Originally published In Press as doi:10.1074/jbc.M001798200 on April 11, 2000
J. Biol. Chem., Vol. 275, Issue 25, 19334-19342, June 23, 2000
Tetramerization of Glycosylphosphatidylinositol-specific
Phospholipase C from Trypanosoma brucei*
Dora Abena
Armah and
Kojo
Mensa-Wilmot
From the Department of Cellular Biology, the University of Georgia,
Athens, Georgia 30602
Received for publication, March 3, 2000
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ABSTRACT |
Glycosylphosphatidylinositol-specific
phospholipase C (GPI-PLC) is an integral membrane protein in the
protozoan parasite Trypanosoma brucei. Enzyme activity
appears to be suppressed in T. brucei, although the
polypeptide is readily detectable. The basis for the apparent
quiescence of GPI-PLC is not known. Protein oligomerization was
investigated as a possible mechanism for post-translational regulation
of GPI-PLC activity. An equilibrium between monomers, dimers, and
tetramers of purified GPI-PLC was detected by molecular sieving and
shown to be perturbed with specific detergents. Homotetramers dominated
in Nonidet P-40, and dimers and monomers of GPI-PLC were the major
species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3.6-20-fold more active than the monomeric enzyme. Tetramer existence was confirmed
by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species in
T. brucei. Assembly of tetrameric GPI-PLC occurred when
parasites were subjected to conditions known to activate the enzyme. In Leishmania where heterologous expression of GPI-PLC causes
a GPI deficiency, the enzyme existed as a tetramer. Hence,
oligomerization of GPI-PLC is associated with high enzyme activity both
in vivo and in vitro.
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INTRODUCTION |
Trypanosoma brucei is the causative agent of sleeping
sickness in humans. In the bloodstream of a vertebrate host, the plasma membrane of the parasite is covered with a variant surface glycoprotein (VSG),1 a
glycosylphosphatidylinositol (GPI)-anchored molecule. These trypanosomes contain a GPI-specific phospholipase C (1-3) that can
cleave the VSG GPI, although the physiological function of the
enzyme remains unclear (4, 5). GPI-PLC is highly specific for GPIs
(kcat, 2.92 × 103
min
1; Km, 360 nM) (1, 6). Turnover of the enzyme-substrate complex is
regulated by thio-myristoylation and palmitoylation of the enzyme (7).
Efficient substrate recognition by GPI-PLC requires a
glucosaminylinositol moiety on the substrate (8).
GPI-PLC activity is detectable only in developmental stages of the
parasite where VSG is present. As revealed by immunoelectron microscopic analysis, GPI-PLC is associated with vesicular structures on the cytoplasmic side of intracellular membranes (9, 10). Little or
no cleaved VSG is released from healthy cells (11). While T. brucei divides every 6-8 h, the half-life of cell-associated VSG
is 32-34 h (11-13). Clearly, VSG is not released actively by GPI-PLC.
This situation is in sharp contrast to the phenotype of
Leishmania major or Trypanosoma cruzi that have
been stably transfected with a cDNA for the T. brucei
GPI-PLC (14, 15). In Leishmania promastigotes (extracellular
insect-stage form), gp63, the major GPI-anchored protein, is secreted
constitutively into the culture medium due to a GPI deficiency (15).
Replication of Leishmania amastigotes (the intracellular
mammalian stage) is severely inhibited by GPI-PLC expression (14). A
similar phenotype has been noted in T. cruzi where division
of the cell nucleus is blocked as a result of a GPI deficiency (16),
and differentiation of amastigotes to trypomastigotes is inhibited (17).
GPI intermediates (e.g. GlcN-PI and
Man1-3GlcN-PI) are found on the cytoplasmic side of the ER
membrane (18). These compounds appear to co-localize with GPI-PLC,
given the data from the in vivo transfection studies with
Leishmania and T. cruzi. Why then in T. brucei are these GPIs spared from cleavage by this potent
membrane-bound phospholipase C? It is estimated that there are 2.4 × 104 molecules of glycolipid A
(ethanolamine-phospho-Man3-GlcN-Ins-phospho-dimyrisotylglycerol), the prefabricated GPI anchor (19), and 3.5 × 104
molecules of GPI-PLC (3); this represents an excess of the enzyme over
the complete GPI anchor. With a turnover number
(kcat) of 2920 min1
(6), GPI-PLC could cleave all the glycolipid A inside T. brucei within seconds. Since the parasites remain capable of
adding GPIs to VSG, it seems clear that GPI-PLC is somehow prevented
from depleting T. brucei of GPI anchors. The absence of the
GPI-negative phenotype observed with transgenic T. cruzi and
L. major expressing GPI-PLC is indirect evidence for
regulation of the enzyme in T. brucei.
In lieu of efforts to understand factors that might control activity of
GPI-PLC in T. brucei, we characterized the native state of
the purified enzyme, and we explored possible contributions of
self-association to enzyme activity both in vivo and
in vitro. Our observations led us to propose a model for
post-translational regulation of GPI-PLC activity in vivo
where prevention of tetramerization might play an important role.
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EXPERIMENTAL PROCEDURES |
Cell Types/Strain
Monomorphic T. brucei strain 427 bloodstream form was
used in this work. Parasites were grown in rodents and harvested by chromatography on DE52 (20).
Materials
Superdex 75 HR10/30 column, Superdex 200 HR10/30 column, the
fast protein liquid chromatography system, and
[35S]methionine were from Amersham Pharmacia Biotech.
Lauryl dimethyl amine oxide (LDAO), Nonidet P-40 (protein grade), and
transferrin were purchased from Calbiochem. Thesit and aprotinin were
obtained from Roche Molecular Biochemicals. GelCode Blue,
disuccinimidyl suberate (DSS), dithiobissuccinimidyl propionate (DSP),
and m-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) were purchased from Pierce. Gel filtration standards were
purchased from Bio-Rad. Sodium deoxycholate (DOC), CHAPS, and all other
reagents were from Sigma.
