From the Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Received for publication, January 19, 2003, and in revised form, February 10, 2003
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ABSTRACT |
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Many eukaryotic proteins are tethered to the
plasma membrane via glycosylphosphatidylinositol (GPI). GPI
transamidase is localized in the endoplasmic reticulum and mediates
post-translational transfer of preformed GPI to proteins bearing a
carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is
a multimeric complex consisting of at least five subunits. Here we
report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T,
form a functionally important disulfide bond between conserved cysteine
residues. GPI8 and PIG-T mutants in which relevant cysteines were
replaced with serines were unable to fully restore the surface
expression of GPI-anchored proteins upon transfection into their
respective mutant cells. Microsomal membranes of these transfectants
had markedly decreased activities in an in vitro
transamidase assay. The formation of this disulfide bond is not
essential but required for full transamidase activity. Antibodies
against GPI8 and PIG-T revealed that endogenous as well as exogenous
proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a
similar intermolecular disulfide bond via its conserved cysteine
residue, suggesting that the trypanosome GPI transamidase is also a
multimeric complex likely containing the orthologue of PIG-T. We also
demonstrate that an inactive human GPI transamidase complex that
consists of non-functional GPI8 and four other components was
co-purified with the proform of substrate proteins, indicating that
these five components are sufficient to hold the substrate proteins.
Many eukaryotic cell surface proteins are tethered to the
plasma membrane via glycosylphosphatidylinositol
(GPI).1 The GPI moiety is
synthesized in the endoplasmic reticulum (ER) by the sequential
addition of sugars and ethanolamine phosphate to phosphatidylinositol
(1, 2). In addition to the amino-terminal signal peptide required for
entry into the ER, precursor proteins destined to be GPI-anchored have
carboxyl-terminal GPI attachment signals that direct GPI modification.
GPI transamidase mediates cleavage of the GPI attachment signal peptide
and en bloc transfer of a preassembled GPI to newly exposed
carboxyl terminus of precursor proteins. During this transamidation
reaction, GPI transamidase forms a carbonyl intermediate with substrate
proteins (1).
GPI anchoring is essential for mammalian development and many specific
cellular functions (3-6) but not for cell survival itself. In
Saccharomyces cerevisiae, GPI is essential for growth (7).
The requirement of GPI in protozoan parasites depends on the species
and stage of life cycle (8, 9). For example, the bloodstream form of
Trypanosome brucei requires GPI; in contrast the
GPI-deficient procyclic form (insect stage) of this parasite is viable
but has decreased infectivity toward its vector, tsetse fly (9, 10).
Thus selective inhibitors of GPI synthesis could be potent therapeutic
drugs for diseases caused by these microorganisms (11), and such drugs
could utilize the differential substrate specificity of enzymes
mediating GPI synthesis and anchoring. It is, therefore, important to
characterize human and parasitic GPI transamidases and investigate
molecular mechanisms conferring specificity.
Human GPI transamidase is a complex consisting of at least five
components, GAA1, GPI8, PIG-S, PIG-T, and PIG-U (12, 13). S. cerevisiae transamidase is composed of the respective orthologues Gaa1p, Gpi8p, Gpi17p, Gpi16p, and Cdc91p (12-14). All these components are essential for the formation of carbonyl intermediates (12, 13, 15).
Several lines of evidence indicate that GPI8/Gpi8p are catalytic
components. (i) They belong to the C13 cysteine peptidase family (16),
one of which, Canavalia ensiformis asparaginyl endopeptidase, catalyzes a transamidation reaction in vitro
(17). (ii) Mutation of cysteine and histidine residues proposed to form a catalytic dyad resulted in complete loss of function (18, 19) and
sensitivity to sulfhydryl-alkylating reagents (20, 21). (iii) The
recombinant GPI8 of T. brucei cleaved a small peptide-based
fluorescent substrate (22). (iv) Finally in vitro translated
model substrate proteins can be cross-linked to GPI8 indicating that
they are situated in close proximity to each other during the
transamidation reaction (23, 24). GPI8 homologues are divided into two
groups, type I transmembrane proteins (human, S. cerevisiae,
and Schizosaccharomyces pombe) and soluble proteins lacking
a corresponding transmembrane region (T. brucei,
Leishmania mexicana, Drosophila melanogaster, and
Caenorhabditis elegans). A human GPI8 mutant lacking its
transmembrane region seemed to be integrated into the complex correctly
because it fully rescued GPI8 mutant cells and was
co-precipitated with GAA1 (18). We have proposed that PIG-T/Gpi16p has
a central role in the formation of the transamidase complex because
stable expression of GPI8/Gpi8p exclusively depends on PIG-T/Gpi16p
(12, 14).
Here we demonstrate that GPI8 and PIG-T form a disulfide bridge that is
required for normal transamidase activity via conserved cysteine
residues not only in mammalian cells but also in T. brucei and provide direct evidence that a transamidase complex consisting of
five components associates with translocated substrate proteins.
Cell Lines--
K562, class K (15), PIG-T knockout
F9 (12), and HeLa cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. Chinese
hamster ovary (CHO) cells were cultured in Ham's F-12 supplemented
with 10% fetal calf serum.
