From the University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom
Received for publication, November 7, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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The addition of glycosylphosphatidylinositol
(GPI) anchors to proteins occurs by a transamidase-catalyzed reaction
mechanism soon after completion of polypeptide synthesis and
translocation. We show that placental alkaline phosphatase becomes
efficiently GPI-anchored when translated in the presence of
semipermeabilized K562 cells but is not GPI-anchored in cell lines
defective in the transamidase subunit hGpi8p. By studying the synthesis
of placental alkaline phosphatase, we demonstrate that folding of the
protein is not influenced by the addition of a GPI anchor and
conversely that GPI anchor addition does not require protein folding.
These results demonstrate that folding of the ectodomain and GPI
addition are two distinct processes and can be mutually exclusive. When
GPI addition is prevented, either by synthesis of the protein in the
presence of cell lines defective in GPI addition or by mutation of the
GPI carboxyl-terminal signal sequence cleavage site, the substrate
forms a prolonged association with the transamidase subunit hGpi8p. The
ability of the transamidase to recognize and associate with GPI anchor
signal sequences provides an explanation for the retention of
GPI-anchored protein within the ER in the absence of GPI anchor addition.
Glycosylphosphatidylinositol
(GPI)1 is a complex
glycolipid that is covalently attached to many eukaryotic cell surface
proteins. GPI provides an alternative anchoring mechanism to a
hydrophobic polypeptide transmembrane domain, enabling stable
association of protein with the lipid bilayer. In mammalian cells, more
than 100 polypeptides are anchored to the plasma membrane via a GPI moiety (1). These encompass a functionally diverse set of proteins including cell surface enzymes, receptors, adhesion molecules, and
differentiation antigens, which have been implicated in a number of
cellular functions such as immune recognition, complement regulation,
and transmembrane signaling (1). In mammalian cells, the GPI anchor is
not essential for survival at the cellular level and cell lines
defective in various stages of GPI backbone assembly, or transfer of
this glycolipid to the polypeptide chain have been established (2, 3).
However, GPI anchoring is essential for embryogenesis (4) and skin
development (5), and defective cell surface expression of GPI-linked
proteins may lead to specific disease states (6, 7). Therefore,
determining the molecular mechanisms involved in the production of
GPI-anchored proteins will provide an insight into the biosynthesis of
proteins that are central to both normal and abnormal cell physiology.
The addition of GPI anchors to proteins entering the secretory pathway
occurs post-translationally. The GPI moiety is synthesized in the
endoplasmic reticulum (ER), and the overall biosynthetic pathway for
the production of the GPI precursor is now well understood (8).
Pre-formed GPI is transferred en bloc to proteins destined to be GPI-anchored. These precursor proteins contain an ER-targeting N-terminal signal sequence and a C-terminal signal sequence for GPI
anchoring (9, 10). This GPI signal sequence consists of a C-terminal
hydrophobic domain, preceded by a short hydrophilic spacer linked to
the GPI attachment ( Genetic approaches have identified two genes, GAA1 and
GPI8, which are required for addition of GPI to proteins in
Saccharomyces cerevisiae (14, 15). Homologues of both these
genes have now been identified in humans (16, 17). Transamidation of
GPI-anchored proteins is therefore thought to depend upon the gene
products Gpi8p and Gaa1p, and it has been suggested that these two
proteins may constitute a transamidase complex (18). In agreement with this finding, mammalian cell lines defective in either Gpi8p or Gaa1p
do not attach GPI to proteins and accumulate complete GPI lipids as
well as GPI precursor proteins (18, 19). Human Gpi8p is a 45-kDa type I
ER membrane protein that belongs to a novel cysteine protease family
and has been postulated to be the catalytic subunit of the transamidase
(18, 20). Human Gaa1p, a 67-kDa ER protein, with several
membrane-spanning domains forms a complex with Gpi8p (18) and therefore
constitutes part of the transamidase. However, the role that Gaa1p
plays in the transamidation reaction remains unknown. The exact
function for both transamidase subunits and their functional
interaction with each other and with the precursor protein and GPI
substrates also remains to be elucidated.
Processing of nascent proteins to GPI-anchored forms has been
extensively studied using cell-free systems. Model GPI-anchored proteins can be translated in vitro and then processed by
rough microsomal membranes (RM) derived from mammalian cells. In early studies translated human placental alkaline phosphatase (PLAP) was
processed by RM to a mature GPI-anchored form, which was identified by
reaction to site-specific antibodies (21). However, PLAP was not ideal
for studying N- and C-terminal signal sequence processing due to the
increased size of the glycosylated protein. Therefore a truncated
version of PLAP, prepromini-PLAP was constructed to act as a
reporter protein in this cell free system (22). Nascent prepromini-PLAP
translated in vitro by microsomal membranes was processed by
cleavage of the N-and C-terminal signal sequences, with subsequent GPI
addition, to GPI-anchored mini-PLAP (22). Studies using this microsomal
cell-free assay system have demonstrated the strict amino acid
requirements at or adjacent to the GPI attachment ( In this report we describe a convenient assay for GPI anchoring based
on a cell-free system using digitonin-permeabilized cells. These
semipermeabilized (SP) cells are capable of replacing microsomal
membranes as a source of intact ER in a conventional in
vitro translation system (26). PLAP was used as a model protein for the biosynthesis, processing, and folding of GPI-anchored proteins
in these experiments. The results indicate that the initial stages of
GPI anchor addition to PLAP can be reconstituted in vitro
using the SP cell system and approximate events seen in vivo. An objective of this work is to determine the role that protein folding has on GPI addition to PLAP. The data presented show
that folding of PLAP is not influenced by the addition of a GPI anchor
and that GPI anchor addition does not require protein folding,
demonstrating that these two processes can occur independently of each
other. Use of a mutant cell line defective in GPI-protein biosynthesis,
combined with this flexible in vitro translation system, has
provided evidence of an association between protein substrate and the
transamidase subunit hGpi8p. The significance of these findings with
regard to possible functions of the hGpi8p subunit of the transamidase
is discussed.
