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J. Biol. Chem., Vol. 277, Issue 15, 12816-12823, April 12, 2002
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From the Graduate Program for Regulation of Biological Signals,
Graduate School of Bioagricultural Sciences, Nagoya University,
Chikusa, Nagoya, 464-8601 Japan
Received for publication, January 8, 2002, and in revised form, January 30, 2002
Wnt is a family of cysteine-rich secreted
glycoproteins, which controls the fate and behavior of the cells in
multicellular organisms. In the absence of Drosophila
segment polarity gene porcupine (porc), which
encodes an endoplasmic reticulum (ER) multispanning transmembrane
protein, the N-glycosylation of Wingless (Wg), one of
Drosophila Wnt family, is impaired. In contrast, the
ectopic expression of porc stimulates the
N-glycosylation of both endogenously and exogenously
expressed Wg. The N-glycosylation of Wg in the ER occurs
posttranslationally, while in the presence of dithiothreitol, it
efficiently occurs cotranslationally. Thus, the cotranslational
disulfide bond formation of Wg competes with the
N-glycosylation by an oligosaccharyl transferase
complex. Porc binds the N-terminal 24-amino acid domain (residues
83-106) of Wg, which is highly conserved in the Wnt family and
stimulates the N-glycosylation at surrounding sites. Porc
is also necessary for the processing of Drosophila Wnt-3/5
in both embryos and cultured cells. Thus, Porc binds the N-terminal
specific domain of the Wnt family and stimulates its posttranslational
N-glycosylation by anchoring them at the ER membrane
possibly through acylation.
Wnt is a family of cysteine-rich secreted glycoproteins and
has been identified in many vertebrates and invertebrates. They have
been shown to have important roles for the decision of cell fate and
behavior at multiple stages during development and cancer. They bind a
family of specific receptors on the cell surface (Frizzled family) and
activate the cell signaling pathways to elicit their effects on, for
example, gene transcription (1, 2). One of the hallmarks of the Wnt
family is the existence of 23 or 24 cysteine residues at conserved
positions in the protein molecules. It has been assumed that these
cysteine residues may have a critical role for folding of Wnt proteins
by disulfide bond formation. However, direct evidence demonstrating
that biologically active Wnt proteins contain any intra- or
intermolecular disulfide bonds has not been shown.
The processing and secretion of Wnt proteins was studied with cultured
cells engineered to express various Wnts (3, 4). The processing of Wnt
is not efficient in most cell types, because multiple processing
intermediates are present. Wnt is therefore not well secreted outside
of the cells. Most of Wnt protein associates with a HSP70 protein, BiP,
and is retained in the ER1
(5). Meanwhile, wg mutants with lesions in the secretion and transport have been identified (6-9), suggesting that Wg processing and secretion is also complex in Drosophila. Caenorhabditis
elegans Mom-3 appears to be necessary for Mom-2 (Wnt) processing
or secretion in addition to Mom-1 (see below). Screening for genes
involved in Wg signaling by Drosophila deficiency kits has
also identified a new gene(s), whose product(s) is required for the
processing or secretion of Wg (10). These results indicate that the
processing and secretion of Wnt are complex and that a number of
specific factors are involved in these events.
One of the Drosophila segment polarity genes,
porcupine (porc) encodes a multipass
transmembrane ER protein, which is required for the normal distribution
of Wg in embryos (11). In porc mutant embryos, Wg is
sequestered in its synthesizing cells and not distributed among the
surrounding cells. Wg signaling components are well conserved in
multicellular organisms, and porc homologs are also present
in other species. C. elegans porc homolog mom-1
was identified in a search for maternal genes necessary for endoderm
formation (12, 13). Mom-1 is necessary in Mom-2 producing cells as Porc is required in Wg-synthesizing cells. Vertebrate (mouse and
Xenopus) homologs of porc have been recently
identified and shown to modify the N-glycosylation of Wg and
mouse Wnt proteins in cultured cells (14). These results demonstrate
that the porc gene family encodes the evolutionary conserved
ER membrane proteins involved in the processing of the Wnt family.
In this study, the N-glycosylation of Wg in
Drosophila S2 cells and imaginal discs was characterized in
detail. We conclude that the cotranslational disulfide bond formation
of Wg competes with the N-glycosylation and that Porc
stimulates the posttranslational N-glycosylation by
anchoring Wg at the ER membrane. Since Porc is suggested to be a member
of the membrane-associated acyltransferase family, it may tether Wg to
the ER membrane through acylation.
Molecular Biology--
Three C-terminal deleted (34, 66, and 110 amino acids) Porc mutants were generated by PCR with PorcHA28
cDNA (encoding Porc tagged with three HA epitopes at amino acid 28)
as a template and the following primers:
5'-CGAGGCCTCTATTCCAGGTCATCGCCCAGCAGCAC-3' ( Transfection, Cell Labeling, and
Immunoprecipitation--
Drosophila S2 cells in six-well
plates were transiently transfected with 0.4 µg of the indicated DNA
by Effectene reagent (Qiagen) according to the manufacturer's
instruction. The expression of transfected genes was induced by
heat-shocking the cells at 37 °C for 45 min. Tunicamycin was added
to the cells at 1 h prior to heat shock and present throughout the
experiment at 15 µg/ml. After heat shock induction, the cells were
washed twice with methionine-free labeling medium and then incubated at
25 °C for 20 min in the same medium (0.5 ml/well). Labeling was
initiated by adding 100 µCi of Redivue Pro-mix
L-35S in vitro cell labeling mix
(Pro-mix; Amersham Biosciences) and continued for 10 min at 25 °C.
When the effect of dithiothreitol (DTT) was analyzed, it was added to
the labeling medium at 30 s or 5 min before the addition of
radioisotope (at the final concentration of 10 mM). For
pulse-chase experiments, cells were washed twice with labeling medium
supplemented with 0.5 mg/ml methionine after 10 min of pulse labeling
and then chased in the same medium at 25 °C. After labeling, the
cells were washed twice with ice-cold phosphate-buffered saline and
then solubilized with radioimmune precipitation buffer containing
protease inhibitor mixture (Roche Molecular Biochemicals). The cell
lysates were centrifuged for 5 min, and then the supernatants were
collected. The antibody was added to the supernatants and incubated for
1 h at 4 °C. Protein A-Sepharose was then added and incubated
for 2 h at 4 °C. The beads were washed five times with
radioimmune precipitation buffer, and then final pellets were suspended
with reducing or nonreducing SDS-PAGE sample buffer.
