From the Division of Molecular Medicine,
International Center for Medical Research and the § Division
of Cardiovascular and Respiratory Medicine, Department of Internal
Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki,
Chuo, Kobe 6500017, Japan, and the
Department of Applied
Biological Chemistry, Graduate School of Agriculture and Life Sciences,
The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
Received for publication, February 23, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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A subfamily of zinc metalloproteases, represented
by Neutral endopeptidase (EC 3.4.24.11) and
endothelin-converting enzyme, is involved in the metabolism of a
variety of biologically active peptides. Recently, we cloned and
characterized a novel member of this metalloprotease family termed
soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive
peptides. Here we report that alternative splicing of the mouse
SEP gene generates two polypeptides,
SEP Mammalian zinc metalloproteases have been implicated in a
diversity of disease states because of their roles in the activation or
inactivation of a variety of biologically active peptides. Therefore,
they provide important therapeutic targets for certain diseases. Within
this large group, neprilysin (M13) constitutes a subfamily in which
seven members have been identified to date, such as Neutral
endopeptidase (EC 3.4.24.11)
(NEP),1 Kell blood
group protein, two different endothelin-converting enzymes (ECE-1 and
ECE-2), PEX, which has been associated with X-linked hypophosphatemic
rickets, endothelin-converting enzyme-like-1, and the recently
identified soluble secreted endopeptidase (SEP). All these members are
type II membrane glycoproteins, which display a single transmembrane
stretch separating a short N-terminal cytoplasmic tail from a large
C-terminal extracellular/luminal domain. This luminal domain bears the
enzyme active site, which includes HEXXH, a highly conserved
pentameric consensus sequence of a zinc binding motif. NEP is a
metalloprotease with wide tissue distribution and is especially
abundant in the brain and kidney. This endopeptidase has been shown to
hydrolyze a wide range of small peptide mediators, such as enkephalins,
substance P, atrial natriuretic peptide, neurotensin, bradykinin,
angiotensin I and II, and endothelins (1). ECE-1 is primarily involved
in the production of the vasoconstrictive peptide ET-1 by the cleavage
of an inactive precursor, big ET-1. Two subisoforms of bovine ECE-1
termed ECE-1a and ECE-1b that differ from each other only in the
N-terminal tip of their cytoplasmic tail showed distinct subcellular
localization (2). ECE-2, which also produces ET-1, has an acidic pH
optimum and may function intracellularly (3). Therefore, clarifying the
precise subcellular localization of the protein would be favorable to
characterize its physiological roles.
SEP, the most recently identified member of this family, shares higher
structural and functional similarities with NEP than with other members
of this metalloprotease family. Structurally, the sequence identity of
SEP with respect to NEP is higher than those of the other members. Two
arginine residues known to constitute the substrate binding sites in
NEP (Arg102 and Arg747 in human NEP) are
conserved in SEP (Arg121 and Arg764 in mouse
SEP). Functionally, both SEP and NEP are promiscuous enzymes that
hydrolyze a variety of physiologically active peptides. SEP has been
implicated in the hydrolysis of angiotensin I, atrial natriuretic
peptide, bradykinin, substance P, leucine-enkephalin, big ET-1,
and ET-1. The activity of SEP is efficiently inhibited by the specific
NEP inhibitor thiorphan but is not completely inhibited by the specific
ECE inhibitor FR901533 (4, 5).
Although SEP shares several important properties with other members of
this metalloprotease family, it exhibits features unique to itself.
First, two isoforms of SEP named SEP and SEP Materials--
Enzymes used in molecular cloning were obtained
from Roche Molecular Biochemicals or from New England Biolabs (Beverly,
MA). Endo- BAC Library Screening--
The mouse SEP gene locus
was cloned by screening a BAC (bacterial artificial chromosome) library
(Genome Systems, Inc., St. Louis, MO). A polymerase chain reaction
(PCR)-generated radiolabeled probe containing sequences within the
mouse cDNA was used to probe a mouse genomic BAC library. The probe
was generated from the following oligonucleotides,
5'-TATTTCCGGCAGGGATTCTC-3' and 5'-CATTATCATCAAAGCCGTGT-3', which were
chosen based on their likelihood to span a region within an
SEP exon according to the genomic structure of
NEP (6) and ECE-1 (7). One positive clone,
pBAC-SEP, containing ~150 kilobase pairs of genomic DNA was
obtained and subjected to sequence analysis. This clone was purified
and digested with several restriction enzymes and was run using a 0.7%
agarose gel. The gel was Southern blotted and probed with
32P-labeled oligonucleotides, which were contained within
the putative insertion exon and its immediate upstream and downstream exons.