Native Gel Electrophoresis of GPI-PLC
Purified recombinant GPI-PLC expressed in Escherichia
coli (1-2 µg) was preincubated in 20-30 µl of 37.5 mM Tris-HCl, pH 9.3, 0.5% Nonidet P-40, containing one of
these reagents as follows: 3-4 M urea, 100 mM
dithiothreitol (DTT), 1% Nonidet P-40, or 0.1-2% CHAPS at 27 °C
for 5 min. Samples were resolved by continuous glycine non-denaturing
polyacrylamide gel electrophoresis (pH 9.5; 5% minigel (Bio-Rad))
(21). To keep track of protein migration, 2 µl of 1% bromphenol blue
was added to each sample prior to loading. Gels were run at 16 A for
either 30 min or 4-5 h at room temperature. Proteins were visualized
with GelCode Blue (Pierce). Standards used were 
-lactalbumin (14.2 kDa, pI = 4-5), carbonic anhydrase (29 kDa, pI = 5.4-5.9),
ovalbumin (43 kDa, pI = 4.6), bovine serum albumin (66-kDa monomer
and 132-kDa dimer, pI = 4.7), and urease (272-kDa trimer and
545-kDa hexamer, pI = 5.0) (Sigma). Seven micrograms of marker
proteins were analyzed.
Gel Filtration Analysis of GPI-PLC
Molecular sieving was performed by fast protein liquid
chromatography on either Superdex 75 HR10/30 at 5 °C or Superdex 200 HR10/30 at 27 °C. For all runs, a 100-µl sample was loaded, and 500-µl fractions were collected at a flow rate of 1 ml/min. The void
volume of the columns was determined with blue dextran, using PBS (140 mM NaCl, 3 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4; pH 7.4) as running buffer. Prior to
loading each sample, the columns were equilibrated with 3 column
volumes of PBS containing the indicated amounts of detergent (see
figure legends). For each running buffer, the columns were calibrated
with thyroglobulin (670 kDa), IgG (160 kDa), transferrin (80 kDa),
ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). Protein peaks were monitored by
A280 absorption.
Recombinant GPI-PLC--
Purified GPI-PLC (620 ng) in 5 µl of
75 mM Tris-HCl, pH 9.3, containing 1% Nonidet P-40 (6),
was added to 95 µl of the indicated running buffer (see figure
legends) and incubated at 27 °C for 5 min before loading onto the
specified column. The elution profile of GPI-PLC was determined by
assaying 5 µl of each fraction for enzyme activity (22).
Enzyme (TbGPI-PLC) from a Crude Lysate of
Trypanosomes--
T. brucei (5 × 108
parasites) was lysed hypotonically in 1 ml of hypotonic lysis buffer
(10 mM sodium phosphate, 1 mM EDTA, pH 8)
containing a protease inhibitor mixture and centrifuged at 14,000 × g at 4 °C for 10 min (7). The membranous pellet obtained was solubilized with 500 µl of PBS containing either 1%
Nonidet P-40 or 1% CHAPS. An aliquot of each sample (107
cell equivalents for Nonidet P-40 and 108 cell equivalents
for CHAPS) was fractionated on the indicated gel filtration column.
Determination of GPI-Phospholipase C Activity
Two µg of SDS-depleted [3H]myristate-labeled
membrane-form variant surface glycoprotein was used as substrate in
buffer AB (50 mM Tris-HCl, pH 8.0, 5 mM EDTA,
1% Nonidet P-40). Reaction mixtures were incubated at 37 °C for 15 min, unless otherwise indicated. Cleaved
[3H]dimyristoylglycerol was quantitated by liquid
scintillation counting (6).
GPI-PLC Activity in Different Detergents
Detergent is needed for solubilization of GPI-PLC under all
conditions because it is an integral membrane protein. Replacement of
Nonidet P-40 in the purified recombinant protein was therefore achieved
by serial dilution into the detergent chosen to replace Nonidet P-40.
Purified enzyme (6.2 ng) in 5 µl of 75 mM Tris-HCl, pH
9.3, containing 1% Nonidet P-40 (6), was diluted 10-fold into buffer
AB lacking Nonidet P-40. A 5-µl aliquot of the diluted enzyme (620 pg) was diluted further into a new detergent by adding 45 µl of
buffer AB containing 1% of the detergent. To measure enzyme activity,
5 µl of each sample was assayed in 25 µl of the corresponding
buffer AB, in which the detergent being tested had replaced Nonidet
P-40. (Residual Nonidet P-40 is now 0.002%.) Enzyme reaction mixtures
were incubated for 15 min at either 5 or 27 °C.
Chemical Cross-linking
Recombinant GPI-PLC--
Purified GPI-PLC (124 ng/µl in 75 mM Tris-HCl, pH 9.3, 1% Nonidet P-40) was diluted 10-fold
into PBS to reduce the concentration of Nonidet P-40 and Tris. A
10-µl aliquot (124 ng) of the diluted enzyme was then placed in a
microcentrifuge tube containing 11.6 µl of deionized water and 3 µl
each of 10× PBS and 10% of the specified detergent. These actions
resulted in a 30-fold dilution of the original enzyme sample. After a
5-min preincubation at 2, 15, or 27 °C, cross-linking was initiated
by addition of 2.4 µl of 3.125 mM disuccinimidyl suberate
(DSS) in dimethyl sulfoxide (Me2SO), and the reactions were
incubated under one of these conditions as follows: 2 °C for 40 min,
15 °C for 40 min, or 27 °C for 10 min. The final concentration of
DSS was 250 µM. In control experiments, 2.4 µl of
Me2SO was added. To quench the reactions, 2.5 µl of 1 M Tris-HCl, pH 7.5, was introduced, and after 5 min
incubation on ice, 10 µl of 5× SDS-PAGE sample buffer added. Samples
were heated at 90 °C for 2-3 min. Proteins were resolved by
SDS-PAGE (12% minigel; Bio-Rad), and GPI-PLC was detected by Western
blotting (7).