The Assay of the Sensitivity of Wg against Exogenous
Trypsin--
The lysate of S2 cells expressing wg was
prepared by gently homogenizing the cells in 0.5 ml of lysis buffer
(0.25 M sucrose, 1 mM DTT, and 10 mM HEPES, pH 7.3) with a glass homogenizer on ice. The cell
lysate was then treated without trypsin or with 0.01% trypsin in the
presence or absence of 1% TX-100 for 3 h on ice.
Phenylmethylsulfonyl fluoride was added at a final concentration of 20 mM, and then the reaction mixtures were transferred into 5 ml of 0.1 M Tris-HCl, pH 8.0, 1% SDS in a boiling water
bath. Total proteins were precipitated with trichloroacetic acid
followed by suspending in the SDS-PAGE sample buffer. Each band on
Western blot was scanned and quantified by NIH Image software.
Western Blot--
After SDS-PAGE, the proteins in the gel were
transferred to nitrocellulose membranes (Hybond ECL; Amersham
Biosciences). The membranes were blocked with TBST containing 5% skim
milk for 1 h at room temperature. They were then incubated with
antibodies at the indicated dilutions at 4 °C overnight and then
washed six times with TBST (each for 10 min). The horseradish
peroxidase-conjugated secondary antibodies were added at 3000-fold
dilution and incubated for 2 h at room temperature. The membranes
were washed as above, and the signal detection was done by an ECL
system (Amersham Biosciences).
The Analysis of the N-Glycosylation of Endogenous Wg Expressed in
Imaginal Discs--
The imaginal discs expressing wg (wing,
leg, and eye-antenna discs) were dissected from 50 wandering third
instar larvae (OreR). They were homogenized in the nonreducing SDS-PAGE
sample buffer and then divided into two equal parts.
RNA Injection into Drosophila Embryos and Immunostaining of
Embryos--
Capped porc, porc3HA28, The N-Glycosylation of Wg in Drosophila S2 Cells and Imaginal
Discs--
Since Drosophila S2 cells were used to produce
biologically active Wg (18) and Xenopus Wnt-8 (19) in the
medium, we analyzed the processing of Wg in this cell line. When
wg is expressed in S2 cells, three forms of Wg with
different molecular sizes are detected (forms I, II, and III). Since
only form I is detectable in the presence of tunicamycin (an inhibitor
of N-glycosylation), forms II and III contain
N-linked glycan chains. In the presence of ectopic
porc, form III is accentuated, and the largest form IV is
also present (Fig. 1A). The
forms II, III, and IV contain one, two, and three N-glycan
chains, respectively (see Fig. 4B). The form III is the
major protein detected in early Drosophila embryos (not
shown) and imaginal discs (Fig. 1D). Thus, Wg is normally
N-glycosylated at two sites, and Porc stimulates this processing in the ER. Furthermore, the overexpression of both wg and porc also induces the ectopic
N-glycosylation of Wg at three sites.
Forms I and II could be inside the ER or the translocational
intermediates through the ER membrane. To distinguish these
possibilities, the cells expressing wg were lysed in an
isotonic buffer by gentle homogenization to disrupt the plasma
membrane. Trypsin was then added with or without TX-100. After
inactivating the protease, Wg was analyzed by Western blot. Forms I,
II, and III are resistant to exogenous protease, demonstrating that
they are inside the ER membrane (Fig. 1B). These results
thus suggest that forms I and II are posttranslationally
N-glycosylated in the ER (see also Fig.
2). As shown by percentage reduction, the
form I is more susceptible against the exogenous protease than forms II
and III, suggesting that some fraction of form I is the
ER-translocating intermediate or the incompletely folded protein
hypersensitive to protease.
We then analyzed the N-glycosylation of endogenously expressed Wg in
wild type, porc hemizygous mutant, and
porc-overexpressing third instar larvae. Some of the
hemizygous porc mutants with maternal porc
contribution can develop to the third instar larvae, in which
wg-dependent gene expression is lost (11). The
overexpression of porc in the third instar larvae was
achieved by crossing UAS-porc and 69B Gal 4 driver lines.
Strong and uniform Gal4 expression in the discs (overlapping with
wg expression domain) was observed with 69B (20). Wing, leg,
and eye-antenna discs were dissected from the larvae, and then the
N-glycosylation of Wg was analyzed by
[35S]methionine labeling followed by immunoprecipitation.
Forms II and III are detected by this analysis. Thus, the
N-glycosylation of form II (resulting in form III) is more
rate-limiting than that of form I in vivo. The ratios of the
form III to II are 1.3, 0.6, and 5.2 in wild type, porc
hemizygous mutant, and porc-overexpressing discs,
respectively (Fig. 1C). The form III is less synthesized in
porc hemizygous mutant discs, indicating that the loss of
the function of porc results in the inefficient
N-glycosylation of endogenously expressed Wg. Meanwhile, the
form III is predominantly synthesized in porc-overexpressing
discs. Thus, the overexpression of porc stimulates the
N-glycosylation of both endogenously (in discs) and
exogenously (in S2 cells) expressed Wg. To address the state of
endogenous Wg in vivo, Wg expressed in the imaginal discs of
the third instar larvae was analyzed by reducing and nonreducing
SDS-PAGE followed by Western blot. Fig. 1D shows that the
endogenously expressed Wg is present as the monomer in vivo. Moreover, Wg migrates faster under nonreducing compared with reducing conditions, indicating that Wg folds by intramolecular disulfide bonds.