Mammalian Expression Plasmids and Selection of
Transfectants--
All expression plasmids were constructed on the
pME18Sf vector, a gift from Dr. K. Maruyama (Tokyo Medical and Dental
University, Tokyo, Japan). Phosphoglycerokinase-driven puromycin,
hygromycin, and blasticidin resistance gene cassettes (18) were cloned
into a unique HindIII site of the pME18Sf vector (designated
as pMEpuro, pMEhyg, and pMEbsd, respectively) and used to establish
stable transfectants. Plasmids for FLAG-GAA1, GST-GPI8, HA-PIG-S, and Myc-PIG-T were used previously (12). The amino- and carboxyl-terminal FLAG and GST tandem (FG) tags used were described previously (12) with
the addition of a single and triple flexible linker
(Gly-Gly-Gly-Gly-Ser) between FLAG and GST as well as between GST and
the tagged protein, respectively, to improve the yield of affinity
purification. Cysteine-to-serine mutants of GPI8 and PIG-T were made
with a QuikChange site-directed mutagenesis kit (Stratagene). Sequences
of all primers used in this study are available upon request. K562 and
class K cells were transfected with pMEpuro-derived plasmids and
selected at 2 µg/ml puromycin. PIG-T knockout F9 cells
were transfected with pMEbsd-CD59. After selection at 4 µg/ml
blasticidin, a clone with high transfection efficiency and high
PIG-T-dependent surface expression of CD59 was isolated.
This clone was further transfected with pMEhyg-PIG-T plasmids
and selected at 500 µg/ml hygromycin.
Purification of Transamidase Complex and Identification of
Associated Proteins--
Affinity purification of the transamidase
complex with anti-FLAG beads (Sigma) and glutathione beads (Amersham
Biosciences) was performed as reported previously (12) except that
SDS-PAGE sample buffer contained 8 M urea to improve
denaturation of PIG-U (13) and 10 mM iodoacetamide for
non-reducing conditions to prevent disulfide rearrangements. The
amino-terminal sequence was determined from Coomassie-stained bands
transferred to a polyvinylidene difluoride membrane with a G1005A
Hewlett-Packard Protein Sequencing System. The bands excised from the
SDS-polyacrylamide gel, which were stained with SYPRO Ruby (Molecular
Probes) or a mass spectrometry-compatible SilverQuest silver staining
kit (Invitrogen), were subjected to in-gel digestion by trypsin or
lysylendopeptidase, analyzed by liquid chromatography and tandem mass
spectrometry with MAGIC 2002 (Michrom Bioresources) and Q-Tof2
(Micromass), and identified by Mascot search (Matrix Science).
Analysis of Protein Complexes--
To analyze the formation of
the disulfide bond between GPI8 and PIG-T, cells were pretreated in
phosphate-buffered saline containing 20 mM
N-ethylmaleimide (NEM) for 30 min on ice and lysed in
Nonidet P-40 lysis buffer (20 mM Tris, 140 mM
NaCl, 1% Nonidet P40, pH 7.4) containing 20 mM NEM. CHO
cells were electroporated with 5 µg each of the indicated plasmids as
reported previously (12). GST-tagged GPI8 was precipitated with
glutathione beads. The precipitates were divided into four aliquots and
Western blotted with anti-FLAG M2 (Sigma), anti-GST (Amersham
Biosciences), anti-Myc (Oncogene Research Products), anti-HA
(Roche Molecular Biochemicals), and horse radish peroxidase-conjugated
protein G (Bio-Rad). Cell lysates of class K cells transfected with
FG-GPI8 constructs and PIG-T knockout cells transfected with
Myc-PIG-T constructs were subjected to precipitation with glutathione
beads and anti-Myc antibody plus protein G beads (Amersham
Biosciences), respectively. Whole cell lysates were prepared as
follows. After treatment with NEM, cell pellets were solubilized in
lysis buffer with NEM at 2 × 107cells/ml for
K562 and HeLa or 4 × 107 cells/ml for class K cells.
Postnuclear lysates were mixed with an equal volume of SDS sample
buffer with iodoacetamide.
In Vitro Translation--
For in vitro translation of
human GPI8, 1.2-kb NcoI-XbaI fragments of
full-length wild-type and C92S mutant GPI8 cDNA were cloned in NcoI and XbaI sites of pSPUTK
(Stratagene). Plasmids were linearized with EcoRI and used
as templates for in vitro transcription. Capped mRNA
synthesized using the AmpliScribe SP6 High Yield Transcription Kit
(Epicentre) and RNA cap structure analog (New England Biolabs) was
in vitro translated using Flexi rabbit reticulocyte lysate
(Promega) and class K microsomal membrane for 90 min in the presence or
absence of 2 mM dithiothreitol in 25 µl according to the
manufacturer's protocol. After incubation, 20 µl of the reaction
mixture was diluted 10-fold with a buffer (50 mM
triethanolamine, 250 mM sucrose, 20 mM NEM, pH
7.5), layered on, and sedimented through 200 µl of a cushion (50 mM triethanolamine, 500 mM sucrose, 20 mM NEM, pH 7.5) by ultracentrifugation with a Beckman
TLA-100.2 rotor at 80,000 rpm for 15 min. Membrane pellets were washed
with phosphate-buffered saline containing 20 mM NEM and
solubilized in sample buffer with iodoacetamide. To reduce the sample,
one-fifth volume of 2-mercaptoethanol was added. For direct analysis by
SDS-PAGE, 5 µl of each reaction was mixed with 15 µl of sample
buffer with iodoacetamide or 2-mercaptoethanol immediately after the
reaction. The in vitro GPI transamidase assay using miniPLAP
(placental alkaline phosphatase) originally developed by Kodukula
et al. (25) was performed as described previously
(18).