Long and Accurate PCR--
Long and accurate PCR was performed
on pBAC-SEP DNA to determine the size and location of the introns using
Takara long and accurate PCR kit as described by the manufacturer.
Oligonucleotides were derived from the mouse SEP cDNA sequence and
were designed to span the putative insertion intron and its immediate
downstream intron. The primers, 5'-GGGAGCCATAGTGACTCTGGGTGTC-3'
and 5'-TCGTTTTACAACCGTCCTCTCATCC-3', were used for the putative
insertion intron amplification, and primers,
5'-GGGAAGCAGCTGCCCCTCTTAACTA-3' and 5'-GCTATCACACAGCTTGGGGTGGTGC-3', were used to amplify an intron immediately upstream of the
insertion intron. The PCR products were digested with restriction
enzyme BamHI, thus resulting in four different fragments.
All four fragments were then sequenced at both the 5' and 3' ends to
verify correct oligonucleotide priming and to deduce the correct
intron-exon boundaries. Automated sequencing was performed with a Model
310 DNA sequencer (Applied Biosystems).
Plasmid Construction--
To construct the fusion genes of
SEP
pME-E/E/S was constructed by fusing the cytoplasmic domain (amino acids
1-52) and the transmembrane domain (residues 53-73) of bECE-1b to the
whole extracellular domain of mouse SEP
The plasmid expressing the E/E/
All mutants were verified by sequencing at the level of the final plasmid.
Mutagenesis of Mouse SEP--
Deletion constructs and amino
acid-substituted constructs were made by site-directed mutagenesis (2)
using the Muta-Gene Phagemid in vitro mutagenesis version 2 kit (Bio-Rad) as described by the manufacturer. All mutants were
verified by DNA sequencing using primers both upstream and downstream
of the insertion region.
Cell Culture--
CHO-K1 cells were cultured as described
previously (4). The coding region of mouse SEP or SEP Immunoblotting--
Conditioned medium and postnuclear lysates
were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to a nitrocellulose membrane. The membranes were probed
with an antibody against the C terminus of mouse SEP (4), bovine ECE-1
(2), or human microsomal triglyceride transfer protein (MTP) (8) and
developed using the ECL Kit (Amersham Pharmacia Biotech) as recommended by the manufacturer.
Fluorescent Immunocytochemistry--
Cells were seeded onto
coverslips and cultured for 2 days. Fluorescent immunocytochemistry was
performed as described previously (2, 4). Intracellular staining was
performed as follows, cells were fixed and permeabilized in methanol
for 5 min at Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation were performed as described previously
(2). However, the soluble proteins in the supernatant were precipitated
by the method of Wessel and Flugge (9). The precipitates were then
dissolved in 500 µl of phosphate-buffered saline and
immunoprecipitated using SEP polyclonal antibody. Immunoprecipitates
were analyzed on 7% SDS-PAGE and developed using the BAS2000 system.
Endoglycosidase Digestion--
Conditioned medium and membrane
fractions were used for digestion. Endo H and PNGaseF digestions were
performed as described previously (4), and endoglycosidase D was done
at 37 °C for 16 h in 0.2 M phosphate buffer, pH
6.5, containing 0.1% Nonidet P-40. The samples were then subjected to immunoblotting.
SEP and SEP
To check whether these two isoforms were produced by the same gene via
an alternative splicing mechanism, the mouse SEP gene was
cloned from mouse BAC genomic library using a PCR-generated 32P-labeled probe spanning a putative exon area of
SEP that we predicted based on the structure of both the
NEP (6) and ECE-1 gene (7). One positive clone,
pBAC-SEP containing ~150 kilobase pairs of genomic DNA was
obtained and subjected to sequence analysis. Exon-specific primers were
designed based on the predicted intron-exon boundaries and used to
directly sequence the BAC clone. All deduced intron-exon boundaries
indicate the canonical consensus splice donor and acceptor sequences in
accordance with the GT/AG rule (10).
