
fluorographs from the HPTLC plates.

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Fig. 5.
Myristate and palmitate are incorporated
post-translationally into GPI-PLC. T. brucei (4 × 109) were metabolically labeled with
[3H]myristate. [3H]GPI-PLC immunoadsorbed
from the lysate was deacylated, as described under "In Gel
Deacylation of GPI-PLC" (see "Experimental Procedures").
Alternatively, T. brucei (1010) were pretreated
with cycloheximide before metabolic labeling with
[3H]myristate. [3H]GPI-PLC was
immunoprecipitated and deacylated as described under "Deacylation of
Native 3H-Acylated GPI-PLC and 3H-Myristoylated
mfVSG" (see "Experimental Procedures"). Methyl esters of the
fatty acids released were analyzed by RP-HPTLC. Lane 1,
fatty acid methyl ester standards; lane 2, methyl esters of
fatty acids in GPI-PLC from untreated parasites; lane 3, methyl esters of fatty acids from GPI-PLC in cycloheximide-treated
parasites. HyperfilmTM (Amersham Pharmacia Biotech) was
developed after the TLC plate was exposed for a month.
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Acylated GPI-PLC Is Enzymatically Active--
Lipid modification
has a potential for regulating the activity of GPI-PLC. For example,
acylation of the protein could inactivate the enzymatic activity.
Alternatively, deacylation might then be required to impart catalytic
activity to the polypeptide. These ideas were tested as described below.
GPI-PLC cleaves the [3H]myristate-labeled GPI of VSG
during hypotonic lysis of T. brucei, releasing the
radioactive lipid as di-[3H]myristoylglycerol (19). If
deacylation of GPI-PLC was necessary for enzyme activity, loss of the
fatty acid from GPI-PLC might be detectable under conditions where
cleavage of VSG is documented, that is loss of [3H]acyl
group from GPI-PLC might precede or be coincident with the loss of
[3H]myristate from the VSG GPI.
We attempted to determine whether any changes in the extent of GPI-PLC
acylation occurred during VSG cleavage. For this purpose, [3H]myristate-labeled trypanosomes were lysed
hypotonically and incubated either at 4 °C (Fig.
6B, lanes 1-4) or 37 °C
(Fig. 6B, lanes 5-8) in an attempt to initiate hydrolysis
of the VSG GPI. GPI-PLC cleaves approximately 70% of
[3H]myristate-labeled VSG during hypotonic lysis of
T. brucei (20). Residual VSG that is not cleaved initially
is the substrate of interest in these experiments because it is lumenal
(21, 22) and therefore inaccessible to GPI-PLC which is
cytoplasmically oriented (10, 12). Detergent is needed to promote
vesicle mixing which brings the enzyme and substrate together.

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Fig. 6.
Acyl-GPI-PLC is active. A,
T. brucei (109) were metabolically labeled with
[3H]myristate and lysed hypotonically. Cell lysates were
processed as illustrated. B, a membranous pellet of a lysate
from [3H]myristate-labeled cells was resuspended in lysis
buffer and incubated for 15 min either at 4 °C (lanes
1-4) or 37 °C (lanes 5-8). In
some experiments, Nonidet P-40 was added to 1% (final concentration)
before incubation (lanes 3, 4, 7, and
8). Lanes 1, 3, 5, and 7 depict immunoprecipitates of GPI-PLC (from 5 × 108
cell eq), and lanes 2, 4, 6, and 8 contain 107 cell eq of total proteins. SDS-PAGE (14%
minigel) was used to resolve the proteins, which were then detected by
fluorography. The dried gel was exposed to HyperfilmTM
(Amersham Pharmacia Biotech) for 5 days.
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Cleavage of VSG was monitored by loss of [3H]myristate
from the protein. Nonidet P-40 was added (to 1% final concentration) in some experiments (Fig. 6B, lanes 3, 4, 7, and
8; see Fig. 6A for outline of protocol) to
facilitate mixing of membrane vesicles. Immunoprecipitated
3H-acylated GPI-PLC (Fig. 6B, lanes
1, 3, 5, and 7) and
[3H]myristate-labeled VSG (Fig. 6B,
lanes 2, 4, 6, and 8) were monitored by
SDS-PAGE/fluorography.