Plasmid Construction--
Plasmids containing human cDNA
coding mini-PLAP (pGEM-4Z/miniPLAP) and full-length PLAP (pGEM-4Z/PLAP)
were kindly provided by Dr. M. E. Medof (Case Western Reserve
University, Cleveland, OH). PLAP was subcloned as an
EcoRI-HindIII fragment into pBluescript SK Transcription in Vitro--
Transcription reactions were carried
out as described by Gurevich et al. (27). Recombinant
plasmids were linearized with HindIII; pBS/PLAP and
pBS/PLAP-D484P were transcribed using T3 RNA polymerase (Promega,
Southampton, UK), and pGEM4Z/miniPLAP was transcribed with T7 RNA
polymerase (Promega).
Preparation of Semipermeabilized Cells--
The human
lymphoblastoid cell line K562 was obtained from the European collection
of animal cell cultures. The class K mutant and class IA mutant K562
cell lines were a gift from Dr. M. E. Medof. Cell lines were
cultured in RPMI 1640 media supplemented with 10% fetal calf serum.
Semipermeabilized (SP) cells were prepared by treatment with digitonin,
as described previously (26).
Translation in Vitro--
RNA was translated using a rabbit
reticulocyte lysate (FlexiLysate, Promega). The translation reaction
(25 µl) contained 17.5 µl of reticulocyte lysate, 40 µM methionine-free amino acid mixture, 45 mM
KCl, 15 µCi of L-[35S]methionine
(PerkinElmer Life Sciences, Houndslow, UK), 1 µl of
transcribed mRNA, and appropriate SP cell preparation (~2 × 105 cells). Hydrazine (10 mM) or DTT (5-10
mM) were included in translations where indicated.
Translations were incubated at 30 °C for 60-90 min and were
terminated by centrifugation (12,000 × g for 3 min at
4 °C) to isolate SP cells. Translation products were then processed for further analysis as detailed below.
Endoglycosidase H Treatment--
After translation, isolated SP
cells were solubilized in dissolution buffer (50 mM
Tris-HCl, pH 8.0, 1% (w/v) SDS) for 5 min at 95 °C. Insoluble
material was removed by centrifugation at 13,000 × g
for 10 min. An equal volume of 150 mM sodium citrate buffer, pH 5.5, containing PMSF (0.5 mM) was added to the
soluble fraction. The sample was divided into two identical aliquots, which were incubated for 12 h at 37 °C with either 1 unit of
endoglycosidase H (Roche Molecular Biochemicals, Lewes, UK) or buffer
alone. Treated and untreated reactions were then subjected to analysis
by SDS-PAGE.
Proteinase Protection Assays--
After translation, isolated
membrane fractions were resuspended in KHM buffer (20 mM
HEPES, pH 7.2, 110 mM potassium acetate, 2 mM
magnesium acetate) supplemented with 10 mM
CaCl2 and were treated with proteinase K (Roche Molecular
Biochemicals) at a concentration of 250 µg/ml for 30 min at 0 °C,
either in the presence or absence of 1% (v/v) Triton X-100. Following
digestion, proteinase K was inhibited by the addition of PMSF to a
final concentration of 1 mM, and samples were then analyzed
by SDS-PAGE.
Triton X-114 Extraction--
After translation SP cells were
isolated and resuspended in 500 µl of TBS (10 mM Tris-HCl
buffer, pH 7.4, 150 mM NaCl) containing 2% (v/v) Triton
X-114. Resuspended cells were sonicated for 3.5 min to solubilize
proteins and incubated on ice for 15 min. Samples were centrifuged for
10 min at 10,000 × g and the supernatant incubated at
37 °C until turbid. The aqueous and detergent phases were then
separated by centrifugation at 10,000 × g for 10 min. The aqueous phase was re-extracted three times with TBS containing 2%
(v/v) Triton X-114, and all aqueous phases were combined. The detergent
phase was re-extracted three times with TBS, and all detergent phases
were combined. The radiolabeled material in the detergent and aqueous
phases was then analyzed by SDS-PAGE.
PLAP Assay--
Following translation for 1 h, isolated
cells were solubilized in KHM buffer (60 µl per 25-µl translation)
containing 0.05% (v/v) Triton X-100 for 30 min at 0 °C. Insoluble
material was removed by centrifugation at 13,000 g for 10 min.
Enzymatic activity of solubilized, translated PLAP (15-µl aliquots)
was measured using a commercial kit (Great Escape SEAP,
CLONTECH, Basingstoke, UK), as described in
manufacturer's instructions for a 96-well format. PLAP activity was
measured by dephosphorylation of the chemiluminescent substrate
3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'chloro)-tricyclo[3.3.1.1]decan-4-yl)phenyl phosphate (CSPD), with concomitant release of light, which was detected
by a luminometer. PLAP activity was measured relative to a standard of
the secreted form of PLAP and is expressed as relative light units
emitted by the sample. The equivalent amount of N-terminally processed
PLAP formed in each assay was estimated by immunoprecipitating an
aliquot (10 µl) of Triton X-100-solubilized PLAP with amino-Ab.
Following immunoprecipitation, PLAP was removed from protein
A-Sepharose by addition of 50 mM Tris, pH 8, 1% (w/v) SDS
with boiling for 3 min. Radioactivity incorporated into processed PLAP
was then estimated by liquid scintillation counting. The specific
activity of PLAP is expressed as relative light units emitted/amount
radioactivity incorporated into N-terminally processed forms of PLAP.
Background values obtained when translation was carried out in the
absence of added RNA were subtracted from both the activity and
radioactivity measurements. The results are the mean values from three
separate experiments ± standard error.
Cross-linking--
Following in vitro translation,
membrane fractions were resuspended in KHM buffer containing
Me2SO (solvent control) or 100 µM
bismaleimidohexane (BMH) (Pierce and Warriner, Warrington, UK), and
incubated at room temperature for 10 min. Cross-linking was then
quenched by the addition of 10 mM DTT, and the samples were
left on ice for 10 min. Samples were then immunoprecipitated, under
either native or denaturing conditions as detailed below.