The sequence analysis clearly showed that the exon encoding the
23-amino acid insertion of SEP is an independent exon separated from
the immediate 5' exon by a ~9.5-kilobase pairs intron and from the
next 3' exon by a 555-base pair intron (Fig.
1A). This transcript resulted
in a 765-amino acid product of SEP. However, when this insertion exon
skipped making its immediate 5' exon join directly to the next 3' exon,
the 742 amino acids of SEP
These results suggest that these two isoforms of SEP mRNA originate
from a single gene of the mouse genome by an alternative splicing mechanism.
SEP and SEP
To further analyze these proteins, we then examined their sensitivity
to Endo H and PNGaseF. Endo H digests N-glycans of a high
mannose type. Resistance to Endo H digestion indicates that a
glycoprotein moved from the ER to the Golgi compartment in which further modification to complex oligosaccharides occurs, whereas PNGaseF completely removes all N-linked oligosaccharides.
Treatment of solubilized membranes from both CHO/SEP and
CHO/SEP
To precisely investigate the subcellular localization of the
membrane-bound protein, we immunostained both CHO/SEP and
CHO/SEP
To examine whether the protein localized in the ER by a retention or a
retrieval mechanism, both SEP and SEP The Luminal Domain of SEP/SEP
Several reports have shown that the cytoplasmic domain of type II
membrane proteins determine ER localization (15-17). Thus, we first
analyzed a chimeric protein S/S/E (SEP
We next asked whether only the luminal domain of SEP
We then confirmed these findings by endoglycosidase digestion. CHO-K1
cells were transfected with cDNAs encoding the wild-type SEP,
bECE-1b, the chimera S/S/E, and E/E/S. After 30 h, the proteins were harvested and treated with Endo H and PNGaseF. SEP and E/E/S gave
a single band of 110-kDa on SDS-PAGE, were sensitive to both Endo H and
PNGaseF, and were converted to a band approximately 20 kDa smaller
(Fig. 3C). On the other hand, bECE-1b and S/S/E gave a
higher molecular mass band (126-kDa) aside from the 110-kDa band. The higher molecular mass bands were sensitive to PNGaseF but
resistant to Endo H, whereas the smaller band was sensitive to both
endoglycosidases as in the case of SEP, E/S/S, and E/E/S, indicating
that proteins with higher molecular mass contain complex-type oligosaccharides (Fig. 3C). Together, these results clearly
demonstrate that the luminal domain of SEP determines the localization
of the SEP protein in the ER.
The ER Localization of Membrane-bound SEP Is Not Attributed to
Misfolding--
The abnormally assembled proteins (misfolded proteins)
in the ER are rapidly destroyed with the half-life of less than 1 h (18). Using CHO/SEP The Minimal Determinant of SEP Cleavage Is Defined within the
Insertion Domain--
Because the SEP and SEP
To determine the sequence in the 23-amino acid insertion of SEP
responsible for its secretion, we initially speculated that the
biologically active soluble form of SEP is proteolytically released
from the membrane-bound SEP by a dibasic processing endoprotease(s) (4). To test this point, we first deleted these dibasic amino acids
(Lys62-Arg63) (
Because we failed to prove that the dibasic residues are the cleavage
site of SEP, we then constructed a series of deletion mutations
spanning the insertion region and expressed them in CHO cells. Of this
deletion mutation series, the deletion of six C-terminal amino acids
( SEP Cleavage Occurs in the ER--
Because we did not observe any
Golgi modification of these glycoproteins during all the chase periods
of membrane-bound SEP/SEP
We established stable transfectants of CHO/secECE-1, CHO/SEP, and
CHO/bECE-1b. These cells were labeled with 35S-amino acids
for 30 min, chased for 0, 1, 2, or 4 h, rinsed, and then cultured
in medium without tracers. Both the cell extracts and medium were
collected and immunoprecipitated using an anti-ECE-1 or anti-SEP
antibody. Half of each sample was digested with Endo H and analyzed by
SDS-PAGE. In the cells transfected with secECE-1, we could not observe
any intracellular protein that received Golgi enzyme modification since
at 4 times of chase periods the protein remained fully sensitive to
Endo H. However, within 1 h, we already detected a significant
amount of secreted protein that is resistant to Endo H in the
conditioned medium, and this increased in a time-dependent manner (Fig. 6A). This means
that before reaching the Golgi compartment, this protein was already
cleaved so that it is easily secreted into the medium. Similar findings
were observed when SEP was transfected into CHO cells. In the cell
lysates, the proteins were sensitive to Endo H, whereas the observed
proteins in the medium were resistant to Endo H during all the chase
periods (Fig. 6B).