In Vivo Cross-linking of T. brucei--
T. brucei(2 × 108 cells) was metabolically labeled with
[35S]methionine (7) and washed with PBS. Parasites were
resuspended in 396 µl of PBS. To initiate cross-linking, 4 µl of
100 mM DSS, DSP, or MBS
(m-maleimidobenzoyl-N-hydroxysuccinimide ester),
all membrane-permeable reagents, was added. In control experiments, 4 µl of Me2SO was introduced. Parasites were incubated at
27 °C for 30 min, after which reactions were quenched by the
addition of 100 µl of 1 M Tris-HCl, pH 7.5. Samples were
incubated on ice for 5 min followed by centrifugation at 14,000 × g for 10 min at 4 °C. The pelleted cells were lysed 1ml
of in 10 mM sodium phosphate, 1 mM EDTA, pH 8. A membranous pellet was recovered by centrifugation at 14,000 × g, 10 min at 4 °C, and solubilized in 1 ml of
immunoprecipitation dilution buffer ((1% v/v) Triton X-100, 200 mM NaCl, 60 mM Tris-HCl, pH 7.5, 6 mM EDTA, 10 units/ml aprotinin).
[35S]Methionine-labeled GPI-PLC was immunoadsorbed to
anti-GPI-PLC monoclonal antibody (mc2A6-6) (7). Radiolabeled proteins
were visualized by phosphorimaging (Personal Molecular Imager FX
(Bio-Rad)). Western blotting with anti-BiP antibody (23) was performed
as described (7) with 10 µl of the detergent-solubilized extract.
In Vivo Cross-linking of Leishmania--
Leishmania
(2.5 × 108 promastigotes) expressing T. brucei GPI-PLC (15) was metabolically labeled with
[35S]methionine (15) and washed with PBS. Parasites were
resuspended in 990 µl of PBS, pH 7.4. Cross-linking was initiated by
the addition of 10 µl of 100 mM MBS. Ten µl of
Me2SO was introduced in control experiments. Parasites were
incubated at 27 °C for 30 min. Reactions were quenched and analyzed
as described under "In Vivo Cross-linking of T. brucei."
Cross-linking of TbGPI-PLC after Hypotonic Lysis of T. brucei--
T. brucei was metabolically labeled with
[35S]methionine, and parasites (2.5 × 108) were lysed hypotonically (7). A membranous pellet
obtained by centrifugation (14,000 × g, 10 min,
4 °C) was resuspended in 450 µl of PBS. Cross-linking was
initiated by the addition of 50 µl of 2.5 mM DSS, or DSP,
followed by incubation at either 27 °C for 5 min or 2 °C for 40 min. In control experiments, 50 µl of Me2SO was added.
Reactions were quenched by the addition of 100 µl of 1 M
Tris-HCl, pH 7.5, followed by incubation on ice for 5 min. Samples were
centrifuged at 14,000 × g for 10 min at 4 °C. The
pellet was solubilized and analyzed as described under "In
Vivo Cross-linking of T. brucei."
Immunoprecipitation of GPI-PLC
Immunoadsorption of GPI-PLC to a monoclonal antibody was
performed as described previously (7).
SDS-PAGE, Western Blotting, and Fluorography
Recombinant GPI-PLC--
A 40-µl aliquot of the cross-linked
reaction mixture containing recombinant GPI-PLC (~120 ng) was
analyzed by Western blotting (7).
TbGPI-PLC--
[35S]Methionine-labeled GPI-PLC,
from intact cells or lysates, was immunoadsorbed to protein
A-Sepharose-mc2A6-6 beads (6) and eluted by heating at 90 °C for
2-3 min in 25 µl of 2.5× SDS-PAGE sample buffer. When preservation
of DSP cross-links was required, a modified 2.5× SDS-PAGE sample
buffer lacking Tris and 
-mercaptoethanol was used. Such samples were
only warmed at 37 °C for 5 min. Eluates were separated from the
Sepharose beads by centrifugation at 14,000 × g for 3 min at 27 °C. A 30-µl aliquot of each supernatant was subjected to
SDS-PAGE (12% minigel) and processed for phosphorimaging or
fluorographic detection with BioMaxTM MR film (Eastman
Kodak Co.).
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RESULTS |
CHAPS Alters Mobility of GPI-PLC during Native Gel
Electrophoresis--
GPI-PLC is a 39-kDa polypeptide (24). In native
gel electrophoresis, it was detected as a diffuse band that barely
entered a 5% minigel (without DTT or SDS) (Fig.
1A, lane 6). Absence of SDS
from the sample and running buffers may explain the diffuse nature of
protein bands in this experiment. Urea (up to 4 M) was added in an attempt to disrupt hydrogen bonds and dissociate what appeared to be a complex of unusual shape and/or size. The mobility of
GPI-PLC did not change (Fig. 1A, lane 7; Fig. 1B, lane
3). Likewise, pretreatment with DTT (100 mM) to reduce
disulfide bonds failed to alter mobility of GPI-PLC in the native gel
(Fig. 1B, lane 2). When the enzyme was treated
with both DTT (100 mM) and urea (4 M), a minor
change occurred in its migration (Fig. 1B, lane 4).
Treatment with 1% SDS caused GPI-PLC to run off the gel (data not
presented), most likely due to denaturation and net negative charge
introduced by the detergent.
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Fig. 1.
Native polyacrylamide gel analysis of
GPI-PLC. Purified GPI-PLC was subjected to non-denaturing gel
electrophoresis without SDS. A, short run, 7 µg each of
protein standards was analyzed: lane 1, -lactalbumin;
lane 2, carbonic anhydrase; lane 3, ovalbumin;
lane 4, bovine serum albumin; lane 5, urease.
GPI-PLC (~1 µg) was preincubated with either Nonidet P-40
(NP-40) or urea prior to analysis: lane 6, 1%
Nonidet P-40; lane 7, 1% Nonidet P-40 and 3 M
urea. Electrophoresis was for 30 min. B, long run, CHAPS
alters electrophoretic mobility of GPI-PLC. Lane 1, carbonic
anhydrase; lanes 2-5, GPI-PLC (~1 µg) preincubated with
several reagents: lane 2, 1% Nonidet P-40 and 100 mM DTT; lane 3, 4 M urea; lane
4, 1% Nonidet P-40, 100 mM DTT, and 4 M
urea; lane 5, 1% CHAPS. Electrophoresis was for 5 h.