Immunoprecipitation--
Polyclonal rabbit antibodies were
commercially produced (SigmaGenosys, Cambridge, UK) against synthetic
peptides conjugated to keyhole limpet hemocyanin. Three site-specific
antibodies (Abs) to human PLAP-513 (endo-antibody, amino-antibody, and
exo-antibody) were generated to specific amino acid sequences in the
PLAP molecule, as described previously (21). Antibodies were also
raised against components of the human transamidase, via peptides in
the amino terminus of the mature form of hGpi8p (SHIEDQAEQFFRSGHTNNW)
and to a peptide sequence representing residues 74-89 in hGaa1p
(ARDFAAHRKKSGALP). Antibody to calreticulin was obtained from Cambridge
Biosciences (Cambridge, UK) and to ERp57 from Dr. T. Wileman (IAH,
Pirbright, UK).
For immunoprecipitations under native conditions, samples were
solubilized in 1 ml of immunoprecipitation buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1%
(v/v) Triton X-100, 0.02% (w/v) azide) and incubated with 50 µl of
protein A-Sepharose (10% (w/v) (Zymed Laboratories
Inc., San Francisco, CA) for 30 min at 4 °C. Samples were
centrifuged to remove protein A binding contaminants and precleared
supernatants incubated overnight with 50 µl of protein A-Sepharose
and 1-10 µl of relevant antisera. In the case of denaturing
immunoprecipitations, samples were denatured for 5 min at 95 °C in
the presence of 1% (w/v) SDS and then diluted with 1 ml of
immunoprecipitation buffer to give a final concentration SDS < 0.05% (w/v). Samples were then precleared and incubated with antibody
as described above. In each case, protein A-Sepharose-bound material
was isolated by centrifugation at 13,000 × g for 1 min, washed three times with immunoprecipitation buffer, and then
subjected to analysis by SDS-PAGE, isoelectric focusing (IEF), or
radioactivity incorporated into protein measured by scintillation counting.
Electrophoresis--
Samples for IEF were immunoprecipitated
under native conditions and material immobilized on protein A-Sepharose
solubilized in IEF solubilization buffer containing 8 M
urea, 0.5% (v/v) Triton X-100, 0.01 g/ml Ampholine, pH 3.5-9.5
(Amersham Pharmacia Biotech, Lower Chalfont, UK), 20 mM
DTT, for 1 h at room temperature. Protein A-Sepharose was then
removed by centrifugation (13,000 × g, 1 min) and
supernatants were loaded onto an Immobiline DryPlate pH 4-7
isoelectric focusing gel (Amersham Pharmacia Biotech), that had been
preswollen overnight in IEF solubilization buffer as described in
manufacturer's instructions. After focusing gels were stained and
fixed in 0.1 (w/v) CuSO4, 0.05% (w/v) R-250 Coomassie Blue, 17% acetic acid, 23% ethanol, destained in 30% (v/v) ethanol, 7% acetic acid, dried, and then visualized by either autoradiography or imaged using a FujiBas 2000 phosphorimager.
Samples for SDS-PAGE were resuspended in SDS-PAGE sample buffer (0.0625 M Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 10% (v/v) glycerol, bromphenol blue) plus 50 mM DTT and were boiled
for 5 min. Proteins were subjected to SDS-PAGE through either 12.5% (mini-PLAP) or 10% gels (PLAP and derivatives). Gels were fixed in
10% (v/v) methanol and 10% (v/v) acetic acid, dried, and then visualized using autoradiography or a phosphorimager as described above.
Reconstitution of GPI Anchor Addition in SP Cells--
We have
previously shown that SP cells can efficiently translate, glycosylate,
and assemble a variety of secretory and membrane-bound proteins (26).
This approach has the advantage over using microsomal membranes in that
the reactions occur within a morphologically intact ER and are
generally more efficient (26). To determine if this system could be
applied to study the translocation and subsequent processing of
GPI-anchored proteins, we utilized the well characterized GPI anchor
substrate, which is a truncated version of human placental alkaline
phosphatase (prepromini-PLAP). The various different processed forms of
this protein can be readily resolved on SDS-PAGE and have been
previously characterized in translations with microsomal membranes
(22). When mRNA coding for prepromini-PLAP was translated in the
absence of SP K562 cells, a single translation product of ~28 kDa was
generated (Fig. 1A, lane 1). In addition to prepromini-PLAP, two
further translation products were generated in the presence of SP K562
cells (Fig. 1A, lane 2). The three
translation products showed essentially the same migration pattern to
those derived from identical translations in the presence of RM (22).
The identity of the translation product synthesized in the absence of
SP cells was confirmed as prepromini-PLAP by the
sensitivity of this product to proteinase K digestion in
the presence of SP cells (Fig. 1A, lane
3). The 27- and 25-kDa species synthesized in the presence
of SP cells were shown to be resistant to proteolysis (Fig.
1A, lane 3), indicating that these
polypeptides were translocated across the ER membrane. Addition of
detergent prior to protease digestion demonstrated the susceptibility
of the translocated portions of the protein to digestion after membrane
solubilization (Fig. 1A, lane 4).
To confirm the identity of the translation products, we generated three
peptide-specific antibodies termed amino-, endo-, and exo-antibodies
that have been used previously (22) to monitor the various stages of
mini-PLAP processing. Amino-Ab was raised to a peptide sequence
corresponding to the N terminus of the mature protein and will only
react with polypeptides lacking the N-terminal signal sequence
i.e. promini-PLAP and mini-PLAP. Endo-Ab was raised to a
peptide sequence corresponding to the C terminus of the mature protein
and reacts with all forms of the protein, whereas exo-Ab was raised to
a peptide sequence corresponding the C-terminal signal for GPI addition
and therefore only reacts with pre- and promini-PLAP. When the
translation products synthesized in the presence of SP K562 cells were
immunoprecipitated with endo-Ab, all three products were
immunoprecipitated (Fig. 1B, lane 1). With amino-Ab the 27- and 25-kDa products, but not the 28-kDa product,
were immunoprecipitated, confirming that the 28-kDa product is
prepromini-PLAP (Fig. 1B, lane 2).