The Effects of Inhibiting Disulfide Bond Formation on the
N-Glycosylation of Wg--
Because Wg contains 23 cysteine residues
and appears to form intramolecular disulfides in the imaginal discs
(Fig. 1D), we tested whether disulfide bond formation has
roles on the N-glycosylation of Wg. The
N-glycosylation of Wg was compared in
wg-expressing S2 cells with or without DTT. DTT has been
used as a reversible inhibitor of disulfide bond formation of newly
synthesized proteins in the ER (21-24). The cells expressing
wg alone and both wg and porc were
treated with 10 mM DTT for 30 s before labeling. After 10 min of labeling with [35S]methionine in the continued
presence of DTT, the cells in one well were washed twice with ice-cold
phosphate-buffered saline containing 20 mM
N-ethylmaleimide, and the cell lysates were prepared with
radioimmune precipitation buffer containing N-ethylmaleimide as above. The cells in two wells were washed and chased for 2 h
either in the presence or absence of DTT. The same sets of cells were
also analyzed by 10-min pulse labeling followed by a 2-h chase in the
complete absence of DTT. The immunoprecipitates were analyzed by 10%
SDS-PAGE under reducing and nonreducing conditions. As shown in Fig.
2A, in the complete absence of DTT, form I decreases, while
forms II and III increase during the chase period. Under the same
condition except with ectopic porc, the form III is
synthesized more during the pulse, and forms I and II decrease while
forms III and IV increase during the chase. In the cells treated with DTT for 30 s before labeling, form III is accentuated and becomes the major protein synthesized during the pulse. The sizes of all forms synthesized in the presence of DTT are larger than those synthesized in the absence of DTT. This is due to the alkylation of
free cysteine residues by N-ethylmaleimide. During a 2-h
chase period without DTT, forms II and III slightly increase, and form I disappears. In addition, the sizes of forms II and III become smaller, indicating that the free cysteine residues of Wg exposed by
DTT form disulfide bonds and are not alkylated by
N-ethylmaleimide (see Fig. 2B). The patterns of
N-glycosylation do not change during the chase with DTT,
except the amount of labeled Wg is reduced. Under the same condition as
above except with ectopic porc, the level of forms I and II
synthesized during the pulse becomes almost undetectable. Thus, the
effects of DTT and Porc on the N-glycosylation of Wg are
additive in the ER with partially reduced condition (treatment of cells
with DTT for 30 s before labeling). The form IV appears during the
chase without DTT but not with DTT, demonstrating that the continued
presence of DTT for a long period (2 h) slows down the
N-glycosylation. The analysis of the same samples by nonreducing 10% SDS-PAGE demonstrates that Wg forms both intra- and
intermolecular disulfide bonds in S2 cells (Fig. 2B). The species of bands with and without ectopic porc are
identical, indicating that Porc is not involved in disulfide bond
formation. Consistent with the above results, Wg synthesized in the
presence of DTT forms both intra- and intermolecular disulfide bonds
during the chase without DTT. Porc does not affect this process.
Meanwhile, wg-expressing S2 cells treated with DTT for 5 min
before 10-min labeling (resulting in the ER with fully reduced
condition) synthesize mostly the form III and the trace amount of forms
I and II. Under this condition, Porc has almost no effects on the
N-glycosylation (Fig. 2C). All of the above
results suggest the following: 1) The N-glycosylation of Wg
occurs posttranslationally; 2) Wg is efficiently
N-glycosylated by a cotranslational manner if disulfide bond
formation is inhibited; 3) Porc stimulates the posttranslational N-glycosylation of Wg without affecting disulfide bond
formation. The effect of Porc on N-glycosylation is specific
to Wg, since it does not modify the processing of partial
Drosophila laminin The Binding of Porc with Wg Is Essential for Its Activity--
As
indicated in Fig. 2, A and B, by the
arrowheads, Porc is likely to bind Wg in the cells. To test
if the binding of Porc with Wg is essential for its activity, the
C-terminal deletion mutants of Porc tagged with HA epitopes were
constructed (HA- Porc Functions on the N-terminal 24-Amino Acid Domain (83-106) of
Wg--
To analyze the domain of Wg responsible for its inefficient
N-glycosylation, the series of four deletion mutants
(containing the N-terminal 367, 282, 196, and 111 amino acids of Wg)
were generated, and their processings in S2 cells were tested. The wild
type and deletion mutants were tagged with triple Myc epitopes at the
arginine 111 of Wg, because an anti-Wg antibody recognizes an 85-amino
acid epitope present at the C-terminal half of Wg. The signal sequence
cleaved but not N-glycosylated forms are present in all
mutants tested. Furthermore, Porc stimulates the
N-glycosylation of all mutants (Fig.
4A). Thus, the
N-terminal 111-amino acid sequence is sufficient to reconstitute both
the inefficient N-glycosylation of Wg and its stimulation by
Porc. This deletion construct (Fig. 4, 111) contains only
two cysteine residues in the mature protein and does not form multimers
by intermolecular disulfide bonds (not shown). Thus, Porc enhances the
posttranslational N-glycosylation of Wg independent of the
types of disulfides (either monomer or multimer) (see also Figs. 1 and
2). Although all deletion mutants have three potential
N-glycosylation sites (Asn49,
Asn103, and Asn108), only a single site appears
to be glycosylated. These results suggest that Wg has two
N-glycosylation sites at Asn414 and either
Asn49, Asn103, or
Asn108.
To determine the N-glycosylation sites of Wg, we constructed
Wg mutants in which the potential N-glycosylation sites
(NX(S/T)) were knocked out by substituting the serine
or threonine residues to alanine. The N-glycosylation of
these mutants is shown in Fig. 4B. The
N-glycosylation pattern of T51A and S110A mutants is
identical to that of wild type, indicating that Asn49 and
Asn108 are not N-glycosylated. Meanwhile, S105A
and T416A mutants result in the forms I and II; in addition, the
S105A/T416A double mutant produces only the form I. These results
demonstrate that Wg is N-glycosylated at Asn103
and Asn414. As shown in Figs. 1 and 2, Wg can be
N-glycosylated at three sites to produce the form IV when
both wg and porc are overexpressed. The
S105A/T416A double mutant gives two additional bands specifically in
the presence of ectopic porc. Thus, Wg could be
N-glycosylated at Asn49 and Asn108
when the normally N-glycosylated sites (Asn103
and Asn414) are knocked out and both wg and
porc are overexpressed.