Cleavage of VSG occurred at both 4 and 37 °C in the presence of
detergent (Fig. 6B, lanes 4 and
8). However, there was no discernible loss of the
[3H]acyl group from GPI-PLC (Fig. 6B, lanes 3 and 7). That loss of the [3H]myristate from
VSG (i.e. GPI cleavage) was not the result of degradation of
VSG was confirmed by Western blotting; full-length VSG was present in
the lysate after loss of the [3H]myristate (data not presented).
We conclude that deacylation is not required for GPI-PLC activity. In
support of this assertion, acylated GPI-PLC is catalytically more
proficient than the deacylated form of the enzyme (Table I). Thus, the possibility that a minute
fraction of unacylated GPI-PLC is responsible for cleavage of VSG in
these lysates is very remote.
Acylation of GPI-PLC Is Reversible in Vivo--
GPI-PLC in
T. brucei and recombinant GPI-PLC expressed in
Escherichia coli are both integral membrane proteins (6, 9), suggesting that membrane binding is not the result of an
eukaryote-specific chemical modification(s). Therefore, thioacylation
(which has been demonstrated only in eukaryotes) seemed unlikely to be
responsible for the ability of the enzyme to bind to membranes. Given
these circumstances, the possibility that thioacylation played a
regulatory role on the activity of the enzyme was entertained.
Regulatory modifications on proteins (e.g. phosphorylation)
are frequently reversible. Accordingly, we tested the possibility that
acylation of GPI-PLC was dynamic.
Acylation of T. brucei GPI-PLC was reversible. When
trypanosomes were labeled with [3H]myristate and then
"chased," lipid detected on the polypeptide decreased with a
half-life of 45 min (Fig. 7). After a
60-min "chase" only ~30% of the acylation signal was detectable
on GPI-PLC.

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Fig. 7.
Acylation of GPI-PLC is dynamic.
[9,10-3H]Myristate-labeled trypanosomes (7 × 109 cells) were resuspended in 14 ml of labeling medium and
incubated for 60 min (37 °C). Two-ml aliquots (109
cells) were harvested every 10 min, immunoprecipitated, and the eluted
GPI-PLC analyzed by SDS-PAGE/fluorography. Dried gels were exposed to
HyperfilmTM (Amersham Pharmacia Biotech) for 12 days.
Images were quantitated using an IS-1000 Digital imaging system. The
value obtained at t = 0 was assigned a value of
100%.
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Reversibility of the acylation reaction and the ability of T. brucei to add the lipids post-translationally indicate that thioacylation of GPI-PLC in T. brucei is dynamic.
Deacylation Reduces Activity of GPI-PLC--
An hypothesis that
thioacylation activated GPI-PLC was next tested, by determining whether
removal of the fatty acid from GPI-PLC would affect the kinetics of GPI
cleavage. For this purpose, a complex consisting of GPI-PLC
immunoadsorbed to protein A-Sepharose (i.e. a
GPI-PLC·mcAb2A6-6·protein A-Sepharose) (see "Experimental Procedures") was treated either with neutral NH2OH or PBS
(as a control) for varying lengths of time. GPI-PLC was then eluted from the column and its enzymatic activity assayed (9).
Deacylation of GPI-PLC under these conditions reduced activity of the
enzyme 4-fold (Fig. 8A). This
decrease in activity was not due to loss of GPI-PLC, since the
full-length polypeptide was detectable by Western blotting following
the hydroxylamine treatment (Fig. 8B). By following the loss
of the [3H] acyl signal from 3H-acylated
GPI-PLC during a similar deacylation experiment, a 4-fold decrease in
signal was obtained after 40 min of neutral hydroxylamine treatment
(Fig. 8C). There is a correlation between decrease in
acylation (Fig. 8C) and loss of enzyme activity (Fig. 8A), suggesting that 100% deacylation might lead to
complete loss of GPI-PLC activity. Exposure of GPI-PLC to
phosphate-buffered saline neither deacylated nor inhibited the enzyme
(data not presented). Finally, when recombinant GPI-PLC expressed in
E. coli was subjected to the above deacylation conditions,
no inhibition of enzyme activity was observed (data not presented).