With exo-Ab only the 28- and 27-kDa products were immunoprecipitated,
confirming the identity of the 25-kDa product as mini-PLAP (Fig.
1B, lane 3). These results are
identical to those obtained with RM (22), and demonstrate that the SP
cells reconstitute GPI signal processing.
Full-length PLAP Is GPI-anchored in SP Cells--
One of the
objectives of this work was to determine the influence of protein
folding on the addition of GPI anchors to proteins and to also
investigate whether GPI addition was required for efficient protein
folding. It has been suggested previously that molecular chaperones
within the ER influence the processing and addition of GPI anchors to
proteins (24). This conclusion was based on studies showing that
removal of soluble proteins from microsomal membranes prevents GPI
addition. These studies were carried out with the model substrate
prepromini-PLAP, an artificial molecule that does not fold correctly
and forms a prolonged interaction with the molecular chaperone BiP
(11). We therefore investigated the influence of protein folding on GPI
addition using authentic full-length prepro-PLAP. PLAP is not ideal for
studying N- and C-terminal signal processing, as the prepro-protein
becomes glycosylated on translocation into the ER, making it difficult
to analyze processed products differing by a few kilodaltons in size by
SDS-PAGE. Hence, before assessing the influence of protein folding on
GPI addition, we needed to prove that PLAP was processed to
GPI-anchored form in our SP cell system.
When PLAP mRNA was translated in the absence of SP K562 cells, a
single labeled product of approximately Mr
58,000, corresponding to prepro-PLAP was formed (Fig.
2, lane 3). In
translations supplemented with SP cells, a second larger form of PLAP
with an approximate Mr of 63,000 was seen (Fig.
2, lane 4), which was protected from proteolysis
by proteinase K (Fig. 2, lane 5). This larger
form was shown to correspond to a glycosylated product, as it was
sensitive to digestion with endoglycosidase H (Fig. 2, lane
2). These results confirm previous work carried out with RM
(21) and indicate that the full-length product was translocated across
the ER membrane and glycosylated following translation in the presence
of SP cells.
As we were unable to demonstrate GPI anchoring of PLAP by differences
in mobility of translation products after SDS-PAGE, we used two
alternative approaches to demonstrate that this modification had indeed
occurred. First, we predicted that GPI-anchored and non-GPI-anchored
proteins would have different isoelectric points and, therefore, show
altered migration patterns on IEF gels. To test this prediction PLAP
mRNA was translated in the presence of either SP parental or mutant
K class K562 cells, and then products were analyzed by IEF (Fig.
3A). The mutant K class cell
line carries a mutation in one of the transamidase subunits Gpi8p,
thereby preventing GPI anchor addition from occurring (16). As can be seen, three radiolabeled translation products were formed in SP K562
cells (Fig. 3A, lane 1). To determine
the identity of these translation products, the three site-directed
antibodies were utilized as described earlier. In SP K562 cells, all
forms of PLAP were immunoprecipitated with the endo-Ab and amino-Ab,
indicating that the N-terminal signal sequence had been cleaved from
these translation products (Fig. 3A, lanes
2 and 3). Exo-Ab did not immunoprecipitate either
translation product (Fig. 3A, lane 4). It should be noted that the amount of the most acidic translation product formed is low in comparison to the more basic product, which
could explain the lack of immunoprecipitation with exo-Ab. When the
translation products generated by translation of PLAP RNA in the
presence of SP K class cells were analyzed by IEF, the major product
was the more acidic form of PLAP that was immunoprecipitated by
amino-Ab and exo-Ab (Fig. 3A, lanes 5 and 6). Taken together these results suggest that the acidic
form corresponds to pro-PLAP, whereas the major more basic form is
C-terminally cleaved PLAP. The identity of the third most basic form is
unknown but could represent C-terminally cleaved non-GPI-anchored PLAP
(22) or different charge forms of PLAP.
From the reactivity of site-specific antibodies with PLAP, it was
evident that the C-terminal signal sequence was cleaved from this
polypeptide during processing in SP K562 cells. However, it was not
clear if this was accompanied by addition of a GPI anchor to the
cleaved PLAP molecule. To determine whether the C-terminally processed
PLAP was GPI-anchored, we used hydrazine as an alternative nucleophilic
agent to GPI. Hydrazine should act as an effective acceptor for an
enzyme-activated
Further indirect confirmation that PLAP was GPI-anchored in SP K562
cells came from experiments with phospholipase D, which specifically
cleaves the lipid moiety from GPI-anchored proteins. Incubation of
putative GPI-anchored PLAP synthesized in SP K562 cells with
phospholipase D produced a cleavage product that was not observed on
incubation with non-GPI-anchored PLAP synthesized in K class cells
(results not shown). Additionally, when subjected to phase partition
between Triton X-114 and water, 40-50% of the translation product
synthesized in K562 cells partitioned into the Triton X-114 phase (Fig.
3C, lane 1). This contrasts to only 10-15% of the translation product synthesized in K class cells partitioning into the detergent phase (Fig. 3C,
lane 3). Therefore, taken together, these
observations indicate that a large proportion of PLAP is GPI-anchored
in SP K562 cells.