The comparison of the N-terminal amino acid sequences of Wg,
Drosophila, and mouse Wnts reveals that the amino acid
sequence surrounding Asn103 (residues 83-105) of Wg is
conserved among them (Fig. 4C). To test whether this domain
is a target for Porc, we constructed the following chimeric proteins.
Each chimeric protein consists of a mature bovine prolactin lacking
N-glycosylation site and 1) Wg signal sequence (amino acids
1-34 of Wg) (PRO), 2) the N-terminal 106 amino acids of Wg (GSM), 3)
Wg signal sequence and amino acids 73-106 of Wg (RDN), and 4) Wg
signal sequence and amino acids 83-106 of Wg (VKG). Three chimeric
proteins (GSM, RDN, and VKG) are N-glycosylated, and the
N-glycosylated forms are predominantly synthesized in the
presence of ectopic porc. Porc does not affect the
unglycosylated PRO chimeric protein. The effect of ectopic porc on the N-glycosylation of VKG appears to be
less than that on the GSM and RDN (Fig. 4D). Furthermore,
co-immunoprecipitation experiments demonstrate that Porc binds the RDN
and VKG (with less extent) but not PRO (Fig. 4E). These
results demonstrate that Porc binds the N-terminal 24-amino acid domain
(residues 83-106) of Wg and stimulates the N-glycosylation
at Asn103.
Porc Is Also Necessary for the Processing of DWnt-3/5--
Porc
functions on the N-terminal domain of Wg, which is conserved among Wnt
family members as described above. It is therefore possible that Porc
functions on the processing of other Drosophila Wnt proteins
in addition to Wg. To address this possibility, we focused on DWnt-3/5,
because its specific antibody was available besides Wg. In wild type
embryos at stage 13, DWnt-3/5 is mainly localized on the commissural
axon tracts of the central nervous system (Fig.
5A) as previously reported
(16). In contrast, DWnt-3/5 appears to be confined in the cell bodies
of neurons at the ventral nerve cord of porc embryos (Fig.
5B), which corresponds to DWnt-3/5 RNA expression
domain (16). The shape and number of axon tracts is somewhat
disorganized in porc embryos, but they are clearly present
based on the staining pattern by monoclonal antibody BP102 (Fig. 5,
C and D). Thus, in porc embryos, both
Wg and DWnt-3/5 are not secreted from the synthesizing cells. In
addition, Porc binds DWnt-3/5 and stimulates its
N-glycosylation in S2 cells (not shown). These results
therefore demonstrate that Porc can function on the
N-glycosylation of multiple Drosophila Wnt
proteins.
The processing and secretion of Wnt family was shown to be
inefficient in many cell types expressing various Wnts.
However, the mechanism of the processing and secretion of Wnts has
never been studied in detail. We therefore addressed the question why the processing of Wnt family is inefficient and how it is modified by
Porc with Drosophila S2 cells expressing wg.
The N-glycosylation of Wg is rate-limiting in S2 cells;
therefore, multiple (three) bands of Wg with zero, one, and two
N-glycan chains are detected. This observation is similar to
that made by mouse Wnt 1 expressed in quail QT6 cells (25). Mouse Wnt 1 has four potential N-glycosylation sites (Asn29,
Asn316, Asn346, and Asn359), and
when expressed in the QT6 cells, three out of four potential N-glycosylation sites (Asn29,
Asn316, and Asn359) were in fact glycosylated.
Multiple bands detected represent Wnt 1 with different numbers (0-3)
of N-glycan chains. Because inefficient
N-glycosylation was also reported with other Wnts (3, 4,
14), this appears to be a common feature of the Wnt family.
The N-glycosylation of endogenous Wg expressed in imaginal
discs is impaired in porc hemizygous mutant larvae (Fig.
1C). However, the small amount of mature form III can be
detected in the absence of zygotic porc. This could be due
to the following: 1) the residual activity of maternal porc
is able to support the N-glycosylation of Wg to some extent;
2) Wg could be inefficiently N-glycosylated by a
Porc-independent mechanism; or 3) porcPB16 is not
completely null. We cannot distinguish these possibilities at this
point. The overexpression of porc enhances the
N-glycosylation of endogenously expressed Wg (Fig.
1C), which exists as a monomer and folds by intramolecular
disulfide bonds (Fig. 1D). These results suggest that Porc
is necessary for the efficient N-glycosylation of both
endogenously and exogenously expressed Wg independent of the types of disulfides.
Oligosaccharyl transferase (OST) complex is localized at the ER
membrane and closely associated with a translocon (26). Therefore, the
OST complex is thought to transfer an oligosaccharide chain from
dolichol pyrophosphate that is anchored to the ER membrane to the
consensus N-glycosylation site of nascent chain when it is
12-13 amino acids apart from the translocon (27). Because the OST
complex and one of its two substrates, the oligosaccharide chain, are
fixed at the ER membrane, they are able to move in only two dimensions.
In contrast, the other substrate, the polypeptide containing
N-glycosylation sites, can freely move in the ER lumen. Thus, it has been believed that the N-glycosylation of
proteins does not occur once their translations are completed. However, Wg appears to be an exception. The positional constraint of OST complex
and dolichol-linked oligosaccharide suggests that Wg is necessary to be
in direct contact with the ER membrane for the posttranslational
N-glycosylation. Consistent with this hypothesis, the
membrane-anchored Wg at its C terminus is more efficiently glycosylated
than the wild type secreted form (not shown). The N-glycosylation of membrane-anchored Wg is further improved
by Porc (not shown), which is consistent with Porc functioning on the
internal sequence of Wg, and, as a result, the
N-glycosylation sites can get access to the OST complex with
higher efficiency. The other examples of the posttranslational
N-glycosylation include a truncated form of a
peptidylglycine Why is Wg posttranslationally N-glycosylated? This is because Wg has 23 cysteine residues and cotranslationally forms disulfide bonds. It
apparently competes with the N-glycosylation. The
competition between N-glycosylation and disulfide bond
formation was also observed with some proteins (e.g. a
tissue-type plasminogen activator (32), carboxypeptidase Y with extra
N-glycosylation sites (33), and hemagglutinin-neuraminidase
glycoprotein of Newcastle disease virus (34)). These proteins were
ectopically N-glycosylated at normally unused sites in the
presence of DTT. Under the fully reduced condition of ER with DTT,
disulfide bond formation is inhibited, and the
N-glycosylation of Wg efficiently occurs cotranslationally. Porc has almost no effects in this case (Fig. 2C). Since the
pattern of the disulfide bond formation of Wg does not change in the
presence of ectopic porc (Fig. 2B), Porc is not
involved in this processing event. However, the possibility cannot be
completely ruled out that Porc enhances the N-glycosylation
of Wg by transiently pausing the disulfide bonds formation (see below).