This finding is in agreement with the pulse-chase result of the
intracellular SEP This study has established that mRNAs encoding the two
isoforms of mouse SEP, the membrane-bound and the soluble forms, are generated by alternative splicing of a single copy gene. After synthesis, the newly synthesized SEP proteins are targeted to the ER as
type II integral membrane proteins. The membrane-bound isoform retained
in this compartment is mediated by the luminal domain. In contrast, the
soluble isoform is cleaved and released into the extracellular
compartment. We have identified and characterized the structural
determinant required for the retention of the membrane-bound form and
the release of the SEP luminal domain and also demonstrated that the
proteolytic cleavage system operates in the ER. Therefore, the
characterization of the membrane-bound and the soluble forms of SEP
reveals a unique mechanism of subcellular localization and protein
trafficking in the secretory pathway.
The finding that SEP can exist in both soluble and membrane-bound forms
led us to ask about how these two forms are produced and how their
production is regulated. There are several membrane-bound proteins that
have soluble counterparts, one example is fibronectin (21). Fibronectin
exists in both insoluble and soluble forms and is present in the
extracellular matrix and plasma, respectively. This diversity is
generated via alternative splicing of cassette exons, alternative
donors and acceptors, and retained introns (22). The incorporation of a
cassette exon in fibroblasts produces cellular fibronectin, whereas its
exclusion in the liver generates plasma fibronectin (22). We
hypothesized that SEP is probably generated by a similar mechanism as
is this protein. The inclusion of a cassette exon immediately following
the exon encoding the putative transmembrane domain of SEP protein
introduces an in frame cleavable exon that allows the intracellular
secretion machinery to cleave and secrete the encoded protein to the
plasma while its exclusion gives rise to membrane-bound
SEP The subcellular localization of neprilysin family members is quite
diverse. NEP (1), Kell (23), bECE-1b (2), and PEX (24)
are expressed on the cell surface where they act as ectoenzymes. Another isoform of ECE-1, bECE-1a, resides in an intracellular compartment that largely overlaps with the Golgi apparatus (2), whereas
ECE-2 seems to function in the trans-Golgi network based on
its acidic activity (3). Valdenaire and Schweizer (25) recently showed
that ECE-like-1 was localized in the ER when transfected into CHO
cells. In the current study, we present the evidence to conclude that
SEP is another member of this family showing ER localization when
transfected in two distinct cell types, CHO cells and COS-7 cells (data
not shown). This conclusion is based on two lines of evidence. First,
indirect immunofluorescence experiments showed that the SEP-staining
pattern was similar to that of MTP, a marker of the ER. Second, the
intracellular form of SEP contained a high mannose-type oligosaccharide
as defined by Endo H sensitivity, whereas the secreted SEP contained an
Endo H resistant oligosaccharide. Nonetheless, the data presented in
this study cannot explain whether the behavior of overexpressed SEP
reflects that of endogenous SEP in mammalian cells. Considering the
fact that the precise in vivo functions of SEP are unknown
so far, the characterization of subcellular localization provides a
clue for the identification of the physiological substrate.