Influence of GPI Addition on PLAP Folding--
As discussed above,
PLAP is an excellent model for investigating the sequence of events
leading to catalytic addition of the GPI anchor to correctly folded
protein. Like many transmembrane proteins, the extracellular domain of
PLAP may contain intrachain disulfide bonds, which contribute to the
stabilization of proteins. The contribution of disulfide bonds to PLAP
biogenesis and the role that PLAP conformational maturation plays on
GPI anchor addition remain undocumented. There is a paucity of
information regarding number and position of disulfide bonds and active
site conformation within PLAP, as the crystal structure of the human
protein remains to be solved. Of the 513 amino acids present in nascent
PLAP, 6 are cysteine residues, several of which are in close proximity to the C-terminal signal sequence. These cysteine residues may participate in the formation of up to two intrachain disulfide bonds
within pro-PLAP (one cysteine residue is contained within the
N-terminal signal sequence). The C-terminal signal sequence in PLAP is
29 amino acids long, implying that translation of the polypeptide has
to be complete in order to expose the
The activity of GPI-anchored PLAP expressed on the surface of mammalian
cells has been measured previously and used to indicate that the active
site was correctly folded (29). To our knowledge the folding of PLAP
within an in vitro translation system has not previously
been determined due to the very low (pmol) amounts of protein
synthesized. The availability of a chemiluminescent assay, designed to
determine as little as 10
To determine whether folding of PLAP into an active conformation
required GPI anchor addition, we measured the activity of PLAP
synthesized and translocated into the ER of the K class cell line that
does not catalyze GPI anchor addition (16). PLAP may be folded as it is
translocated across the ER, or alternatively the polypeptide may only
attain the correctly folded conformation after GPI addition and release
from the transamidase. The translation product synthesized in the
presence of K class cells was enzymically active (3.4 ± 0.23 RLU/dpm), indicating that absence of a GPI anchor does not lead to
improper protein folding. We observed variability between individual
experiments, making it difficult to draw comparisons between the
efficiency of folding in K562 and K class cells; however, the enzyme
synthesized in K562 cells was consistently more active than the enzyme
synthesized in K class cells. This could reflect subtle differences in
the conformation of the protein when the C-terminal signal sequence is
still present. These data show that PLAP activity measured within the
ER of SP cells was derived from both the pro- and the GPI-linked
protein and that PLAP was able to fold into an enzymically active
conformation even in the absence of GPI addition.
Influence of PLAP Folding on GPI Addition--
Having established
that preventing disulfide bond formation in PLAP prevented protein
folding, we then went on to investigate the effect of preventing
polypeptide folding on GPI anchor addition. PLAP mRNA was
translated in either SP K562 or K class cells in the presence or
absence of reducing agent as described above. Formation of GPI-anchored
PLAP and pro-PLAP translation products were then monitored by IEF. As
expected, in the absence of added reducing agent during translation, SP
K class cells synthesized essentially only pro-PLAP (Fig.
4A, lane
3), whereas K562 cells produced both pro- and GPI-anchored
PLAP (Fig. 4A, lane 1). This pattern
of PLAP products remained unaffected in either cell type by the
inclusion of reducing agent during translation at concentrations that
we had established prevent correct protein folding (Fig. 4A,
lanes 2 and 4). We also assessed the
ability of the transamidase to catalyze the formation of the hydrazide
form of free PLAP by carrying out translation of PLAP mRNA in the
presence of hydrazine. When translation was carried out in the presence
of K562 SP cells, the hydrazide form of free PLAP was formed in the
presence of added DTT (Fig. 4B, lane
2). Hence, the GPI anchor or hydrazine was added to PLAP,
whether the protein was in either the folded or the unfolded state.
These results also agree with the observation that promini-PLAP, an
engineered and probably incorrectly folded protein, undergoes GPI
addition in this SP cell system. Folding of the extracellular domain of
PLAP may therefore be independent from events leading to GPI addition.
The fact that the transamidase recognizes both folded and unfolded PLAP
means that the only requirement for substrate recognition may be the
conformation of the C-terminal signal sequence. This may be governed
largely by its hydrophobicity, rather than by the conformation of
the entire protein, as changes in overall hydrophobicity of this signal
sequence have been shown to prevent protein GPI anchoring (30).
It remains to be determined whether the hydrophobic C-terminal signal
sequence must traverse the ER membrane prior to recognition by the
transamidase (30) or if the signal sequence triggers its own
integration into the lipid bilayer, enabling subsequent presentation of
the Interaction of PLAP with Transamidase Subunits--
Two gene
products (Gpi8p and Gaa1p) have recently been identified by genetic
analysis to be essential for GPI anchor addition in yeast (14, 15) and
in mammalian cells (16, 18) and probably constitute subunits of the
transamidase. To determine whether either of these subunits interacts
with pro-PLAP synthesized in our SP cell system, we used the
bifunctional cysteine-specific cross-linking reagent BMH to cross-link
proteins that were in close proximity to the nascent polypeptide chain.
As mentioned previously, PLAP contains several cysteine residues, two
of which are in close proximity to the
Discrete cross-linking products were obtained after the addition of BMH
to translated PLAP constructs in all conditions studied (Fig.
5, A-D, lane
2). Several of these remain unidentified, but at least three
can be attributed to an association of the folding ectodomain with
calreticulin and ERp57 (Fig. 5E). This observation is not
unexpected, as PLAP is a glycoprotein and most glycoproteins interact
with calnexin, calreticulin, and ERp57 at an early stage during their
biosynthesis (35). The appearance of the same cross-link products after
immunoprecipitation of either calreticulin or ERp57 is due to an
interaction between these two proteins as described previously (35).