Porc binds the N-terminal 24-amino acid (residues 83-106) domain of Wg
and stimulates the posttranslational N-glycosylation at
Asn103. In this domain, the
GX6ECQXOFRX2RWNC
motif is conserved in many Wnt family members. Apparently, this motif
has important roles for Wg processing or secretion, since the
substitution of the last cysteine (Cys104) to alanine
(wgIL114 allele) impairs the secretion of Wg at the
restrictive temperature (6). Most of the Wnt proteins including Wg (but
not all) have a single N-glycosylation site in this domain.
If the cysteine residue at 104 (Cys104) forms a disulfide
bond, it is likely that the asparagine at 103 (Asn103)
cannot be N-glycosylated. If this is the case, Porc may
delay disulfide bond formation at Cys104, thereby enhancing
the N-glycosylation at Asn103. However, the
effect of Porc does not seem to be restricted to the
N-glycosylation site in its binding domain. As shown in Fig. 4B, both Asn49 and Asn108 can be
N-glycosylated in the presence of ectopic porc if
the usually N-glycosylated Asn103 and
Asn414 are knocked out. These results therefore suggest
that Porc binds the N-terminal conserved domain of Wnt family and
anchors Wnt proteins at the ER membrane. The posttranslational
N-glycosylation of Wnt proteins is then accelerated at the
surrounding sites of the ER membrane anchored region by the increased
access of NX(S/T) sequence to the OST complex.
Consistent with the data demonstrating that Porc functions on the
N-terminal conserved domain of the Wnt family, Porc is also found to be
necessary for the processing of DWnt-3/5 in both embryos (Fig. 5) and
cultured cells (not shown). DWnt-3/5 protein is restricted in its
synthesizing cells and not distributed at the axon tracts in the
central nervous system of porc embryos. As revealed by anti-BP102 monoclonal antibody staining, porc embryos have
the axon tracts. Thus, the mislocalization of DWnt-3/5 protein does not
result from the loss of localization targeting structures. This
observation is similar to that made with Wg (6). However, it remains to
be answered whether Porc is necessary for the processing of other
Drosophila Wnt proteins.
Finally, we propose the following model for the
N-glycosylation of Wnt family. Wnt proteins
cotranslationally form intramolecular disulfide bonds in
vivo, and as a result, the N-glycosylation is
inhibited. Porc then binds the N-terminal specific domain (including GX6ECQXQFRX2RWNC
motif) of Wnt and stimulates the N-glycosylation at the
surrounding sites. Porc has been recently suggested to be a member of
the membrane-associated acyltransferase family (35, 36) and therefore
may transfer fatty acid onto one of the amino acids of Wg. The covalent
attachment of fatty acid might allow Wg to be tightly associated with
the ER membrane, and as a result, the access of NX(S/T)
sites to the OST complex would be accelerated. Because Wg and DWnt-3/5
are not secreted from the synthesizing cells in porc
embryos, the role of Porc is different from that of Skinny
hedgehog, which is an acyltransferase required for the
palmitoylation and signaling activity but not secretion of
Hedgehog. We would propose that the acylation of Wg by Porc is
primarily necessary for the posttranslational
N-glycosylation of Wg. It remains to be established whether
Porc functions as a bona fide acyltransferase for Wg.
Quality control aides the folding of proteins by retaining them in the
specialized compartment of ER. Various ER chaperones are involved in
the quality control, and calnexin and calreticulin require
glucose-trimmed N-linked oligosaccharides for their
recognition of unfolded glycoproteins (37). Since the loss of Porc
results in the hypoglycosylation of Wg, calnexin, and calreticulin may fail to bind Wg efficiently in the ER. As a result, Wg remains unfolded
and incompetent for exit from the ER. The processing of the Wnt family
in the secretory pathway is very complex. It requires not only Porc but
also other proteins (e.g. C. elegans Mom-3) (13).
The molecular analysis of their functions will be essential to
understand how Wnt signaling is regulated at the level of ligand presentation.
We thank S. Cumberledge for anti-Wg antibody,
T. Niimi for Drosophila lam *
This work was supported by a Research Fellowship from the
Japan Society for the Promotion of Science for Young Scientists (to
T. K.) and a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (to Y. K. and T. K.).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.
Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M200187200
The abbreviations used are:
ER, endoplasmic
reticulum;
DTT, dithiothreitol;
OST, oligosaccharyl transferase;
HA, hemagglutinin.
Drosophila Segment Polarity Gene Product Porcupine
Stimulates the Posttranslational N-Glycosylation of
Wingless in the Endoplasmic Reticulum*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
34),
5'-CGAGGCCTCTAGGACAGGGAGTTCAATCGCCGTTT-3' (66),
5'-CGAGGCCTCTAGGCTAGGAACGCCAGCGAGATGAG-3' (110), and M13 reverse
primer. The PCR products were then cloned in pCaspeR-hs. To construct a
Myc epitope-tagged Wg, the DNA fragment encoding triple Myc epitopes
was first PCR-amplified with pKK-1 (carrying an insert DNA encoding
triple Myc epitopes) as a template and the following two primers:
5'-TACAGCTCGAGGGGTGAACAAAAGTTGATTTCTGAA-3' and
5'-AATAGCTCGAGAAGGATCCGTTCAAGTCTTCTTCTG-3'. The PCR product was
digested with XhoI and cloned in the same restriction enzyme site (at amino acid 111) of wg cDNA. This was then used
as a template for PCR as described below. Deletion mutants of Myc
epitope-tagged Wg were constructed by PCR with the following sets of
primers: 5'-GTTATGCGGCCGCATGGATATCAGCTATATCTTCGTCATCTGCCTG-3' and
either 5'-CGTCTAGATTATCTATTATGCTTGCGTCCCTGACG-3' (367),
5'-CGTCTAGATTAAGCGTTGGTGGCCCGGAGACTGTTGGTCAC-3' (282),
5'-CGTCTAGATTAGGAGAACTTGAACCCGAATCCGATGTTGTC-3' (196), or
5'-CGTCTAGATTACCTCGAGAAGGATCCGTTCAAGTCTTCTTC-3' (111). The PCR products
were digested with NotI and XbaI and cloned in
the same restriction enzyme sites of pCaspeR-hs. The Wg mutants (T51A, S105A, S110A, and T416A) were constructed with the TaKaRa LA PCR in vitro mutagenesis kit and the following mutagenic
primers: 5'-GTCCATGTACATGATGGGCGCAATGTTGTTGGGTTCGCCGA-3'
(T51A), 5'-CCTCGAGAAGTTTCTCGTCGCGCAGTTCCAGCGGCGATTTC-3' (S105A), 5'-CGAATAGATTTTTGCCCCTCGCGAAGTTTCTCGTCG-3'
(S110A), and 5'-GCCGTCGACGCCCAGCGAGGCCTCATTGCACTGGCGGCCAT-3' (T416A).