C, optimization of CHAPS-induced changes in mobility of
GPI-PLC. Two µg of GPI-PLC was incubated with detergent prior to
electrophoresis. Lane 1, 1% Nonidet P-40; lane
2, 0.1% CHAPS; lane 3, 0.5% CHAPS; lane 4,
1% CHAPS; lane 5, 2% CHAPS. The gel was run for 4 h.
Protein standards ran off the gel. Proteins were detected with GelCode
blue. S, slow; F, fast;  .
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CHAPS caused a significant change in the mobility of GPI-PLC, resolving
it into two species (Fig. 1B, lane 5). A titration of the
detergent revealed that 0.5% was sufficient for optimal migration of
GPI-PLC (Fig. 1C, lanes 2-5). The major species of GPI-PLC
detected during electrophoresis in CHAPS is labeled F (Fig.
1C).
Two conclusions can be drawn from these observations. First, the size,
shape, or net charge of GPI-PLC can be modulated by CHAPS. (The
detergent has no net charge (Fig. 8).) Second, the quaternary structure
of GPI-PLC is not dependent on disulfide bonds.
Detergents Modulate the Size and/or Structure of GPI-PLC--
One
hypothesis to explain the altered mobility of GPI-PLC in the native gel
after addition of CHAPS was that the detergent changed the quaternary
structure of the enzyme. To examine this proposal, GPI-PLC was analyzed
by gel filtration in a buffer containing different detergents, some
with structures similar to CHAPS. A Superdex 75 HR10/30 column employed
for this purpose had a void volume of 9 ml (fraction 18) and a bed
volume of 24 ml (fraction 48). The experiment was performed at 5 °C.
Proteins of 100 kDa and higher were excluded from the column.
In 0.1% Nonidet P-40, the peak of GPI-PLC activity was in fraction 22, close to the elution position of an 80-kDa standard (Fig.
2A). The migration of enzyme
activity is consistent with that of a dimer (78 kDa). Twelve percent of
the enzyme activity was detected in the void volume (Fig.
2A), suggesting one of two possibilities. First, GPI-PLC
forms complexes (e.g. trimers) larger than 100 kDa.
Alternatively, GPI-PLC may be associated with micelles of the detergent
that have a molecular mass of 90 kDa such that a monomer-micelle
complex will have a mass of 130 kDa, causing it to be excluded from the
column. In 1% Nonidet P-40 GPI-PLC was predominantly dimeric (data not
shown).
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Fig. 2.
GPI-PLC is a tetramer in Nonidet P-40 but
monomeric in CHAPS. Purified GPI-PLC was preincubated in the
appropriate running buffer (see below) before loading onto the column.
A, GPI-PLC (620 ng) was analyzed in PBS containing Nonidet
P-40 (0.1%) or Thesit (1%) on Superdex 75 HR10/30 column at 5 °C.
B, GPI-PLC (620 ng) was analyzed in PBS containing CHAPS
(2%) on Superdex 75 HR10/30 column at 5 °C. C, GPI-PLC
(124 ng) was analyzed in PBS containing Nonidet P-40 (NP40)
(0.1%) on Superdex 200 HR10/30 column at 27 °C. D,
GPI-PLC (620 ng) was analyzed in PBS containing CHAPS (2%) on Superdex
200 HR10/30 column at 27 °C. A 5-µl portion of each fraction (500 µl) was assayed for enzyme activity with 3H-membrane-form
variant surface glycoprotein as substrate in buffer AB containing 1%
Nonidet P-40 ("Experimental Procedures"). No activity was detected
when CHAPS replaced Nonidet P-40 in the assay buffer. Migration
position of the standards, thyroglobulin (670 kDa), IgG (160 kDa),
transferrin (80 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) are indicated.
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To determine whether the hydrophilic head group or hydrophobic tail of
Nonidet P-40 contributed to dimerization of GPI-PLC, Thesit (65-68
kDa) whose hydrophilic head is identical to that of Nonidet P-40 was
tested. In 1% Thesit, most of GPI-PLC was a dimer and 29% of the
enzyme eluted in the void volume (Fig. 2A). More
importantly, detection of dimers suggest that the polymeric oxyethylene
((CH2-CH2-O)9-10) head (present in
both Nonidet P-40 and Thesit) may be sufficient to cause
oligomerization of purified GPI-PLC.
In CHAPS, the majority of the enzyme migrated with a molecular mass
greater than the 39-kDa monomer but less than the 80-kDa dimer (Fig.
2B). This finding is consistent with the assignment of a
47-kDa molecular size to the enzyme, after adsorption to a 7-kDa CHAPS
micelle (1). Only 23% of the total activity migrated as a dimer (Fig.
2B). About 13% of active enzyme eluted in fraction 19 (Fig.
2B), suggesting the presence of larger oligomers. Analysis of the enzyme in 2% LDAO produced similar results. The majority of
GPI-PLC behaved as monomer, with about 23% dimers and 11% of oligomers (data not presented).
To test the effects of temperature on interactions between GPI-PLC
monomers, gel filtration was performed at 27 °C. Initial observations indicated that the peak of enzyme activity was outside the
optimal separation range of Superdex 75 HR10/30 column (i.e. larger than 80 kDa) (data not presented). Therefore Superdex 200 HR10/30 with an optimal separation range up to 600 kDa was used. In
0.1% Nonidet P-40, the peak of enzyme activity was found in fraction
22, indicating that GPI-PLC was a tetramer (Fig. 2C). About
17% of the activity migrated as a larger oligomer in fraction 19 (Fig.
2C). In contrast, CHAPS (2%) converted GPI-PLC into
monomers and dimers predominantly (63% of activity) (Fig.
2D). Tetramers comprised 26% of the activity (Fig.
2D).