This result is consistent with our inability to detect acylation of
GPI-PLC in the bacterium. Furthermore, the result indicates that the
deacylation conditions per se do not inhibit GPI-PLC.

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Fig. 8.
Deacylation reduces activity of
GPI-PLC. A, GPI-PLC was immunoprecipitated from 2 × 109 trypanosomes with 100 µl of mc2A6-6·protein
A-Sepharose complex, which was then treated with neutral hydroxylamine
(see "Deacylation of GPI-PLC and Determination of Phospholipase C
Activity" under "Experimental Procedures") for the indicated
periods. GPI-PLC was eluted, and activity of the enzyme was determined.
Activity obtained at time 0 was assigned a 100% value. B,
decrease in activity of GPI-PLC (A) is not due to loss of
the polypeptide. Western blot analysis was performed on the deacylated
samples using R18B3, a polyclonal anti-GPI-PLC antibody. Lane
1, purified GPI-PLC expressed in E. coli; lane
2, mc2A6-6 monoclonal antibody; lanes 3-6,
T. brucei GPI-PLC deacylated for 0, 10, 30, and 40 min,
respectively. C, time course for deacylation of
[3H]GPI-PLC. [3H]Myristate-labeled
GPI-PLC was deacylated with neutral hydroxylamine for the indicated
periods. Protein eluted from the complex was analyzed by
SDS-PAGE/fluorography.
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We conclude that deacylation reduces the enzymatic activity of GPI-PLC
significantly and infer that thioacylation activates the enzyme.
Turnover Number of GPI-PLC Is Reduced by Deacylation--
A
kinetic basis for the reduced rate of GPI cleavage by deacylated
GPI-PLC (Fig. 8A) was sought. Rate of the reaction could be
decreased by altering either kcat or
Km; therefore, both parameters were determined for
acylated GPI-PLC and the deacylated form of the enzyme (Table I).
During enzyme-catalyzed reactions, product formation is preceded by
assembly of an enzyme·substrate complex (E·S). At steady
state, the Michaelis constant (Km) equals
(k
1 + k2)/k1, where
k1 is the kinetic constant for formation of an
E·S complex. k
1 is the reaction
rate constant for breakdown of the E·S complex into free
enzyme (E) and substrate (S) (i.e. GPI-PLC and
VSG, respectively). k2 is the rate constant for
conversion of an E·S complex to product (P) and free
enzyme; k2 equals kcat
when k
1
k2.
Deacylation of GPI-PLC did not have a profound effect on VSG binding by
the enzyme, since Km was not altered significantly (Table I). However, when the maximal reaction velocity was corrected for amount of enzyme added (to obtain the turnover number,
kcat), the two enzymes had strikingly different
properties. Turnover number decreased 18-30-fold for deacylated
GPI-PLC, as compared with the acylated enzyme (Table I). The properties
of deacylated T. brucei GPI-PLC appear to be similar to
E. coli-expressed recombinant GPI-PLC which is not acylated
and whose turnover number was 10-fold less than GPI-PLC isolated from
T. brucei (15).
In essence, acylation alters the properties of the E·S
complex. The reduced reaction velocity of the deacylated enzyme is traceable to the diminished rate of converting the GPI-PLC·VSG complex to products. We surmise that thioacylation of GPI-PLC promotes
rapid conversion of an E·S complex to product (P) and free
enzyme (E).
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DISCUSSION |
Thiomyristoylation, a Novel Modification of a Phospholipase
C--
The use of myristate in thioacylation (i.e.
S-myristoylation) of GPI-PLC is unique. Frequently,
palmitate is the fatty acid attached to cysteines on proteins (reviewed
in Ref. 1). Myristate is typically linked to polypeptides through an
amide bond (N-acylation) (1). In T. brucei,
myristate is a rare fatty acid (23, 24), with most of it found on
mature GPIs of VSG (23). S-Myristoylation may confer
some unusual properties on GPI-PLC.