In this report, we also demonstrated that ER localization of SEP
appears to be mediated by the luminal domain, independent from its
cytoplasmic domain and transmembrane domain. Thus, the luminal
domain functions as an intracellular sorting signal. An important
criterion to define an intracellular sorting signal is the ability of
that signal to function in a heterologous system, i.e.
whether it is transferable. Indeed, we showed that the luminal domain
of SEP protein is obviously sufficient for inducing ER localization of
the cell surface protein, bECE-1b. To our knowledge, the only type II
membrane protein with an ER localization signal in the luminal domain
is the one bearing the His-Asp-Glu-Leu (Lys-Asp-Glu-Leu in
eukaryotes) motif. This motif is recognized by a receptor in the Golgi
apparatus, which results in the recycling of KDEL proteins back to the
ER (26). Thus, it acts as a retrieval signal. Because the luminal
domain of SEP lacks a KDEL motif, it must contain a new ER localization
determinant and/or motif. Therefore, the detailed characterization of
this ER localization signal is a challenging work for further
experiments aimed at understanding the molecular basis by which ER
localization is achieved.
Both the retention and retrieval mechanisms are thought to be involved
in keeping ER resident proteins from escaping down the secretory
pathway. In the current paper, we suggest that the mechanism for ER
localization of the SEP protein is retention rather than retrieval.
Several arguments support this idea. Although we used a strong SR There are two kinds of proteins retained in the ER, the misfolded
and/or incompletely assembled proteins and the ER resident proteins.
The misfolded proteins associate with ER chaperones and folding
enzymes, such as calnexin or BiP, and are rapidly destroyed with a
half-life of less than 1 h (11, 18). We feel that this is not the
case for SEP since the membrane-bound SEP was fairly stable for at
least 4 h with a half-life of more than 2 h (Fig. 4,
A and B). A more attractive explanation for this localization is that the membrane-bound SEP is the ER resident protein,
and the luminal domain is directly responsible for ER residency. A
strong argument in favor of this view is our previous observation that
not only the soluble form but also the membrane-bound form of SEP show
enzymatic activity to hydrolyze big ET-1 (4). Thus, the maintenance of
the enzymatic activity of the ER proteins strongly indicates that
membrane-bound SEP is a properly folded protein (18, 28, 29).
The present results show that the insertion domain of SEP is obviously
responsible for secretion, and the WDERTVV region (residues 55-61) is
the critical part of the cleavage determinant. This finding is in
conflict with the data published by Ghaddar and co-workers (5) who
postulated that SEP was converted into a soluble form by the action of
pro-hormone convertases at the dibasic residues (KR). There are at
least two possible explanations for this discrepancy, the difference of
the cell types and/or overexpression. We believe that the former is not
the case for SEP because the In our preliminary attempts to identify the class of protease(s)
responsible for SEP secretion, we examined the effect of the inhibitors
of three classes of proteases. Phosphoramidon (a metalloprotease
inhibitor) up to 1 mM, MDL 28170 (a calpain inhibitor) up
to 100 µM, and TLCK (a serine protease inhibitor) up to
100 µM did not affect the rate of SEP
secretion.2 These findings
raised at least two possibilities. First, proteases other than
phosphoramidon-sensitive metalloproteases, MDL 28170-sensitive calpains, and TLCK-sensitive serine proteases may be involved in the
cleavage of the insertion domain of SEP. Second, the proteases may be
resistant to the standard treatment in a culture cell system as is the
case for angiotensin-converting enzyme secretase (34), and certain
treatments and/or systems may be needed to examine the direct
inhibition of the secretase activity, e.g. the cell-free membrane system (35) or prewashing the membrane fraction with detergent
(36).
The retention of SEP in the ER indicates that proteolytic cleavage
occurs in this compartment in agreement with the observation of similar
trafficking and turnover between SEP and secECE-1 proteins. Pulse-chase
experiments demonstrated that the membrane-bound forms of both of these
proteins had never acquired Endo H resistance, indicating that these
proteins did not gain Golgi-type glycosylation. Therefore, it appears
likely that the posttranslational proteolytic processing of SEP is
accomplished in the ER, and without further cleavage, this protein is
then secreted into plasma. This finding makes SEP quite unique among
secretory proteins, which are otherwise proteolytically cleaved in the
cell surface or Golgi compartment. For instance, angiotensin-converting
enzyme (31), colony-stimulating factor-1 (33), and kit ligand (32) were
secreted by the action of cell surface secretase, whereas cysteine
array matrix metalloproteinase (37) was cleaved in the
trans-Golgi network prior to secretion. Prohaptogobin was
indeed cleaved in the ER, but this was only partial cleavage, and the
complete cleavage took place in the plasma membrane (38). In
conclusion, the discovery that SEP is proteolytically cleaved in the
endoplasmic reticulum before secretion provides a new model for protein
trafficking and processing within the secretory pathway.
and SEP. After synthesis, both isoforms are inserted
into the endoplasmic reticulum (ER) as type II membrane proteins.