Flow Cytometric Analysis--
Cells were stained with
biotinylated anti-CD59 5H8 followed by
phycoerythrin-conjugated streptavidin (Biomeda) and analyzed on a
FACScaliber (BD Biosciences).
Polyclonal Antibodies against Human GPI8 and PIG-T
Proteins--
We immunized rabbits with affinity-purified,
carboxyl-terminally His-tagged human GPI8
(Ser28-Ser264) and human PIG-T
(Ile308-Leu578). Human GPI8
(Ser28-Ser264) and human PIG-T
(Leu22-Leu578), respectively, were expressed
as maltose-binding protein (MBP) fusion proteins and coupled to HiTrap
N-hydroxysuccinimide-activated HP columns (Amersham
Biosciences). An IgG fraction of antisera was isolated with
HiTrap rProtein A FF columns (Amersham Biosciences), preadsorbed with
MBP-coupled columns, and finally affinity-purified with MBP-GPI8 or
MBP-PIG-T columns.
Trypanosome Manipulation--
We disrupted trypanosomal
GPI8 (TbGPI8) gene in procyclic form of
T. brucei as described previously (9). An episomal plasmid, pTMCSzeo, was constructed by replacing the neomycin resistance gene
with a bleomycin resistance gene and inserting a multiple cloning site
(nine restriction enzyme sites) flanked by aldolase splice acceptor
signal and polyadenylation signal into a unique SmaI site of
pT11-bs (26). A 1-kb fragment encoding FLAG-tagged wild-type or C76S
mutant TbGPI8 was amplified by PCR and cloned into KpnI and
BamHI sites of pTMCSzeo. TbGPI8 knockout
procyclics were transfected with the resulting plasmids by
electroporation and selected at 2-10 µg/ml phleomycin. Transfectants
were stained with monoclonal anti-EP-procyclin (Cedarlane
Laboratories) plus fluorescein isothiocyanate-labeled secondary
antibody and analyzed in a FACScaliber. For immunoprecipitation, cells
of a confluent 10-ml culture (~108) were pretreated,
lysed in 1 ml of lysis buffer with NEM, and subjected to
immunoprecipitation with anti-FLAG beads.
GPI8 and PIG-T Are Linked via a Disulfide Bond--
We previously
reported that GPI transamidase components GAA1, PIG-S, PIG-T, and PIG-U
were co-purified with FLAG- and GST-tagged GPI8 (FG-GPI8) by a two-step
affinity purification using anti-FLAG and glutathione beads. Under
reducing conditions, FG-GPI8, GAA1, PIG-S, and PIG-T migrate at 60-70
kDa (12), while PIG-U migrates as a diffuse band at 35 kDa (13) (Fig.
1C, left lane).
Under non-reducing conditions, however, most of the FG-GPI8 migrates at
about 160 kDa. Western blotting with anti-GST antibody detected an
intense 160-kDa band with more slowly migrating minor bands in addition
to a faint 70-kDa monomeric FG-GPI8 band (Fig. 1A). These
high molecular mass bands disappeared when the sample was analyzed under reducing conditions (data not shown), suggesting that a
major fraction of GPI8 is ligated to other proteins through a disulfide
bond. To see whether GPI8 is linked to other GPI transamidase components or to unknown proteins, we analyzed the same sample by
silver staining. Consistent with the Western blotting results, several
bands appeared at and over 160 kDa, and only two intense bands remained
at around the 70-kDa position (Fig. 1B), suggesting that a
covalent complex was formed between GPI8 and another 60-70-kDa component of transamidase. To test this, the same sample was analyzed by two-dimensional electrophoresis (Fig. 1C). The first
dimension of electrophoresis was performed under non-reducing
conditions as in Fig. 1B, and then a gel strip was reduced
and subjected to a second dimension of electrophoresis under reducing
conditions. Two spots derived from the 160kDa bands in the first
dimension ran side by side below the gel diagonal (Fig. 1C),
suggesting that formation of several 160-kDa bands may be due to
multiple conformations of a single GPI8-containing complex. The two
60-70-kDa bands in the first dimension gave two spots on the diagonal
(Fig. 1C), indicating that the 160-kDa bands are
intermolecular disulfide-bonded complexes of two 60-70-kDa proteins.
While one of these proteins is FG-GPI8, the partner protein could not
be identified based on mobility because the difference in molecular
weight between GAA1, PIG-S, and PIG-T is quite small and the two
dimensional gel was slightly distorted.
To determine the partner protein linked to GPI8, we transfected
differentially tagged GPI8, PIG-T, GAA1, and PIG-S into CHO cells.
Transfectants were pretreated and lysed in the presence of 20 mM N-ethylmaleimide, a membrane-permeable
alkylating reagent, to trap disulfides formed within the cells and to
prevent their rearrangement during and after lysis (27, 28). The GPI
transamidase complex was precipitated with glutathione beads and
Western blotted with anti-tag antibodies (Fig.