Thiomyristoylation in T. brucei has properties that appear
to be derived from two distinct types of lipid modifications, namely S-palmitoylation and N-acylation, found in animal
cells. First, similar to S-palmitoylation, cysteine is the
acceptor amino acid in GPI-PLC myristoylation. Second, the chemical
link between fatty acid and the protein is an ester bond. Third, a
significant proportion of the S-myristoylation occurs
post-translationally. Fourth, S-myristoylation is
reversible. Other features of S-myristoylation mimic
N-acylation as follows: 1) a fatty acid of short/medium
chain length is used, and 2) a large fraction of the myristate on
GPI-PLC is incorporated co-translationally, in sharp contrast to
S-palmitoylation.
Given that the parasites were originally labeled with myristate,
detection of palmitate on GPI-PLC was unexpected. The observation indicates that some myristate was elongated to palmitate prior to
incorporation into the enzyme. This conclusion is consistent with the
ability of T. brucei to convert myristate into palmitate and
stearate (23). Finally, as compared with the extent of myristoylation, the degree of palmitoylation of GPI-PLC could have been underestimated; [3H]palmitoyl-CoA, the presumed activated donor of the
fatty acid, could be competing with a large intracellular pool of
unlabeled palmitoyl-CoA for incorporation into GPI-PLC. Further work
will be needed to resolve this issue, since the relative levels of myristoyl-CoA and palmitoyl-CoA in T. brucei are unknown.
S-Acylation May Regulate Activity of Proteins Positively or
Negatively--
Thioacylation has the potential to modulate activity
of proteins in a positive or negative manner, much like
phosphorylation. Since deacylation reduces activity of GPI-PLC (Fig. 8
and Table I), the fatty acid(s) appear to be positive modulators of the activity of the enzyme. In contrast, S-palmitoylation of
Gz decreases its interaction with the corresponding
GTPase-activating protein (25), and active site cysteine acylation
inhibits activity of some mitochondrial enzymes (26). Hence, reversible
thioacylation could have a general role of positive and negative
control of protein function.
We speculate that GPI-PLC activity is controlled by reversible
thioacylation in T. brucei. In this model (Fig.
9), acylated GPI-PLC is presumed to be
the state in which most of the enzyme exists. Deacylation of the
GPI-PLC by a myristoylthioesterase, possibly in response to
extracellular signals, would suppress enzyme activity. Reacylation of
GPI-PLC by S-myristoyltransferase would restore optimal
activity to the phospholipase C. Endogenous regulators of T. brucei origin are not known yet, although some aminoglycoside
antibiotics (e.g. G418) can activate GPI-PLC in vitro (27). In addition, protein kinase C inhibitors promote cleavage of VSG by GPI-PLC (19). Whether these events can be tied into
a cycle of acylation/deacylation in vivo awaits
exploration.

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Fig. 9.
Proposed model for lipid regulation of
GPI-PLC. The activity of GPI-PLC may be controlled in
vivo by reversible acylation. The balance between the activity of
a myristoyltransferase and a myristoylthioesterase might determine
the predominant form of the enzyme. S-Myristoylated GPI-PLC
is the hyperactive form of the enzyme, whereas the deacylated form is
less active.
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Phosphatidylinositol-specific phospholipases C (PI-PLCs) act at the
interface between a lipid bilayer and the aqueous cytosol. In general,
these enzymes are soluble, only making transient contact with membranes
to hydrolyze their membrane-bound substrates, usually in response to
extracellular signals (reviewed in Ref. 28). Lipid modification could
be used by phosphatidylinositol-specific PLCs as a means of gaining
access to membranes. However, no acylation of a
phosphatidylinositol-specific PLC has been reported yet.
T. brucei GPI-PLC is the only eukaryotic PLC known to be
stably integrated into a lipid bilayer (6-8). Could modification with
lipid be responsible for the membrane association of this enzyme?
Current evidence suggests
otherwise.2 When expressed in
E. coli, GPI-PLC remains tightly associated with membranes
(15), but attempts to label the enzyme metabolically with fatty acids
have yielded negative results. Hence, membrane binding by GPI-PLC does
not appear to require an eukaryote-specific modification.