SEP
then becomes an ER resident, whereas SEP, which
differs by only the presence of 23 residues at the beginning of its
luminal domain, is proteolytically cleaved by membrane secretase(s) in
the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEP
and bovine
endothelin-converting enzyme-1b (bECE-1b) demonstrated that the
retention of SEP
in the ER is mediated by the luminal
domain. In addition, the dissection of the chimeric bECE-1b/SEP
insertion showed that its insertion domain is obviously responsible for
its secretion. A series of mutagenesis in this region revealed that the
minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the
secretory pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
differ from
each other in the presence of a 23-amino acid insertion region flanking
the transmembrane domain of SEP. This feature was not found in other
members of this metalloprotease family. Second, the membrane-bound SEP
seems to localize in the early secretory pathway, which is unusual for
these metalloprotease family members. Third, although all the other
members discovered so far are membrane-associated proteins, SEP exists
not only as a membrane-bound form but also as a circulating soluble
form. This observation suggests that a proteolytic cleavage event
occurred during the intracellular transport of SEP. These features make SEP unique among the members of this neprilysin family. Therefore, we
designed the current study to investigate these special characteristics of SEP. In this report, we present evidence that two isoforms of SEP
are generated via an alternative splicing mechanism. The membrane-bound
SEP localized in the endoplasmic reticulum, and this ER localization is
not attributed to misfolding. In addition, by making chimeric proteins
between SEP and bovine endothelin-converting enzyme-1b (bECE-1b),
another member of this metalloprotease family that is normally
localized in the cell surface, the luminal domain of the SEP protein
was identified as being important for retention. Furthermore, we also
identified a specific motif in the SEP insertion region that is
necessary for the cleavage process of this protein in the ER. This
unique mechanism of localization and processing of SEP defines an
interesting model for protein trafficking within the secretory pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylglucosaminidase H (Endo H) and
peptide-N-glycosidase F (PNGaseF) were from Roche Molecular
Biochemicals, Endo D was from Seikagaku Co. Ltd. (Tokyo),
rProtein A-Sepharose Fast Flow beads were from Amersham
Pharmacia Biotech AB (Uppsala, Sweden).
and bECE-1b, cDNAs for mouse SEP
(2) and bECE-1b (3) were used. For the generation of the pME-S/S/E
chimera, the cytoplasmic domain (amino acids 1-17) and the
transmembrane helix (residues 18-40) of mouse SEP
were
fused in frame to the entire extracellular domain of bECE-1b (residues
78-758). Initially, site-directed mutagenesis was performed to
introduce Eco47III and SmaI sites at the
beginning of the extracellular domain of mouse SEP
and
bECE-1b, respectively. The resulting pME-SEP
plasmid was
digested with Eco47III and NotI to remove its
luminal domain, whereas the mutant pME-bECE-1b plasmid was digested
with SmaI and NotI to only give a fragment
containing its luminal domain. These two fragments were then ligated in
the correct reading frame.
(residues
40-742) in two steps. The beginning of the extracellular domain of
pME-bECE-1b and pME-SEP
was first mutagenized to contain
EcoRV and Eco47III sites, respectively. The
mutant pME-bECE-1b plasmid was digested with EcoRV and
XbaI to remove its luminal domain, whereas the mutant
pME-SEP
plasmid was digested with Eco47III
and XbaI to only isolate its luminal domain. These two
fragments then were ligated to yield pME-E/E/S.
/E chimeric protein was prepared by
locating the insertion region (amino acids 41-63) of mouse SEP between
the transmembrane and the extracellular domain of bECE-1b as follows.