2A). Anti-GST and anti-Myc but not anti-FLAG or anti-HA antibodies detected the 160-kDa band, indicating that PIG-T is the partner protein. The anti-GST- and anti-Myc-reactive 160-kDa band was not detected when the samples were
reduced (data not shown).
Human GPI8 has five cysteines, but only Cys92 and
Cys206 are conserved in other orthologues. To determine the
cysteine residue involved in this disulfide linkage, we constructed
cysteine-to-serine mutants of GPI8. Cys206 is a catalytic
site of GPI transamidase as reported previously (18). Mutant GPI8
proteins were stably expressed in class K cells. Expression of
C51S was very low, but this mutant clearly generated the 160-kDa band
(Fig. 2B). Only the C92S mutant was incapable of forming the
160-kDa band, indicating that Cys92 of GPI8 is used for the
disulfide bond.
Cys139 and Cys182 of human PIG-T are conserved
between species. These cysteine residues were mutated to serines and
stably expressed in PIG-T knockout cells. The C182S mutant
of PIG-T did not form a covalent complex, whereas the C139S mutant
formed the covalent complex (Fig. 2C). Therefore,
Cys92 of GPI8 and Cys182 of PIG-T are involved
in formation of the disulfide bond. Notably the disulfide linkage is
not essential for the generation of the tetrameric complex since the
C182S mutant of PIG-T, GAA1, and PIG-S were co-precipitated with GPI8,
and conversely the C92S mutant of GPI8 co-precipitated GAA1, PIG-T, and
PIG-S normally when expressed in CHO cells (data not shown).
Functional Importance of the Disulfide Bond for Transamidase
Activity--
To examine whether the disulfide linkage is functionally
important for GPI transamidase activity, we determined the abilities of
GPI8 and PIG-T mutants to restore the surface expression of GPI-anchored proteins on their respective mutant cells (Fig.
3). Wild-type GPI8 restored the surface
CD59 expression on class K cells to a level similar to parental K562
cells (data not shown). The C206S mutant had no complementation
activity as previously reported because this cysteine is an active site
of GPI transamidase (18). The C51S and C275S/C280S mutants restored
CD59 expression at levels similar to wild-type GPI8, whereas the C92S
mutant only partially restored CD59 expression even in this
overexpression experiment (mean fluorescence intensities (MFIs) for
wild type, C51S, C275S/C280S, and C92S were 893, 678, 873, and 187, respectively) (see below for a slightly lower MFI for C51S mutant).
We also assessed the activities of PIG-T mutants in PIG-T
knockout F9 cells, which express a high level of CD59 on the cell surface when rescued with PIG-T. In stable transfectants, the difference in MFI between wild type (MFI = 1269), the C139S mutant (MFI = 1222), and the C182S mutant (MFI = 935) transfectants
was relatively small, probably due to a compensation of low activity inherent to stable overexpression systems. We therefore tested transient expression of PIG-T proteins using the same cells.
While wild type (MFI = 1141) and the C139S mutant (MFI = 1042) restored the surface expression of CD59 at comparable levels, the
C182S mutant restored it at a significantly lower level (MFI = 434).
The surface expression of GPI-anchored marker proteins is not a direct
measure of the rate of GPI modification but rather measures the
accumulation of GPI-anchored proteins on the cell surface. Therefore,
we used an in vitro GPI
transamidase assay for more direct evaluation (Fig. 4). Again the C206S
mutant GPI8 showed no transamidase activity. The
C275S/C280S mutant processed miniPLAP at a level similar to wild
type. C51S had a significantly lower activity, but this might be due to
its low expression. When specific activity was calculated by dividing
the intensity of the GPI-anchored and free form bands by the GPI8
protein level, the C51S mutant had 95% of the activity of wild type.
The C92S mutant was expressed well; however, it had only 17% of the
wild-type activity (Fig. 4, left). Similarly the C182S
mutant of PIG-T restored only a trace amount of GPI-anchored miniPLAP
in both stable and transient expression systems (Fig. 4,
right). Thus, the disulfide linkage between GPI8 and PIG-T
is important but not essential for normal GPI transamidase
activity.
Endogenous GPI8 and PIG-T Form a Complex--
To further rule out
the possibility that disulfide formation is restricted to
overexpression experiments and does not reproduce in an in
vivo situation, it is important to demonstrate that endogenous GPI8 and PIG-T form an intermolecular disulfide bond. First we tested
disulfide bond formation in an in vitro translation system that can reproduce co- and post-translational modifications occurring in the ER. We have previously shown that a trimeric complex of GAA1,
PIG-T, and PIG-S is stably formed in the absence of GPI8; thus class K
cells might contain this trimeric complex. Microsomal membranes of
class K cells were, therefore, expected to process a disulfide bond
formation between in vitro translated GPI8 and pre-existing
endogenous PIG-T. As shown in Fig. 5, the
proform of wild-type GPI8 was converted to the mature form by cleavage of the signal peptide and was able to form a 160-kDa complex when the
translation reaction was performed in the absence of dithiothreitol (lane 1). This allowed synthesis of functional proteins with
native disulfide bonds such as major histocompatibility complexes
within the ER (29). This complex was destroyed when the sample was reduced (lane 5). Under the same conditions, the C92S mutant
failed to form this complex (lane 3). Formation of this
disulfide was sensitive to the redox status during translation. When
switched to reducing conditions by dithiothreitol addition, complex
formation with wild-type GPI8 was abolished (lane 2).