Taken together, these observations indicate that (i) GPI-PLC can
oligomerize, and (ii) temperature as well as detergents can influence
the distribution between monomers and oligomers of the enzyme. At
5 °C, CHAPS and LDAO promote formation and/or stabilization of
monomers, whereas Nonidet P-40 and Thesit enable detection of dimers.
At 27 °C, GPI-PLC is a tetramer in Nonidet P-40 but exists primarily
as dimers and monomers in CHAPS. Finally, from a comparison of the gel
filtration data with that obtained from the native gel analysis, it
seems likely that the slow species detected during native gel
electrophoresis in Nonidet P-40 (S, Fig. 1C)
represents the tetramer of GPI-PLC. The faster-moving species in CHAPS
(F, Fig. 1C) may correspond to dimers and monomers.
GPI-PLC Is Most Active in Its Tetrameric Form--
Capitalizing on
the ability of detergents and temperature to modulate self-association
of GPI-PLC, we investigated whether different oligomers of the enzyme
possessed varying activity. When an enzyme assay was performed in
Nonidet P-40 at 5 °C where GPI-PLC is predominantly dimeric (Fig.
2A), the activity was 1.7-fold higher than the monomer (Fig.
3) found in CHAPS (Fig. 2B).
This implies that dimeric GPI-PLC may be slightly more active than the
monomer.
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Fig. 3.
Stabilization/formation of tetrameric GPI-PLC
correlates with a 4-fold increase in enzyme activity. Equal
amounts of recombinant GPI-PLC were assayed in buffer AB containing 1%
of Nonidet P-40 (NP-40), CHAPS, Thesit, LDAO or DOC as
described under "GPI-PLC Activity in Different Detergents" (see
"Experimental Procedures"). Enzyme assays were performed for 15 min
at 5, 27, or 37° C.
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To compare the activity of dimeric GPI-PLC with that of the tetramer,
enzyme was assayed in Nonidet P-40 at 27 °C (Fig. 2C) and
compared with its activity in CHAPS at 27 °C (Fig. 2D).
Tetrameric GPI-PLC was 3.6-fold more active than dimers and monomers
(Fig. 3). An attempt to isolate tetramers generated by cross-linking for the purposes of activity determination was not feasible, since DSS,
DSP, and MBS were all found to inhibit the activity of the enzyme (data
not presented). Replacement of Nonidet P-40 with Thesit or LDAO reduced
GPI-PLC activity 2-4-fold at 5 °C (Fig. 3). Deoxycholate (1%)
completely inhibited GPI-PLC activity at both 5 and 27 °C (Fig. 3).
When the assay was performed at 37 °C in 1% of detergent, GPI-PLC
was 20-fold more active in Nonidet P-40 than in CHAPS, Thesit, LDAO, or
DOC (Fig. 3).
These observations indicate that GPI-PLC activity is increased under
conditions where tetramer formation or stabilization is enhanced.
Cross-linking of GPI-PLC--
Additional evidence for
self-association of GPI-PLC was obtained by chemical cross-linking.
Purified GPI-PLC migrates normally as a 39-kDa protein after
SDS-polyacrylamide gel electrophoresis (Fig.
4A, lane 1). Following
cross-linking with DSS, the major product formed was a doublet, most
likely tetramers and (possibly) pentamers (Fig. 4A, lanes
2-6). Interestingly, denaturation of GPI-PLC with SDS inhibits
cross-linking by DSS, leaving the monomer as the predominant species
(Fig. 4A, lanes 7 and 8). Aberrant (possibly
intramolecular) cross-linking might also have occurred in the presence
of SDS, since a ladder of bands with apparent molecular masses of 80 kDa and larger is visible (Fig. 4A, lanes 7 and
8).
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Fig. 4.
Native GPI-PLC can be cross-linked into
oligomers. A, GPI-PLC forms oligomers. Purified
recombinant GPI-PLC (~120 ng) was incubated at 27 °C for 10 min in
the presence of either Me2SO (lane 1) or 250 µM DSS (lanes 2-8). Reactions were performed
in the presence of detergents: lanes 1 and 2,
Nonidet P-40 (NP-40) (1%); lane 3, Thesit (1%);
lane 4, CHAPS (1%); lane 5, LDAO (1%);
lane 6, DOC (1%); lane 7, SDS (1%); lane
8, SDS (2%). GPI-PLC was detected by immunoblotting after
SDS-PAGE. B, dimers of GPI-PLC are detectable at lower
temperatures. GPI-PLC (120 ng) was incubated in 1% of the indicated
detergents for 40 min at 2 (lanes 1-3) or 15 °C
(lanes 4 and 5) in the presence of
Me2SO (lane 1) or 250 µM DSS (lanes 2-5). GPI-PLC was
detected by Western blotting. C, dimers are the major
product of cross-linking at 27 °C in excess Tris-HCl. GPI-PLC (1.24 µg) was diluted 10-fold into 50 mM Tris-HCl, pH 8, 5 mM EDTA. Ten microliters of the diluted solution was made
up to 30 µl with deionized water (i.e. final
concentrations of 16.7 mM Tris-HCl, pH 8, 1.7 mM EDTA) and 1% Nonidet P-40. After addition of DSS or MBS
(to a final concentration of 1 mM), the mixture was
incubated at 27 °C for 30 min, quenched, and analyzed
("Experimental Procedures"). Lane 1, Me2SO;
lane 2, DSS; lane 3, Me2SO; and
lane 4, MBS.
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To test whether intermediates between monomers and tetramers could be
detected, cross-linking was performed (i) at lower temperatures, or
(ii) in the presence of Tris (16.7 mM). At 2 °C, dimers
of GPI-PLC were detected in both Nonidet P-40 and CHAPS (Fig. 4B, lanes 2 and 3). In addition, a small proportion of
trimers was detected. The species of GPI-PLC marked with an
asterisk in Fig. 4B (lanes 2 and
3) could arise from intramolecular monomer cross-linking that produced a knotted molecule that migrates faster than a monomer. Some dimers were detected at 15 °C, but the major product was the
tetramer (Fig. 4B, lane 4). As observed previously,
tetramerization was inhibited by SDS (Fig. 4B, lane 5).