These mutated wg cDNAs were then cloned in pCaspeR-hs.
The S105A/T416A mutant was constructed by substituting the
HpaI-BglII fragment of T416A mutant with that of
S105A mutant. To construct bovine prolactin with Wg signal
sequence (PRO), the DNA fragment encoding Wg signal sequence (amino
acids 1-34 of Wg) was PCR-amplified with the wg cDNA as
the template and the following primers:
5'-GGGGTACCATGGATATCAGCTATATCTTCGTC-3' and
5'-CCGCTCGAGCCGGCCCCTTCCGGATTTCTGTTT-3'. The PCR product was digested
with KpnI and XhoI. The DNA fragment encoding
mature prolactin was PCR-amplified with prolactin cDNA as a
template and the following primers:
5'-TAACTCTCGAGACCCCCGTCTGTCCCAATGGGCCTGGCAAC-3' and
5'-TAACTGATATCGCAGTTGTTGTTGTAGATGATTCTGCAATT-3'. The PCR product was
digested with XhoI and EcoRV. The above two PCR
products were then ligated with KpnI- and
EcoRV-digested pUC19 carrying DNA encoding the single
Myc epitope. To construct RDN and VKG chimeric prolactin, the DNA
fragments encoding amino acids 73-106 (RDN) and 83-106 (VKG) of Wg
were PCR- amplified with the wg cDNA as the template
and the following primers:
5'-TAACTCTCGAGCGTCGAGCAGTTCCAGCGGCGATTTCTGAA-3' and either
5'-TAACTCTCGAGAGGGACAATCCCGGTGTACTGGGAGCCCTG-3' (RDN) or
5'-TAACTCTCGAGGTCAAGGGCGCCAACTTGGCCATTAGCGAG-3' (VKG). Each PCR product
was digested with XhoI and cloned at the same restriction enzyme site of the above plasmid in a correct orientation. To construct GSM, the DNA fragment encoding amino acids 1-106 of Wg was
PCR-amplified with the wg cDNA as the template and the following primers: 5'-GGGGTACCATGGATATCAGCTATATCTTCGTC-3' and 5'-TAACTCTCGAGCGTCGAGCAGTTCCAGCGGCGATTTCTGAA-3'. The PCR product was
digested with KpnI and XhoI and cloned at the
same restriction enzyme sites of the above plasmid to replace the DNA
fragment encoding the Wg signal sequence. All DNA fragments encoding
the chimeric prolactins were cloned in the pCaspeR-hs. Myc
epitope-tagged DWnt-3/5 was generated by PCR with DWnt-3/5
cDNA as a template and the following primers:
5'-GGGGTACCATGAGTTGCTACAGAAAAAGGCAC-3' and
5'-TACGCGATATCTTTACATGTGTGCTCCTCGAGTAC-3'. The PCR product was then
cloned in pUC19 carrying a DNA fragment encoding the single Myc
epitope. The Myc epitope-tagged DWnt-3/5 cDNA was
finally cloned in the pCaspeR-hs. All PCR products were confirmed by
DNA sequencing.
-Mercaptoethanol was added to the one part at the final
concentration of 2%. The lysates were analyzed by 10% SDS-PAGE
followed by Western blot with anti-Wg mouse monoclonal antibody (4D4)
as above, except 3% bovine serum albumin was used as a blocking
reagent. 30 green fluorescent protein-positive (wild type) and negative
(porc) third instar larvae were selected from a stock of
FM7act-GFP/y w f porcPB16 and dissected in S2
medium. Similarly, 30 third instar larvae from a cross of
UAS-porc and 69B were also dissected. The isolated discs
were suspended in S2 medium and washed twice with methionine-free labeling medium and then suspended in 485 µl of the same medium. Labeling was initiated by adding 180 µCi of Pro-mix and continued for
1 h at 25 °C. The labeling medium was then removed, and the discs were suspended with radioimmune precipitation buffer containing protease inhibitor mixture. The cell lysates were prepared by homogenizing with plastic pestle in Eppendorf tubes. The
immunoprecipitation was performed as described above. The
radioactivities of forms II and III in each sample were measured by a
Bioimage analyzer BAS2000 (Fuji), and their ratios were calculated.
This experiment was repeated twice.
34,
66, and 110 RNA was prepared by in
vitro transcription. The size and quantity of each capped RNA was
analyzed by gel electrophoresis. The same amount of RNA was injected
into precellular blastoderms. The embryos were derived from females
with homozygous svbYP17bporcPB16
germ line clones crossed with FM7ftz-lacZ males.
porc mutant embryos rescued as a result of RNA injection
showed the svb phenotype (15). The injected embryos were
allowed to develop at 18 °C, and their cuticles were examined after
3 days. The embryos derived from females with homozygous
porcPB16 germ line clones crossed with
FM7ftz-lacZ males were immunostained with anti-DWnt-3/5
antibody (16) or BP102 (developed by C. S. Goodman and obtained
from the Developmental Studies Hybridoma Bank) along with
anti--galactosidase antibody. The signal was detected by rhodamine
and horseradish peroxidase (for -galactosidase)-conjugated secondary
antibodies. In these experiments, germ line clones were generated with
the FLP-DFS technique (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
The N-glycosylation of Wg in
Drosophila S2 cells and imaginal discs.