Next rabbit polyclonal antibodies were raised against human GPI8 and
PIG-T using bacterially expressed recombinant proteins. To confirm
antibody specificities we used purified endogenous proteins.
Transamidase complexes purified with FG-GPI8 or FG-PIG-T contained
endogenous PIG-T or GPI8, respectively, and FG-PIG-S co-purified both
proteins (Fig. 6A, compare
lane 1 and lanes 2 and 3). Western
blotting with anti-GPI8 antibodies detected endogenous GPI8
(lanes 4 and 6) as well as FG-GPI8 (lane
5). Similarly anti-PIG-T antibodies reacted with both endogenous
PIG-T (lanes 7 and 8) and FG-PIG-T (lane
9). Neither of these antibodies cross-reacted with other
transamidase components. With these antibodies, we tested the in
vivo formation of a disulfide bond between endogenous GPI8 and
PIG-T (Fig. 6B). Anti-GPI8 antibody detected the 160-kDa band in K562 (lane 1) and HeLa cells (lane 3) but
not in class K cells (lane 2), consistent with class K cells
being mutated for GPI8. Similarly K562 and HeLa cells
(lanes 4 and 6) but not class K cells (lane
5) contained the anti-PIG-T-reactive 160-kDa protein complex. In
contrast to the experiment with overexpressed FG-GPI8 (see Figs.
1A and 2B), monomeric GPI8 was not detected in
those wild-type cells. In addition, neither K562 nor HeLa cells contained monomeric PIG-T. These results suggest that all of the PIG-T
and GPI8 are complexed within the wild-type cells. These results
demonstrated that endogenous GPI8 and PIG-T are covalently linked
within normal cells.
Trypanosome GPI8 Forms a Similar High Molecular Weight Complex via
a Disulfide Bond--
Cys76 of TbGPI8 corresponds to
Cys92 of human GPI8. TbGPI8 might, therefore, form an
intermolecular disulfide bond via Cys76. Expression
plasmids for FLAG-tagged wild type and the C76S mutant were transfected
into the TbGPI8 knockout procyclic form of T. brucei. TbGPI8 was immunoprecipitated and analyzed by Western blotting with anti-FLAG antibody under non-reducing conditions. As
shown in Fig. 7A, the
wild-type transfectant generated a 160-kDa band in addition to a very
faint 35-kDa band corresponding to a predicted size of monomeric TbGPI8
(lane 2). In contrast, the C76S mutant produced only the
35-kDa band (lane 3). When the samples were reduced, the
160-kDa band disappeared, and only the 35-kDa band was detected in
wild-type transfectants (lane 5).
We next examined the surface expression of EP-procyclin, a major
GPI-anchored coat protein of the procyclic form of trypanosome (Fig.
7B). The TbGPI8 knockout trypanosome does not
express surface procyclin. Wild-type TbGPI8 rescued procyclin
expression on 70% of transfectants at a level comparable to that of
wild-type cells, whereas only 30% of transfectants were obtained with
the C76S mutant. Therefore, TbGPI8 also forms a functionally important disulfide bond with its conserved cysteine residue.
GPI Transamidase Consisting of the Five Components Binds the
Proform of Substrate Proteins--
Because of a lack of a
membrane-free assay of GPI transamidase, it is unclear whether five
components are sufficient. We predicted that the noncatalytic C206S
mutant of GPI8 should terminate the reaction immediately after the
recognition of GPI attachment signals and that the five-protein complex
with C206S GPI8 would be able to hold the substrate protein. A
representative result is presented in Fig.
8. When proteins co-purified with
FG-tagged wild-type and C206S mutant GPI8 proteins were
compared, two additional bands at 97 kDa (closed arrowhead)
and 31 kDa (open arrowhead) were observed in the mutant
transfectant (lane 2), and only the 97-kDa band was observed
in the wild-type transfectant (lane 1). We identified 97- and 31-kDa bands as calnexin and UL16-binding protein 2 (UL16BP2), respectively, by mass spectrometry. UL16BP2 is a GPI-anchored major
histocompatibility complex class I-related protein with N-glycan. Calnexin may have been associated with UL16BP2.
Amino-terminal sequencing of UL16BP2 showed that the amino-terminal
signal peptide had been cleaved off, therefore it had been translocated
into the ER lumen as a proform. Most of the UL16BP2 proteins were
eluted by a Nonidet P-40 wash from the complex purified with digitonin (lanes 4 and 6), suggesting that interaction of
its GPI attachment signal and the transamidase complex is relatively
weak. Consistent with this, UL16BP2 was not co-purified in the presence
of Nonidet P-40 (lane 8). These results demonstrate that the
affinity-purified transamidase complex comprised of five components was
sufficient to hold the proform of substrate GPI-anchored proteins.
Functional Importance of the Disulfide Bond between GPI8 and
PIG-T--
We found that the majority of GPI8 is linked to PIG-T via a
disulfide bond in the GPI transamidase complexes. Formation of a
disulfide linkage between GPI8 and PIG-T requires these proteins to be
in close proximity and is therefore consistent with our previous result
showing that expression of GPI8 was dependent on and stabilized by
PIG-T probably through a direct interaction (12).