Two cross-linking products to the Gpi8 subunit of the transamidase were
identified with relative molecular masses of ~120 and 130 kDa when
PLAP was translated in SP mutant IA cells (Fig. 5C,
lane 3). The presence of two cross-linking
products with differing mobility probably reflects cross-linking from
different positions within the respective proteins, although they may
represent an additional interaction with a third, as yet unidentified,
protein. These results indicate that PLAP was in close proximity to the Gpi8p subunit of the transamidase. We assume that the processed form of
PLAP associate either directly with this subunit of the transamidase or
indirectly via other transamidase subunits/cofactors. An interaction
with the Gpi8p transamidase subunit was also seen when
These results demonstrate that, where the transamidase is functional
but PLAP does not undergo processing to GPI-PLAP, an adduct to Gpi8p
was formed. This is probably due to accumulation of the pro-form of the
protein interacting directly with the Gpi8p transamidase subunit. The
Gpi8p has sequence homology to cysteine proteases and has been
suggested to be the catalytic component that cleaves the GPI attachment
signal peptide (20). The GPI signal sequence may be directly recognized
by Gpi8p or may be presented to this putative catalytic subunit of the
transamidase by another interacting subunit of the enzyme. It has been
suggested that Gaa1p subunit of the transamidase is involved in the
recognition of the protein substrate (14, 18), although the functions
of Gaa1p cannot be predicted from the primary amino acid sequence of
this protein. In the present study, we did not identify any
cross-linking products that immunoprecipitated with antiserum
recognizing the Gaa1p subunit of the transamidase (Fig. 5,
A-D, lane 4). The antiserum used in
these studies was able to react with Gaa1p on Western blots and by
immunoprecipitation (results not shown). The failure to detect a
cross-linking product to Gaa1p may be due to the fact that cysteine
residues within PLAP and Gaa1p were not in close enough proximity for
chemical linkage (BMH has a spacer arm of 16.1 Å). Therefore the
absence of PLAP-Gaa1p intermediates was not necessarily due to the lack of molecular interactions between these two proteins. Alternatively, it
may be that pro-PLAP may be transferred directly to the Gpi8p subunit
of the transamidase. The Gaa1p subunit of the transamidase may bind
GPI, recruiting this glycolipid to the pro-PLAP-Gpi8p complex and have
a correspondingly brief interaction with protein destined to be
GPI-anchored which cannot be detected using this approach. The fact
that we were able to observe cross-links of PLAP with Gpi8p, but not
with Gaa1p, may mean that the Gpi8p subunit of the transamidase has a
very slow reaction rate and may catalyze the rate-limiting step of the
transamidation reaction.
Quality control of post-translational modification is an important
facet of protein biogenesis. GPI quality control is probably mediated
by polypeptide retention in the ER followed by degradation via the
cytoplasmic proteasome (33). Misfolding of PLAP monomers within the ER
may not play an important role in this process, as both GPI-anchored
and non-GPI-anchored PLAP were folded in the SP cell system.
Alternatively, the direct interaction of precursor proteins with the
transamidase demonstrated here may explain how precursor GPI-anchored
proteins are retained in the ER. Modification of proteins by GPI
addition would then release the protein from the transamidase and allow
subsequent transport through the secretory pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
) site (10). The removal of the C-terminal
signal sequence and replacement with pre-formed GPI at the
-site is
catalyzed by a transamidase that resides within the ER membrane (11).
The GPI transamidase is proposed to bind to the GPI signal sequence and
attack the carbonyl group of the
-site amino acid. Cleavage of the
signal sequence results in formation of a carbonyl intermediate between
precursor protein and enzyme. The amino group on the terminal
ethanolamine of GPI then attacks this intermediate to produce
GPI-anchored protein (12, 13). Although this proposed reaction
mechanism for GPI addition is now widely accepted, isolation of an
active enzyme capable of such anchor attachment has not been achieved
to date.
) site (22, 23)
and have shown that soluble components of the ER lumen are needed for
formation of GPI-mini-PLAP (24). These cell-free systems have also
provided evidence that GPI anchoring proceeds via a transamidation
mechanism, as small nucleophiles such as hydrazine can replace the GPI
anchor (13, 25), and that the process is energy-independent (12).
However, these in vitro studies have not directly identified
molecular components of the processing machinery and are not
appropriate for a study of whether protein folding has a role on the
overall process of GPI anchor addition.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(Stratagene, Cambridge, United Kingdom (UK)) to
generate pBS/PLAP. Introduction of the mutation D484P into pBS/PLAP was
achieved by site-directed mutagenesis using a Quickchange mutagenesis
kit (Stratagene) with the following primers:
5'-GCCCCCCGCCGGTACCACCCCCGCCGCGCACCCG-3' and
5'-CGGGTGCGCGGCGGGGGTGGTACCGGCGGGGGGC-3', creating the plasmid pBS/PLAP-D484P.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Co-translational processing of mini-PLAP in
the presence of SP K562 cells. Panel A,
mini-PLAP mRNA was translated using rabbit reticulocyte lysate in
the presence (lanes 2-4) or absence
(lane 1) of SP K562 cells for 1 h at
30 °C. Isolated cells were treated with proteinase K (250 µg/ml)
(lanes 3 and 4) with addition of 1%
(w/v) Triton X-100 as indicated (lane 4). After
30 min at 0 °C, the protease was inhibited by addition of PMSF (1 mM). Labeled translation products were analyzed by SDS-PAGE
and autoradiography. Panel B, mini-PLAP mRNA
was translated in vitro for 1 h at 30 °C in a rabbit
reticulocyte lysate system supplemented with SP cells prepared from
K562 cells (lanes 1-3). Processed forms of
mini-PLAP were immunoprecipitated with three site-directed antibodies
raised to specific peptides in PLAP: endo-Ab (lane
1), amino-Ab (lane 2), and exo-Ab
(lane 3). The samples were analyzed by SDS-PAGE
and autoradiography. Identities of processed mini-PLAP forms in
panels A and B are shown according to
Kodukula et al. (22).
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Fig. 2.
Translocation of PLAP into the ER of SP K562
cells. PLAP mRNA was translated using rabbit reticulocyte
lysate in the presence (lanes 1, 2,
and 4-6) or absence (lane 3) of SP
K562 cells for 1 h at 30 °C. Isolated cells were incubated with
endoglycosidase H (1 unit) at 37 °C for 12 h (lane
2) or treated with proteinase K (250 µg/ml)
(lanes 5 and 6) with addition of 1%
Triton X-100 as indicated (lane 6). After 30 min
at 0 °C, the protease was inhibited by addition of PMSF (1 mM). Labeled translation products were then analyzed by
SDS-PAGE and autoradiography.
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Fig. 3.
C-terminal processing of PLAP.