A, three forms (I-III) of Wg with different numbers of
N-glycan chains (none, one, and two) are synthesized in S2
cells. In the presence of tunicamycin (Tun), only form I is
produced. In the presence of ectopic porc (Porc),
form III is synthesized most, and a form IV with three
N-glycan chains is also detected. B, the
sensitivity of forms I, II, and III against the exogenous protease. The
lysates of cells expressing wg were treated with trypsin and
TX-100 where indicated ( 
/+), and then Wg was analyzed by Western
blot. All forms are protected against trypsin in the absence of TX-100.
The level of each form susceptible to the protease is shown by
percentage reduction. C, the N-glycosylation of
endogenous Wg expressed in imaginal discs of wild type (wt),
porc hemizygous mutant (porc), and
porc-overexpressing (UAS-porc X 69B) third instar
larvae was analyzed by pulse labeling with
[35S]methionine and immunoprecipitation. The exogenously
expressed Wg in S2 cells (S2) was included for the
identification of the three different forms of Wg. D, the
endogenous Wg expressed in imaginal discs of wild type third instar
larvae was analyzed by SDS-PAGE under either reducing (R) or
nonreducing (NR) condition followed by Western blot. The
exogenously expressed Wg in S2 cells (S2) was included for
the identification of the three different forms of Wg (under reducing
conditions). The nonreducing sample was loaded three lanes away from
the reducing samples to avoid interference by diffused
-mercaptoethanol.
View larger version (38K):
[in a new window]
Fig. 2.
The inhibition of disulfide bond formation
results in the efficient N-glycosylation of Wg.
A, the N-glycosylation of Wg in S2 cells was
analyzed by pulse (0 h)-chase (2 h) experiments with (+) or without
( ) DTT. When the cells were treated with DTT, it was added at 30 s before labeling and present throughout the 10-min pulse. The 2-h
chase was carried out either in the absence or presence of DTT. All
samples were analyzed by 10% SDS-PAGE under reducing conditions. See
"Experimental Procedures" for details. The
arrowhead indicates Porc co-immunoprecipitated with Wg.
Three upper bands above Wg are nonspecifically immunoprecipitated
proteins, which are also detected in S2 cells not expressing
wg. B, the same samples of A were
analyzed by 10% SDS-PAGE under nonreducing condition. Wg forms both
intra- and intermolecular disulfide bonds under normal conditions. The
arrowhead indicates Porc co-immunoprecipitated with Wg.
C, S2 cells expressing wg were pretreated with
DTT for 5 min before labeling, and DTT was present throughout the
10-min pulse labeling.
chain, which also forms both intra-
and intermolecular disulfide bonds (not shown).
34, -66, and -110 lack the C-terminal 34, 66, and 110 amino acids of Porc, respectively), and their effects on the
N-glycosylation of Wg were analyzed. As shown in Fig.
3A, the amount of forms I and
II increases with deletion mutants, and the synthesized ratios of three
forms become similar to those in the absence of ectopic
porc. This demonstrates that all deletion mutants have reduced activity on the N-glycosylation of Wg compared with
wild type PorcHA28 (full-length Porc tagged with HA epitope),
indicating that the C-terminal domain of Porc is essential for its
function. We then analyzed if the C-terminal deleted Porc mutants can
bind Wg. PorcHA28 is co-immunoprecipitated with Wg irrespective of the
presence of tunicamycin. By contrast, the binding of C-terminal deletion mutants to Wg is reduced (Fig. 3B). These results
suggest that Porc binds the Wg and that its C-terminal domain is
essential for efficient binding. The intracellular localization of
deletion mutants is not affected based on the indirect
immunofluorescent analysis (not shown), excluding the possibility that
their targeting to the ER membrane is impaired. The full-length
PorcHA28 is functional in embryos, since it can rescue the phenotypes
of porc embryos lacking both maternal and zygotic
contribution of porc (referred as porc embryos
hereafter) by RNA injection. By this assay, all deletion mutants fail
to rescue the phenotypes of porc embryos (not shown). Thus,
the binding of Porc with Wg appears to be necessary for the stimulation
of the N-glycosylation of Wg.
View larger version (36K):
[in a new window]
Fig. 3.
The C-terminal domain of Porc is essential
for the enhancement of the N-glycosylation of Wg and
binding with Wg. A, the N-glycosylation of
Wg is analyzed in the absence (None) and presence of ectopic
wild type Porc (Porc), HA epitope tagged Porc
(PorcHA28) and three C-terminal deletion mutants of
PorcHA28 ( 34, 66, and 110) by
Western blot. The increase of forms I and II is detected by the
deletion mutants compared with the wild type. Control is the lysate of
cells without expressing wg. B, the amount of
PorcHA28 and the deletion mutants bound with Wg is analyzed by
immunoprecipitation (IP) with anti-Wg antibody followed by
Western blot with anti-HA monoclonal antibody (12CA5) in the absence
() and presence (+) of tunicamycin (Tun). The binding of
the deletion mutants with Wg is less efficient compared with the wild
type. The amount of each HA epitope-tagged Porc in the cell lysates
prior to immunoprecipitation is constant based on Western blot analysis
with 12CA5 as shown in the lower panel.
View larger version (30K):
[in a new window]
Fig. 4.
Porc binds the N-terminal 24-amino acid
domain (residues 83-106) of Wg and stimulates the
N-glycosylation at Asn103.
A, the N-glycosylation of the wild type
(wt) and four deletion mutants (containing N-terminal 367, 282, 196, and 111 amino acids) of Myc epitope-tagged Wg was analyzed. Signal sequence cleaved but
not glycosylated forms, which are synthesized in the presence of
tunicamycin (Tun), are present in all cases. Wild type Wg is
N-glycosylated at two major sites, whereas deletion mutants
have a single N-glycan chain. Wild type, 367, and 282 proteins were separated by 12% SDS-PAGE, whereas 196 and 111 proteins
were separated by 15% SDS-PAGE. B, the processing of Wg
mutants, in which the potential N-glycosylation sites were
knocked out, and wild type Wg (wt) was analyzed in the
absence ( ) and presence (+) of ectopic porc. The serine or
threonine residues at the position number indicated in the Wg
were substituted to alanine. C, the amino acid sequence near
Asn103 (indicated by an arrowhead) of Wg is
aligned with those of Drosophila and mouse Wnts. The
conserved amino acids are indicated by asterisks.