The disulfide linkage per se is not required for generation
of the tetrameric complex because co-precipitation of GAA1 and PIG-S
with GPI8 occurred normally with the C182S mutant of PIG-T. In
contrast, the disulfide linkage between GPI8 and PIG-T is important for
normal activity of the GPI transamidase. We have previously reported
that Cys92 of GPI8 is important for its function; an
alanine mutant restored the surface expression of GPI-anchored proteins
on class K cells to only 10% of the wild-type level (18). We confirmed
this using the C92S mutant that restored only 20% of the surface CD59
expression (Fig. 3). A 10% difference in this activity could be due to
different expression levels of GPI8 with higher expression levels in
stable transfectants than in transient transfectants. This disulfide bond also affects the activity of PIG-T in the GPI transamidase holoenzyme since the C182S mutant PIG-T has a considerably lower activity (35%) in restoring the surface expression of CD59 on the
transient transfectants of PIG-T knockout cells (Fig. 3), although it has 75% of the activity in stable transfectants. Moreover, in an in vitro transamidase assay, the C92S GPI8 mutant had
17% of the wild-type activity; transient and stable transfectants of
the C182S PIG-T mutant had 16 and 37% of the wild-type activity, respectively (Fig. 4). It seems, therefore, that the complex formed by
non-covalent interaction has a very weak activity.
These analyses were performed using overexpressed proteins. To address
how covalent and non-covalent association of GPI8 and PIG-T occurs in
the endogenous GPI transamidases, we raised antibodies against GPI8 and
PIG-T and demonstrated that the majority of GPI8 and PIG-T are
covalently linked in two wild-type cell lines (Fig. 6B).
Taken together, these data clearly demonstrate that a disulfide bond
between GPI8 and PIG-T is formed within normal cells and is critical
for full transamidase activity probably through holding the proper
position of components and stabilizing the complex.
Trypanosome GPI Transamidase Complex Has an Architecture Analogous
to but Different from Mammalian Enzyme--
The disulfide bond
is formed between Cys92 of GPI8 and Cys182 of
PIG-T (Fig. 2, B and C). Both residues are
conserved in GPI8 and PIG-T homologues of S. cerevisiae,
S. pombe, C. elegans, D. melanogaster, Anopheles gambiae, and
Arabidopsis thaliana. In addition, T. brucei, L. mexicana, and Plasmodium falciparum GPI8
contain a cysteine residue corresponding to Cys92. It is,
therefore, possible that a similar disulfide bridge is present in the
GPI transamidases of those organisms. Indeed we demonstrated that
trypanosome GPI8 formed an intermolecular disulfide bond through this
conserved cysteine because the C76S mutant failed to form a high
molecular weight complex (Fig. 7A). Although the partner
protein was not identified, PIG-T homologue is a likely candidate
because its molecular mass seemed to be similar to human PIG-T
(43-kDa human and 35-kDa trypanosome GPI8 proteins formed the
160-kDa disulfide-bonded complex). We recently identified a T. brucei homologue of
PIG-T.2
It has been reported that GPI anchoring ability is lost when the
microsomal membranes of T. brucei are washed at high pH
(20), whereas a similar treatment of the mammalian microsomal membranes has no effect (30). Moreover, the GPI anchoring can be restored by
adding the high pH extract of trypanosomal membranes or recombinant GPI8 protein of the related protozoan L. mexicana to the
washed membranes (20). These results suggest that trypanosomal but not
mammalian GPI8 is a soluble protein. In fact, T. brucei and L. mexicana GPI8 lacks the transmembrane domain (8, 10, 22). Our results suggest either that TbGPI8 is released from the complex due
to a disulfide exchange that might be caused by protein denaturation at
high pH or that endogenous TbGPI8 binds its partner protein mainly
non-covalently, whereas transfected TbGPI8 mainly uses covalent
binding. Identification of the partner protein in trypanosome would not
only resolve this issue but also lead to an understanding of the
molecular mechanisms that determine the specificity of GPI attachment signals.
Requirements for GPI attachment signals are different between mammalian
cells and parasitic protozoa. The larger size of amino acids at the GPI Transamidase Comprising Five Components Captures Substrate
Proteins--
We found that a proform of UL16BP2 was co-purified with
an inactive transamidase complex. The members of the human UL16BP family, consisting of three molecules, are GPI-anchored ligands for
natural killer cell receptor NKG2D (33). Although it was reported that UL16BP2 is expressed well in K562 cells, the parent line
of class K cells, many other proforms of GPI-anchored proteins were not
co-purified. There are some explanations for this. (i) Within the cells
a number of proform proteins bound to the complex, but most of them
were lost during affinity purification of the complex. (ii) UL16BP2
predominantly occupied the complex already in the ER due to its higher
affinity. (iii) UL16BP2 is extremely abundant in this cell line, and
while many other proproteins co-purified with the complex, they are
below our detection limit.