Finally, a minor product that failed to enter the stacking gel was
observed in all cases where tetramers were the major product (Fig. 4,
A and B).
Dimers were the major product when Tris was present during
cross-linking at 27 °C (Fig. 4C). Both DSS (Fig.
4C, lane 2) and MBS (Fig. 4C, lane 4) gave
similar results. Since approximately 50% of GPI-PLC remained as a
monomer, the accumulation of dimers may have resulted from reduced
efficiency of cross-linking.
These observations have three implications. First, monomers of purified
GPI-PLC can form tetramers that are detectable by chemical
cross-linking. Dimers accumulate only when cross-linking is
ineffective. Second, effective oligomerization requires the native
conformation of GPI-PLC, since SDS, which inhibits enzyme activity (8),
blocks tetramerization. Finally, Nonidet P-40, Thesit, CHAPS, LDAO, and
DOC are unlikely to denature GPI-PLC, even if they cause tetramers to
dissociate. Unlike SDS the detergents did not suppress tetramerization.
The oligomerization state of native GPI-PLC from T. brucei
was determined. GPI-PLC from detergent extracts of T. brucei
lysates (TbGPI-PLC) migrated during gel filtration at
5 °C on Superdex 75 HR10/30 column in Nonidet P-40 as a dimer;
in CHAPS, it was predominantly a monomer (Fig.
5). These results are in agreement with
data on the purified enzyme (Fig. 2). When the effect of self-association on activity of TbGPI-PLC was determined at
27° C, a 2-fold drop in activity occurred in CHAPS compared with
Nonidet P-40 (data not presented). This result suggests that oligomeric TbGPI-PLC is more active than the monomer. We conclude that
GPI-PLC produced in T. brucei can have its properties
altered by detergents in a manner similar to that obtained for E. coli-expressed GPI-PLC (22). Recombinant GPI-PLC is not modified
by lipid, whereas the enzyme expressed in T. brucei is
thio-myristoylated and palmitoylated (7). Hence, lipid modification is
unlikely to play a role in regulating oligomerization of GPI-PLC.
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Fig. 5.
Oligomeric properties of GPI-PLC solubilized
from T. brucei membranes. T. brucei
membranes from a hypotonic lysate were solubilized in PBS containing
1% of either Nonidet P-40 (NP-40) or CHAPS. An aliquot was
analyzed on Superdex 75 HR10/30 column at 5 °C in PBS containing the
solubilizing detergent. A 5-µl portion of each eluate was assayed for
GPI-PLC activity as described in the legend to Fig. 2.
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Quaternary Structure of GPI-PLC in Vivo--
Although GPI-PLC
polypeptide is detectable in bloodstream form T. brucei, the
enzyme appears to be largely inactive in vivo (see
Introduction). This fact raises the possibility that activation of the
enzyme involves post-translational mechanisms (e.g. covalent modifications or protein-protein interactions). This idea was pursued
by an investigation of the oligomeric state of GPI-PLC in vivo.
T. brucei were incubated with membrane-permeable reagents to
cross-link GPI-PLC to its nearest neighbors. Products were immunoadsorbed to an anti-GPI-PLC monoclonal antibody and analyzed (Fig. 6A).
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Fig. 6.
Oligomers of GPI-PLC are not detected in
living parasites. A, cross-linking of GPI-PLC in intact
T. brucei. [35S]Methionine-labeled T. brucei (2 × 108 intact parasites) were treated
with Me2SO (DMSO, lane 1),
DSS (lane 2), or DSP (lanes
3). Parasites were lysed hypotonically and solubilized, and
GPI-PLC was immunoadsorbed. GPI-PLC was detected by SDS-PAGE and
phosphorimaging. B, an aliquot of the membrane fraction of
T. brucei used in A was analyzed by Western
blotting with anti-BiP antibodies. DSP cross-links were either left
intact (lane 3) or reversed by heating in
-mercaptoethanol (-ME) (lane 4).
C, cross-linking of recombinant GPI-PLC in
Leishmania. [35S]Methionine-labeled
Leishmania (2.5 × 108 cells) were treated
with Me2SO (lane 1) or MBS (lane 2).
As a control, [35S]methionine-labeled intact T. brucei were treated with MBS (lane 3) or DSS
(lane 4). GPI-PLC was then analyzed as described for
A.
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In intact T. brucei the majority 80% (by PhosphorImager
quantitation) of GPI-PLC was monomeric (Fig. 6A, lanes
1-3). DSS (Fig. 6A, lane 2) and DSP (Fig. 6A,
lane 3) produced a high molecular weight complex, amounting to 5 and 11%, respectively, of the total GPI-PLC (Fig.
7B), which did not enter the
stacking gel.
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Fig. 7.
Tetramers of GPI-PLC are detected in a
hypotonic lysate of T. brucei. A,
cross-linking of TbGPI-PLC in membranes from a hypotonic lysate of
T. brucei. Parasites (2.5 × 108
[35S]methionine-labeled) were lysed hypotonically. The
lysate was treated with Me2SO (DMSO, lane
1), DSS (lane 2), or DSP
(lanes 3 and 4). DSP cross-links were
reversed with -mercaptoethanol (-ME)
(lane 4). GPI-PLC was adsorbed from the solubilized
membranes, resolved by SDS-PAGE, and the gel exposed to
BioMaxTM MR film at  80 °C for 2-3 days. B,
quantitation of tetramers and GPI-PLC complex (from A).
C, components of GPI-PLC complex and tetramers." DSP
cross-links were performed and analyzed as described for A
(except that 3 × 109 cell equivalents of lysate were
used). The bands corresponding to GPI-PLC complex or tetramers were
excised from an SDS-polyacrylamide gel, cut into pieces (100 µl
total), and an equal volume of 2.5× SDS-PAGE sample buffer containing
12.5% -mercaptoethanol added. The polyacrylamide pieces were heated
at 90 °C for 10 min. Radioactivity present in the sample buffer was
analyzed by SDS-PAGE (12% minigel)/phosphorimaging. Lane 1, gel-purified tetramers; and lane 2, gel-purified GPI-PLC
complex.