Panel A, PLAP mRNA was translated for 1 h at 30 °C with rabbit reticulocyte lysate in the presence of SP
K562 cells (lanes 1-4) or SP K class cells
(lanes 5 and 6). An aliquot of each
translation was removed and cells isolated by centrifugation. Remaining
translation products were immunoprecipitated with endo-Ab
(lane 2), amino-Ab (lanes 3 and 5), or exo-Ab (lanes 4 and
6). Isolated cells or immunoprecipitates were then
resuspended in IEF solubilization buffer prior to focusing on an
Immobiline DryPlate, pH 4-7. Differently charged forms of PLAP were
identified using autoradiography and are indicated on the
right. Panel B, PLAP mRNA was
translated in vitro using rabbit reticulocyte lysate and
either SP K562 cells (lanes 1 and 2)
or SP K class cells (lanes 3 and 4)
with (lanes 2 and 4) or without
(lanes 1 and 3) 10 mM
hydrazine for 90 min at 30 °C. Following translation, samples were
immunoprecipitated with amino-Ab and samples were separated by SDS-PAGE
and visualized by autoradiography. The putative PLAP-hydrazide product
is indicated. Panel C, PLAP mRNA was
translated using a rabbit reticulocyte lysate and either SP K562 cells
(lanes 1 and 2) or SP K class cells
(lanes 3 and 4) for 90 min at
30 °C. Translation products were partitioned into aqueous and
detergent phases by Triton X-114 extraction as described under
"Materials and Methods." Samples were separated by SDS-PAGE and
visualized by autoradiography.
-carbonyl intermediate, and has been shown to
promote formation of the hydrazide of free mini-PLAP in translations
with RM (28). Mobility by SDS-PAGE of translation products synthesized
in SP K class cells was unaffected by inclusion of hydrazine (Fig.
3B, lanes 3 and 4). This
confirms previous studies, which showed that RM isolated from K class
cell line efficiently produced the pro-form of mini-PLAP, but
additional COOH processing of this product did not occur either in the
presence or absence of hydrazine (19). In the present study, incubation
of SP K562 cells with hydrazine resulted in production of a faster
migrating product (Fig. 3B, compare lanes 1 and 2). In these experiments, the hydrazide
form of free PLAP was the major translation product and may arise
directly from either processed pro-PLAP or via cleavage of the GPI
moiety from GPI-anchored PLAP. Cleavage of C-terminal signal sequence
at the
-site of pro-PLAP by the transamidase must therefore be
accompanied by GPI anchor addition to the protein, as there was no
evidence for the formation of free PLAP in translations with SP K562
cells (Fig. 3B, lane 1). From Fig.
3B it can be seen that on SDS-PAGE mature PLAP appeared to
co-migrate with pro-PLAP (lane 1 compared with
lane 3), and it is logical to suggest that the
difference in size between mature PLAP and non-GPI-anchored, free PLAP
(lane 1 versus lane
2) is due to GPI anchor attachment. Therefore, the additional translation product identified in K562 cells compared with K
class cells by IEF must be due to GPI anchor addition to C-terminally
cleaved PLAP. These results, combined with cross-reactivity of various
forms of PLAP with site-specific antibodies, suggest that in SP K562
cells the majority of PLAP was fully processed and also
GPI-anchored.
-site to the transamidase for
GPI attachment. Hence, PLAP folding, especially within the vicinity of
the C-terminal signal sequence, and the assumption of the correct
protein conformation could play a part in positioning pro-protein at
the
-site for optimal recognition by the transamidase. Therefore,
the location and kinetics of disulfide bond formation may play an
important role in GPI addition to PLAP.
13 g of a secreted
form of PLAP protein (see "Materials and Methods") has enabled us
to study folding of this hydrolytic enzyme using the SP cell system. To
examine the role of disulfide bond formation on the folding of PLAP
synthesized in the SP cell system, we translated PLAP mRNA under
reducing (in the presence of 5 mM DTT) and non-reducing conditions and measured the specific activity of products formed. The
rabbit reticulocyte lysate used in these experiments contains no added
DTT and supports the formation of native disulfide bonds in proteins
translocated across the ER membrane (26). When PLAP was synthesized in
the absence of added DTT, the translation product was enzymically
active (9.24 ± 2.04 RLU/dpm), indicating that it had folded
correctly. However, when the translation was carried out in the
presence of added DTT, essentially no enzyme activity was detected
(0.13 ± 0.05 RLU/dpm). The activity was normalized to take into
account any variation in the amount of protein synthesized in the
presence and absence of DTT. Small shifts in mobility of PLAP monomer
on non-reducing compared with reducing SDS-PAGE were also observed when
the translations were carried out in the absence of added DTT,
indicating the formation of intrachain disulfide bonds (results not
shown). Thus, it appears that, in our SP cell system, PLAP contains at
least one intrachain disulfide bond that must be formed to provide
enzymically active protein.
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Fig. 4.
Effect of reducing agents on GPI anchor
addition. Panel A, PLAP mRNA was
translated in vitro using rabbit reticulocyte lysate and
either SP K562 cells (lanes 1 and 2)
or SP K class cells (lanes 3 and 4)
with (lanes 2 and 4) or without
(lanes 1 and 3) 10 mM DTT
for 60 min at 30 °C. Following translation samples were
immunoprecipitated with amino-Ab and were evaluated by isoelectric
focusing and autoradiography. The different forms of PLAP are
designated on the right. Panel B, PLAP
mRNA was translated for 90 min at 30 °C using a rabbit
reticulocyte lysate and either SP K562 cells (lanes
1 and 2) or SP K class cells (lanes
3 and 4) in the presence of 5 mM DTT
(lanes 1-4) with the addition of 10 mM hydrazine (lanes 2 and
4). Translation products were separated by SDS-PAGE and
visualized by autoradiography.