D, four Myc epitope-tagged chimeric proteins consisting of
bovine prolactin (containing no N-glycosylation site) and
either Wg signal sequence (amino acids 1-34 of Wg) (PRO) or the
N-terminal 106 amino acids of Wg (GSM) or the Wg signal sequence plus
amino acids 73-106 of Wg (RDN) or the Wg signal sequence plus amino
acids 83-106 of Wg (VKG) were constructed. Three chimeric proteins
(GSM, RDN, and VKG) but not PRO are N-glycosylated, and Porc
enhances this processing event. E, the binding between Porc
and chimeric proteins (PRO, RDN, and VKG) was analyzed by
immunoprecipitation. The cell lysates expressing PorcHA28 and either
PRO, RDN, or VKG were immunoprecipitated with anti-Myc antibody, and
then the immunoprecipitates were analyzed by Western blot with anti-HA
rat monoclonal antibody to detect the co-immunoprecipitated Porc. Porc
is co-immunoprecipitated with RDN and VKG (with a lesser amount) but
not PRO. The part of cell lysates was directly Western blotted with
anti-HA rat monoclonal antibody to confirm that PorcHA28 is expressed
at the same level in all cell lysates.
View larger version (148K):
[in a new window]
Fig. 5.
DWnt-3/5 protein localization in
porc embryos. Wild type (A) and
porc (B) embryos were immunostained with
anti-DWnt-3/5 antibody. DWnt-3/5 is mainly localized at the commissural
axon tracts of the central nervous system in wild type embryos, but it
remains in the cell bodies of neurons at the ventral nerve cord of
porc embryos, where DWnt-3/5 RNA is expressed.
The immunostaining of wild type (C) and porc
(D) embryos with BP102 reveals that the axon tracts of the
central nervous system are present in porc embryos but with
irregular patterns.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amidating monooxygenase (28), small acceptor
tripeptides (29), an aglycoinsulin receptor generated by tunicamycin
treatment (30), and a calreticulin under heat shock (31).
However, the precise mechanisms of their N-glycosylation
have not been elucidated.
ACKNOWLEDGEMENTS
cDNA, R. Nusse for
anti-DWnt-3/5 antibody and DWnt-3/5 cDNA, V. R. Lingappa for bovine prolactin cDNA, and G. Fink for pKK-1. The
BP102 and 4D4 monoclonal antibodies were purchased from the
Developmental Studies Hybridoma Bank developed under the auspices of
the NICHD, National Institutes of Health, and maintained by the
Department of Biological Sciences, University of Iowa.
FOOTNOTES
To whom correspondence should be addressed. Tel.: 81-52-789-5237;
Fax: 81-52-789-5237; E-mail: emi@nuagr1.agr.nagoya-u.ac.jp.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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T. I. Cesena, J.-R. Cardinaux, R. Kwok, and J. Schwartz CCAAT/Enhancer-binding Protein (C/EBP) beta Is Acetylated at Multiple Lysines: ACETYLATION OF C/EBPbeta AT LYSINE 39 MODULATES ITS ABILITY TO ACTIVATE TRANSCRIPTION J. Biol. Chem., January 12, 2007; 282(2): 956 - 967. [Abstract] [Full Text] [PDF]
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D. Coudreuse and H. C. Korswagen The making of Wnt: new insights into Wnt maturation, sorting and secretion Development, January 1, 2007; 134(1): 3 - 12. [Full Text] [PDF]
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R. M. Goodman, S. Thombre, Z. Firtina, D. Gray, D. Betts, J. Roebuck, E. P. Spana, and E. M. Selva Sprinter: a novel transmembrane protein required for Wg secretion and signaling Development, December 15, 2006; 133(24): 4901 - 4911. [Abstract] [Full Text] [PDF]
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 D. Y. M. Coudreuse, G. Roel, M. C. Betist, O. Destree, and H. C. Korswagen Wnt Gradient Formation Requires Retromer Function in Wnt-Producing Cells Science, May 12, 2006; 312(5775): 921 - 924. [Abstract] [Full Text] [PDF]
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I. T. Struewing, A. Toborek, and C. D. Mao Mitochondrial and Nuclear Forms of Wnt13 Are Generated via Alternative Promoters, Alternative RNA Splicing, and Alternative Translation Start Sites J. Biol. Chem., March 17, 2006; 281(11): 7282 - 7293. [Abstract] [Full Text] [PDF]
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 G. Bolt, C. Kristensen, and T. D. Steenstrup Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII Glycobiology, May 1, 2005; 15(5): 541 - 547. [Abstract] [Full Text] [PDF]
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 A. J. Zhu and M. P. Scott Incredible journey: how do developmental signals travel through tissue? Genes & Dev., December 15, 2004; 18(24): 2985 - 2997. [Abstract] [Full Text] [PDF]
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L. Zhai, D. Chaturvedi, and S. Cumberledge Drosophila Wnt-1 Undergoes a Hydrophobic Modification and Is Targeted to Lipid Rafts, a Process That Requires Porcupine J. Biol. Chem., August 6, 2004; 279(32): 33220 - 33227. [Abstract] [Full Text] [PDF]
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M. E. Linder and R. J. Deschenes Model organisms lead the way to protein palmitoyltransferases J. Cell Sci., February 15, 2004; 117(4): 521 - 526. [Abstract] [Full Text] [PDF]
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R. Nusse Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface Development, November 15, 2003; 130(22): 5297 - 5305. [Abstract] [Full Text] [PDF]
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D. M. Kamikura and J. A. Cooper Lipoprotein receptors and a Disabled family cytoplasmic adaptor protein regulate EGL-17/FGF export in C. elegans Genes & Dev., November 15, 2003; 17(22): 2798 - 2811. [Abstract] [Full Text] [PDF]
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