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As a control for the in vivo cross-linking experiments, the
lysates analyzed in Fig. 6A were examined by Western
blotting with antibody against the ER chaperone BiP (Fig.
6B). In the absence of cross-linker, a single 80-kDa band
corresponding to the size of monomeric BiP was detected (Fig. 6B,
lane 1). Higher molecular weight complexes containing BiP appeared
as a result of cross-linking with either DSS (Fig. 6B, lane
2) or DSP (Fig. 6B, lane 3). Since BiP binds to many
different proteins in the lumen of the ER in its role as a chaperone,
one does not expect to produce discrete bands from cross-linking of the
protein to its binding partners. The DSP-dependent
complexes were cleaved by -mercaptoethanol (Fig. 6B, lane
4), as expected. These data demonstrate that DSP traverses the
cytosol and enters the ER lumen in T. brucei to cross-link
full-length BiP. Therefore, if oligomers of GPI-PLC existed on the
cytosolic side of ER membranes in vivo, they would not have
been missed. Further evidence for this last assertion comes by way of
studies of the enzyme heterologously expressed in Leishmania
(15).
Leishmania lack a GPI-PLC-like activity. Interestingly, the
parasites become GPI-deficient when T. brucei GPI-PLC is
expressed in them (14, 15). Based on these observations, it was
hypothesized that GPI-PLC could be constitutively activated in that
parasite. To test whether assembly of GPI-PLC into oligomers, possibly
a contributory factor to activation of the enzyme, occurred in
Leishmania, the organization of GPI-PLC was determined.
Cross-linking of GPI-PLC was attempted in intact Leishmania.
MBS proved to be the best reagent in this trypanosomatid. Tetramers and
the so-called "GPI-PLC complex" comprised most of the enzyme in
Leishmania (Fig. 6C, compare lane 2 to
lane 1). By using MBS in T. brucei monomers were
the major species detected (Fig. 6C, lane 3; also see Fig. 6A). As compared with the monomers detected with DSS (Fig.
6C, lane 4), the MBS monomers migrated at a position
suggestive of a smaller size (compare lanes 3 and
4). We presume that the lower molecular weight species of
GPI-PLC arises from intramolecular cross-linking. Although this latter
species is detected in Leishmania, it is efficiently
cross-linked into oligomers in that cell.
Two conclusions may be drawn from the work with Leishmania.
First, the chemical probes are able to detect oligomers of GPI-PLC in
cells where multimers of the enzyme exist. That is, had there been a
significant proportion of tetramers in T. brucei, we would have detected them. Second, in Leishmania where GPI-PLC is
constitutively active against GPIs in vivo (14, 15),
tetramers and the GPI-PLC complex are the predominant form of the enzyme.
Activation of GPI-PLC Is Accompanied by Increased
Oligomerization--
Although GPI-PLC may be largely inactive against
GPIs in T. brucei (see Introduction), it cleaves VSG after
hypotonic lysis of T. brucei (1), implying that the enzyme
can be activated under such conditions (2, 3). The oligomerization
status of activated GPI-PLC was therefore examined by cross-linking
after hypotonic lysis (Fig. 7). By using DSS, a significant proportion of GPI-PLC (33% of the total GPI-PLC, from densitometric analysis) was
detected as an aggregate with mobility corresponding to a tetrameric
complex (Fig. 7A, lanes 1 and 2).
Residual monomers and a larger complex (the "GPI-PLC complex") were
also observed (Fig. 7A, lane 2). A proportion of
protein, possibly derived from intramolecular cross-linking and
migrating faster than the monomer, was detectable. In the presence of
DSP, the major product was the GPI-PLC complex (Fig. 7A, lane
3).
The relative proportion of enzyme in tetramers and in the "GPI-PLC
complex" from intact parasites was determined. Similar quantitation
was performed for enzyme present in the hypotonic lysates, and ratios
of the two were obtained. In DSS cross-linking the quantity of
tetrameric GPI-PLC increased 2.3-fold upon activation of the enzyme.
The fraction in the GPI-PLC complex rose 3-fold (Fig. 7B).
When DSP was used, an 8-fold increase in the amount of the GPI-PLC
complex was observed after activation (Fig. 7B). Concurrently, the proportion of GPI-PLC in tetramers doubled (Fig. 7B). Similar proportions of products were obtained when
cross-linking was performed at 2 °C (data not presented).
Constituents of the GPI-PLC complex and the "tetramer" (Fig.
7A) were examined (Fig. 7C). For this purpose,
the two complexes were generated with DSP, resolved by and obtained
from a polyacrylamide gel. Cross-links were cleaved in-gel with
-mercaptoethanol, and the components of each complex were examined
after SDS-PAGE and phosphorimaging (Fig. 7C). From the
tetramer, monomeric GPI-PLC was the major protein released (Fig.
7C, lane 1). The GPI-PLC complex contained three
major polypeptides of 160, 117, and 55 kDa (marked with
asterisks) (Fig. 7C, lane 2) in
addition to GPI-PLC. Since these polypeptides are not associated with
GPI-PLC prior to activation (Fig. 6A), we hypothesize that
they are recruited into proximity of GPI-PLC during or after activation
of the phospholipase C.
In summary, monomers of GPI-PLC predominate in "quiescent" T. brucei. Following activation of the enzyme, GPI-PLC is found either in a tetrameric complex or in association with other proteins in
a very large complex. Thus, activation of GPI-PLC both in
vitro and in vivo entails increased protein-protein
interactions of which tetramerization is a central part.
| |
DISCUSSION |
Monomer-Oligomer Equilibrium of GPI-PLC May Be Modulated by
Detergents--
GPI-PLC is an integral membrane protein (1, 22).
Consequently, in most biochemical studies a detergent is needed to keep the enzyme solubilized. Although widely used to study membrane proteins, secondary effects of detergents frequently go unrecognized. In this work, we show that several "mild" detergents can perturb the equilibrium of an integral membrane protein between different oligomeric forms, presumably by inserting between dimerization interfaces of the subunits. This observation has been exploited to
study aspects of GPI-PLC biochemistry that were not amenable to
investigation otherwise.