The interactions of substrate proteins with transamidase components
have been reported previously (23, 24). These studies were carried out
with in vitro translation of model proteins followed by
chemical or photo cross-linking, demonstrating that substrate proteins
are positioned in close proximity to GPI8. Co-purification of UL16BP2
with the complex rendered inactive by a mutation of the catalytic site
cysteine of GPI8 is expectable and is consistent with a prolonged
association of substrate proteins with GPI8 when GPI attachment signal
was uncleavable or GPI was not available, which is supported by the
successful chemical cross-linking of these proteins (24). Our results
clearly demonstrate that the complex consisting of five components is
sufficient for physical association with the substrate proteins, at
least UL16BP2. Release of UL16BP2 from the complex by Nonidet P-40
implies that the interaction between GPI attachment signals and the
transamidase complex is relatively weak.
Composition of GPI Transamidase Subunits--
The difference
between the molecular mass of GPI transamidase complex determined
experimentally in digitonin extract of HeLa cells and S. cerevisiae (about 460 and 430-650 kDa, respectively) (14, 34) and
the mass calculated from the five known human components (280 kDa) has
several potential explanations: a non-globular shape of the complex,
bound detergent, multiple molecules of components, or unidentified
components that are lost during affinity purification of the complex.
The association of tubulin and the transamidase complex may partially
account for this difference (34).
GPI transamidase contains one copy of GAA1 because endogenous GAA1 is
not found in the complex containing FLAG-tagged GAA1 (34). Similarly
our results using antibodies against GPI8 and PIG-T suggest that one
molecule each of GPI8 and PIG-T are contained within this complex
because neither endogenous GPI8 nor PIG-T co-purified with FG-tagged
GPI8 or FG-PIG-T, respectively (Fig. 6A), although the
number of molecules for PIG-S and PIG-U remains to be determined in a
similar way. Under the conditions used, five components were
consistently purified, but variability of the minor bands accompanying
the complex was observed between the individual preparations and
conditions used (Fig. 8). Purification of the complex under various
conditions including various detergents, ionic strength, and so on
could identify such weakly associated proteins. Development of the
assay for GPI anchoring using a purified transamidase complex will
clarify these issues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GPI8 forms an intermolecular disulfide bond
with another transamidase component. A and
B, a GPI transamidase complex was purified from class K
cells expressing FLAG/GST-tagged GPI8 by a two-step affinity
purification and electrophoresed under non-reducing conditions.
A, Western blotting with anti-GST antibodies; B,
silver staining. C, a gel strip as shown in B was
excised before silver staining, reduced, and overlaid on another gel
for the second dimensional electrophoresis under reducing conditions.
The gel was then silver-stained. A reduced sample of the purified
complex was also loaded in the left lane as reference
components. The identities of bands are indicated on the
left.
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Fig. 2.
GPI8 and PIG-T form a disulfide-bonded
complex through specific cysteine residues. A, CHO
cells were transiently transfected with a mixture of GST-GPI8,
FLAG-GAA1, HA-PIG-S, and Myc-PIG-T. Two days later cells were lysed,
and the transamidase complex was precipitated with glutathione beads.
The precipitates were divided into four aliquots, electrophoresed under
non-reducing conditions, and Western blotted with the indicated
antibodies. B, class K cells were stably transfected with an
empty vector, FG-tagged wild-type GPI8, or cysteine-to-serine mutant
GPI8 proteins. The transamidase complexes precipitated with
glutathione beads were electrophoresed under non-reducing conditions
and Western blotted with anti-GST antibodies. C,
PIG-T knockout F9 cells expressing CD59 were stably
transfected with an empty vector, Myc-tagged wild type, or
cysteine-to-serine mutant PIG-T proteins. The transamidase
complexes immunoprecipitated with anti-Myc antibody plus protein G
beads were electrophoresed under non-reducing conditions and Western
blotted with anti-Myc antibody. The asterisk indicates a
nonspecific band.
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Fig. 3.
A disulfide bridge between GPI8 and PIG-T is
important for the surface expression of GPI-anchored proteins.
Surface expression of CD59 on class K cells and PIG-T
knockout (KO) CD59 transfectants was measured by flow
cytometry. The stable transfectants of class K and PIG-T
knockout cells used in Fig. 2, B and C, were
used. For the transient transfection of PIG-T knockout
cells, cells were transfected with the same plasmids used to establish
stable transfectants except for the absence of a
phosphoglycerokinase-hygromycin resistance gene and stained 2 days
after transfection. Bold and dotted lines
indicate anti-CD59 and control staining, respectively.
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Fig. 4.
GPI8 and PIG-T, which are unable to form a
disulfide linkage, have decreased transamidase activities in
vitro. Transfectants analyzed for the surface
expression of CD59 in Fig. 4 were assayed for in vitro
transamidase activity. Top, miniPLAP mRNA was translated
in vitro using rabbit reticulocyte lysates and microsomal
membranes prepared from the indicated transfectants. Radiolabeled
miniPLAP proteins were immunoprecipitated with anti-PLAP antibody,
electrophoresed, and visualized by autoradiography. Identities of the
bands according to Kodukula et al. (25) are shown on the
left. Bottom, transfectants analyzed in the
upper panel for transamidase activity were evaluated for
expression levels of the proteins. Precipitated proteins were
electrophoresed under reducing conditions and Western blotted with the
indicated antibodies. KO, knockout.
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Fig. 5.