-site to the transamidase (31). It has been shown that, in
comparison with the expression of correctly processed GPI-anchored PLAP
on the cell surface, non-GPI-anchored PLAP proteins became localized
intracellularly, probably in association with the ER membrane (32). The
retention of improperly processed GPI-anchored proteins within the ER
has been attributed to determinants contained within the hydrophobic
C-terminal signal sequence (33, 34). Our results suggest that folding
of unprocessed precursor proteins is unlikely to account for the
retention of these polypeptides by quality control mechanisms within
the ER. We can therefore conclude that GPI anchoring at least for PLAP
is not conformation-specific, and that protein folding and GPI addition
constitute two distinct processes that are independent of one another.
-site allowing cross-linking
at or near to the C-terminal signal sequence. PLAP mRNA was
translated either in the presence of SP K562, K class, or mutant IA
K562 cells and then treated with BMH. Mutant IA cells do contain a functional transamidase but are not able to synthesize the GPI moiety
destined to be attached to processed PLAP (3). PLAP-D484P mRNA,
which contains an Asp
Pro mutation at the
-site that prevents
GPI anchor addition (10), was also translated in the presence of SP
K562 cells and cross-linked with BMH as above. Cross-linking partners
to N-terminally processed forms of PLAP were identified by
immunoprecipitation with amino-Ab, and PLAP cross-links to subunits of
the transamidase were identified by immunoprecipitation with antibodies
raised to either the hGpi8p or hGaa1p.
-site mutant
mRNA was translated in SP parental K562 cells (Fig. 5D,
lane 3). This form of PLAP contains an
uncleavable C-terminal signal sequence and is therefore not
GPI-anchored. Therefore, in two independent experiments in which PLAP
was unable to undergo GPI addition, either because of a defect in GPI
biosynthesis (mutant IA cell line) or because the C-terminal signal
sequence was not cleaved from the protein (
-site mutant),
cross-linked products to Gpi8p were observed. The specificity of these
cross-linked products was underlined by the finding that radiolabeled
PLAP did not form an interaction with this transamidase subunit in mutant K cells. This cell line contains a non-functional Gpi8p, which
we can conclude is needed for interaction with proteins destined to be
GPI-anchored. No cross-linked product to PLAP translated in SP parental
K562 cells was observed (Fig. 5A, lane
3). The lack of cross-linked product with K562 cells is
likely due to the fact that cross-linking was studied after 1 h of
translation and the majority of PLAP was processed and GPI-anchored
after this time with SP K562 cells.
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Fig. 5.
Interaction of in vitro
synthesized PLAP with transamidase components. mRNA
encoding PLAP (panels A-C and E) and
site mutant of PLAP (panel D) were translated
in a rabbit reticulocyte system for 60 min in the presence of either SP
K562 cells (panels A, D, and
E), SP K class cells (panel B), and SP
mutant IA cells (panel C) for 60 min at 30 °C.
After termination of translation, cells were washed and, where
indicated, treated with 100 µM BMH (panels
A-E, lanes 2-4). The samples were
denatured with 1% SDS prior to immunoprecipitation with PLAP amino-Ab
(lanes 1 and 2), anti-Gpi8p
(lane 3), and anti-Gaa1p (lane
4) for panels A-D and with amino-Ab
(lane 1 and 3) or anti-calreticulin
(lane 2) or anti-ERp57 (lane
4) for panel E. All samples were
analyzed by SDS-PAGE and autoradiography. The cross-link products
immunoprecipitated with Gpi8p antibodies are indicated with a
star and to calreticulin and ERp57 with a filled
circle.
-site mutant contained a C-terminal signal sequence that was not
processed in SP K562 cells (results not shown), and so probably
remained in a "dead-end" complex with the transamidase. In the
mutant IA cell line, GPI is not available to react with the
carbonyl-enzyme intermediate. Therefore, a pro-PLAP-Gpi8p complex must
have been formed, which was not released or was very slowly released
from the transamidase, in comparison to the situation in K562 cells.
From these results we can infer that nucleophilic attack by GPI is
required for release of the pro-PLAP intermediate from the Gpi8p
subunit of the transamidase. These data also demonstrate that, in the
mutant IA cells, pro-PLAP interacts with Gpi8p in the absence of GPI.
Therefore, direct or indirect recognition of pro-PLAP by Gpi8p occurs
independently of GPI binding to the transamidase and may occur in
either the presence or absence of GPI. It has been shown previously
that, when prepromini-PLAP was translated in the presence of RMs
derived from GPI-deficient cells, far smaller amounts of both
GPI-linked and free mini-PLAP were synthesized compared with
translation products synthesized in wild-type RMs (25). These workers
suggested that GPI might act as an allosteric effector of the
transamidase such that an activated complex between the pro-form of the
protein and the transamidase only occurs in the presence of GPI.
However, the fact that hydrazide-mini-PLAP was generated in microsomes
derived from a GPI-deficient cell line provided evidence that a
carbonyl intermediate forms in the absence of GPI (12). The
identification of a pro-PLAP-Gpi8p transamidase intermediate formed in
the absence of GPI in the present study is entirely consistent with the
action of a transamidase.
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ACKNOWLEDGEMENT |
---|
We thank Dr. M. E. Medof for providing PLAP clones and the K and IA class cell lines.
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FOOTNOTES |
---|
* This work was supported by Medical Research Council Grants G9722981 and G9722026 and by a grant from the Royal Society.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. Tel.: 44-61-275-5103;
Fax: 44-61-275-5082; E-mail: neil.bulleid@man.ac.uk.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010128200
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ABBREVIATIONS |
---|
The abbreviations used are: GPI, glycosylphosphatidylinositol; BMH, bismaleimidohexane; CSPD, 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'chloro)-tricyclo[3.3.1.1]decan]-4-yl)phenyl phosphate; DTT, dithiothreitol; ER, endoplasmic reticulum; IEF, isoelectric focusing; PAGE, polyacrylamide electrophoresis; PLAP, human placental alkaline phosphatase; RM, rough microsomal membrane; SP, semipermeabilized; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; RLU, relative light unit(s); Ab, antibody.
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