By gel filtration analysis, GPI-PLC can be fractionated into various
oligomeric species in a detergent- and
temperature-dependent manner (Fig. 2). GPI-PLC forms
tetramers at 27 °C but exists as dimers and monomers at 5 °C
(Fig. 2). These data suggest that hydrophobic interactions play a role
in oligomerization of GPI-PLC, since hydrophobic interactions are
facilitated by increased temperature. Activity of the enzyme was
highest when tetramers were the predominant form of the enzyme (Figs. 2
and 3). Loss of GPI-PLC activity after chemical cross-linking (in a
failed bid to obtain active tetramers) may have been due to reaction of
the reagents with residues in or close to the active site of the
enzyme. Recent experiments indicated that a modification of Cys-80 and
Gln-81 inhibit activity of GPI-PLC (25).
All detergents that converted GPI-PLC into monomers (e.g.
CHAPS, LDAO, and DOC) (Fig. 2) strongly inhibited enzyme activity (Fig.
3). This observation rules out the contention that the major effect of
the detergents is allosteric. Instead, production and/or stabilization
of monomers may suffice to inhibit GPI-PLC activity.
As monitored by chemical cross-linking, only native GPI-PLC forms
tetramers. The protein loses its ability for efficient tetramerization when denatured with SDS (Fig. 4A), a potent inhibitor of
enzyme activity (8). Although tetramers were the predominant product when cross-linking was performed at 15 °C and above (Fig.
4B), dimers were found when cross-linking was performed at
2 °C (Fig. 4B) in both CHAPS and Nonidet P-40. Detection
of dimers at 2 °C (Fig. 4B) but not at 27 °C (Fig.
4A) could be due to (i) a slower rate of cross-linking
and/or (ii) stabilization of GPI-PLC dimers at the lower temperature.
The former possibility is supported by the demonstration of dimers in
the presence of Tris (Fig. 4C), whereas the latter is
substantiated by the stabilization of dimers in Nonidet P-40 at 5 °C
(Fig. 2A) compared with tetramers at 27 °C (Fig.
2C).
CHAPS increased the migration of GPI-PLC in a native gel (Fig. 1,
B and C). Two explanations may account for this
phenomenon. First, CHAPS might alter the quaternary structure by
interacting with the oligomerization interfaces. Second, a positive
charge on CHAPS (see Fig. 8) may form a
salt bridge with an acidic residue on the enzyme. This would leave an
extra negative charge on the GPI-PLC·CHAPS complex (i.e.
per molecule of detergent bound) that causes the enzyme to migrate
faster toward the cathode. (Since GPI-PLC is a basic protein with an
isoelectric point of 8 (22), introduction of the new negative charge
may cause a significant change in net charge.) Although data from the
gel filtration experiments (Fig. 2B) supports the first
hypothesis, these two explanations are not mutually exclusive.
Oligomerization May Trigger Activation of GPI-PLC in
Vivo--
Most GPI-PLC molecules in intact trypanosomes remain
monomeric (Fig. 6A), unlike the purified enzyme that is
predominantly tetrameric (Figs. 2C and 4A). Since
monomers of purified GPI-PLC are less active than oligomers (Fig. 3),
we speculate that prevention of tetramerization in T. brucei
is one of the mechanisms by which the activity of GPI-PLC is held in
check (Fig. 9). This action would avert
excessive cleavage of GPIs in T. brucei. In line with our
hypothesis, activation of GPI-PLC is associated with assembly of the
enzyme into tetramers and a larger complex in T. brucei (Fig. 7C). In Leishmania where heterologously
expressed GPI-PLC is constitutively active, the enzyme exists
predominantly as an oligomer (Fig. 6C). Thus, both in
vivo and in vitro, the quaternary structure of GPI-PLC
has a striking effect on enzyme activity (Fig. 9).
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Fig. 9.
Proposed model for post-translational
regulation of GPI-PLC activity. In T. brucei, GPI-PLC
appears to be enzymatically quiescent. Under these conditions the
enzyme exists predominantly as a monomer. Upon activation, as occurs
during or after hypotonic lysis, GPI-PLC forms homotetramers and
hetero-oligomers (i.e. GPI-PLC complex). In
vitro, detergent-solubilized GPI-PLC exists as monomers, dimers,
or tetramers. The equilibrium between these species can be shifted with
detergents. In CHAPS monomers and dimers (the less active forms) are
the major species. In Nonidet P-40, tetramers (the activated form of
the enzyme) are the predominant species. Enzyme that is inactive in
CHAPS can be activated by replacing the detergent with Nonidet P-40.
When GPI-PLC is expressed in Leishmania, the enzyme is
active in vivo and is found to exist predominantly as
tetramers. We propose that in healthy T. brucei GPI-PLC is
maintained predominantly in its monomeric state, in order to prevent
the enzyme from cleaving GPIs.
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|
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant AI 33383 (to K. M.-W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Cellular Biology, the University of Georgia, 724 Biological Sciences Bldg., Athens, GA 30602. Tel.: 706-542-3355; Fax: 706-542-4271; E-mail:
mensawil@cb.uga.edu.
Published, JBC Papers in Press, April 11, 2000, DOI 10.1074/jbc.M001798200
| |
ABBREVIATIONS |
The abbreviations used are:
VSG, variant surface
glycoprotein;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
Me2SO, dimethyl sulfoxide;
DOC, deoxycholate;
DSS, disuccinimidyl suberate;
DSP, dithiobis-succinimidyl propionate;
DTT, dithiothreitol;
LDAO, lauryl dimethyl amine oxide;
MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester;
PBS, phosphate-buffered saline;
GPI-PLC, glycosylphosphatidylinositol-specific phospholipase C;
TbGPI-PLC, Trypanosoma brucei GPI-PLC;
BiP, binding protein;
ER, endoplasmic reticulum;
PAGE, polyacrylamide gel
electrophoresis;
Man, mannose;
GlcN, glucosamine;
PI, phosphatidylinositol.
| |
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ABSTRACT
INTRODUCTION