In vitro translated GPI8 forms a
disulfide-bonded high molecular weight complex in the presence of class
K microsome membranes. Wild-type (WT) or mutant
(C92S) GPI8 mRNA was translated in rabbit reticulocyte
lysate using class K microsomes in the presence (+) or absence ( ) of
2 mM dithiothreitol (DTT). After incubation,
microsomes were pelleted by ultracentrifugation through a sucrose
cushion, solubilized in a sample buffer, and electrophoresed
under non-reducing (left) or reducing (right)
conditions. Radiolabeled proteins were visualized by autoradiography.
An arrowhead indicates a high molecular weight complex. Note
that direct treatment of samples in a sample buffer without isolation
of microsome membranes gave a similar result (data not shown).
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Fig. 6.
Endogenous GPI8 and PIG-T form an
intermolecular disulfide bond. A, the transamidase
complex was purified from K562 cells expressing FG-tagged PIG-S
(lanes 1, 4, and 7) or PIG-T
(lanes 3, 6, and 9) and class K cells
expressing FG-tagged GPI8 (lanes 2, 5, and
8) and electrophoresed under reducing conditions. Proteins
were visualized by silver staining (left) or Western blotted
with anti-GPI8 (middle) or anti-PIG-T (right)
antibodies. Identities of bands on a silver-stained gel are indicated
on the right. B, whole cell lysates of K562
(2 × 105), class K (4 × 105), and
HeLa (2 × 105) cells were electrophoresed under
non-reducing conditions and Western blotted with anti-GPI8
(left) or anti-PIG-T (right) antibodies.
Asterisks indicate nonspecific bands.
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Fig. 7.
Trypanosome GPI8 forms a disulfide-bonded
protein complex through a conserved cysteine residue.
A, the TbGPI8 knockout procyclic trypanosome was
transfected with an empty vector (lanes 1 and 4),
FLAG-tagged wild-type TbGPI8 (lanes 2 and 5), or
its C76S mutant (lanes 3 and 6) and selected by
phleomycin. Cell lysates were prepared and subjected to
immunoprecipitation with anti-FLAG antibodies plus protein G beads.
Immunoprecipitates were electrophoresed under non-reducing
(left) or reducing (right) conditions and
analyzed by Western blotting with anti-FLAG antibody. A band at about
the 50-kDa position is nonspecific. B, surface expression of
GPI-anchored procyclin. Transfectants used in A were stained
for procyclin and analyzed by flow cytometry. Bold and
dotted lines indicate anti-procyclin and control staining,
respectively. KO, knockout.
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Fig. 8.
The transamidase complex is physically
associated with a proform of the substrate protein. The
transamidase complex was purified from lysates of class K cells
expressing FG-tagged wild-type (W) or C206S mutant
(206) GPI8. The lysates were prepared in a buffer containing
1% digitonin (lanes 1-6) or 1% Nonidet P-40
(NP-40, lanes 7 and 8). The protein
complex bound to glutathione beads at the second purification step was
directly treated with sample buffer (lanes 1, 2,
7, and 8) or further washed in a 1% Nonidet P-40
buffer (lanes 3-6). Proteins still bound to the beads
(lanes 3 and 4) and eluted into a wash
(lanes 5 and 6) were separated, and eluted
proteins were precipitated by trichloroacetic acid and sodium
deoxycholate. The bands specifically co-purified with the C206S mutant
GPI8 and eluted by Nonidet P-40 are indicated by closed and
open triangles (lanes 2 and 6). Their
identities, digested peptides identified by mass spectrometry (in
plain letter), and amino-terminal sequence determined by
protein sequencing (in bold letter) are shown on the
left. It is unclear whether the broad faint bands appearing
after Nonidet P-40 treatment and in the samples prepared from Nonidet
P-40 extract (indicated by dots in lanes 3,
4, 7, and 8) correspond to
calnexin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to
+ 2 sites in parasitic protozoa as compared with those in human
suggests that parasite GPI transamidase accommodates and tolerates
larger amino acids in its catalytic pocket (31). It would, therefore,
be possible to design inhibitors that specifically inhibit parasitic
transamidase, leading to potent chemotherapeutics for diseases caused
by trypanosomes and potentially other protozoan parasites (11). A
candidate gene for T. brucei and Leishmania major
GAA1 homologues with a similar hydrophobic structure was suggested, but its function was not demonstrated (22, 32). Molecular
cloning of the partner proteins of GPI8 will reveal whether
transamidase complexes of those parasitic protozoa have an analogous
architecture with similar components.
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ACKNOWLEDGEMENTS |
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We thank Fumiko Ishii and Keiko Kinoshita for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.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.
Present address: Advanced Medical Discovery Inst. of the
University Health Network and Ontario Cancer Inst., University of Toronto, Toronto, Ontario M5G 2C1, Canada.
§ To whom correspondence should be addressed: Dept. of Immunoregulation, Research Inst. for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8328; Fax: 81-6-6875-5233; E-mail: tkinoshi@biken.osaka-u.ac.jp.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300586200
2 K. Nagamune, K. Ohishi, Y. Maeda, and T. Kinoshita, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: GPI, glycosylphosphatidylinositol; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; GST, glutathione S-transferase; HA, hemagglutinin; FG, FLAG and GST tandem; NEM, N-ethylmaleimide; PLAP, placental alkaline phosphatase; MBP, maltose-binding protein; MFI, mean fluorescence intensity; UL16BP2, UL16-binding protein 2.
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