First, pME-bECE-1b was mutagenized to create SalI site at
the beginning of its extracellular domain. Second, PCR amplification of
the insertion region of SEP was performed using a sense primer
containing a SalI site (underlined),
5'-GTCGACAGGGAAGCAGCTGCC-3', and an antisense primer,
5'-GTCGACCGTTTTACAACCGTC-3', including a SalI
site in the 5' end. The PCR product was digested with SalI and inserted into the plasmid pME-bECE-1b digested with the same enzyme.
was subcloned into the pME18Sf(
) expression vector under the control
of the SR
promoter (4). Stable transfection of CHO cells and
isolation of the transfectant clones (CHO/SEP and
CHO/SEP
) were performed as described previously (2).
Transient transfections of SEP, SEP
, and mutant cDNA
were carried out using LipofectAMINE Plus (Life Technologies, Inc.) as
described by the manufacturer. The cells were cultured for 30 h
after transfecting the plasmid into the cells.
20 °C. After washing and blocking, the cells were
probed with a polyclonal antibody directed against the mouse SEP
C-terminal peptide (1:100), the bovine ECE-1 C-terminal peptide
(1:200), or the human MTP (1:50). The cells were again washed before
incubation with normal goat serum/phosphate-buffered saline containing
7.5 µg/ml fluorescein isothiocyanate-goat anti-rabbit IgG
(Zymed Laboratories, Inc.). Finally, the coverslips
were mounted on microscope slides with 90% (v/v) glycerol, 50 mM Tris-HCl, pH 9.0, and 2.5% (w/v)
1,4-diazadicyclo-[2.2.2]octane.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Are Derived from a Single Gene by
Alternative Splicing--
Previously, we have isolated and
characterized two isoforms of mouse SEP (4). SEP (765 residues) and
SEP
(742 residues) differ only in the absence of a
23-amino acid insertion immediately following the transmembrane domain
in SEP
, but both share the same N termini transmembrane
domain and C-terminal residues.
were produced (Fig.
1B). It then became apparent that this insertion exon
coincided with an intron-exon boundary.
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Fig. 1.
Organization of the region around the
insertion exon of the mouse SEP gene.
A, the partial nucleotide sequences and intron-exon
junctions of the 5' region of the mouse SEP gene. The
insertion exon (dotted box), its immediate 5' exon
(closed box), and its next 3' exon (open box) are
shown. Intron-exon boundaries were determined by sequencing the SEP-BAC
clone oligonucleotides as shown under "Experimental Procedures."
Upper and lowercase letters represent exon and
intron sequences, respectively. Consensus splice donor and acceptor
sites are underlined. The intron immediately located 5' to
the encoding insertion exon is ~9.5 kilobase pairs, and the length of
its immediate 3' intron is 555 base pairs. B, generation of
SEP and SEP isoform mRNAs.
Expression Patterns Show ER
Localization--
To characterize the features of both SEP and
SEP
, we generated transfectant cells CHO/SEP and
CHO/SEP
by transfecting expression constructs driven by
the SR
viral promoter. Immunoblot analysis with an anti-SEP
C-terminal peptide antiserum showed that only CHO/SEP and not
CHO/SEP
cells release its soluble form with an apparent
molecular mass of ~126 kDa into the culture medium, whereas both
membrane-bound forms are expressed as an approximate 110-kDa protein in
the membrane preparation of these cells (4).
cells with either Endo H or PNGaseF reduced the
apparent molecular mass from 110- to 89-kDa, which corresponds to the
calculated molecular mass of SEP, whereas N-linked
glycosylation of the SEP soluble form was removed by PNGaseF and found
to be resistant to Endo H (Fig.
2A). These observations
confirm that the 110-kDa species observed in the membrane fraction of
the cells is the partially glycosylated protein present in the early
secretory pathway.
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Fig. 2.
ER localization of the membrane-bound
SEP. CHO cells were stably transfected with an expression
construct containing the SEP, SEP , or MTP expression
constructs. A and C, glycosidase sensitivity of
soluble and membrane-bound forms of SEP. The soluble form of SEP in the
supernatant as well as the membrane-bound SEP was digested with Endo H
(H), or PNGaseF (F), and Endo D (D) as
described under "Experimental Procedures." Control samples (
)
were incubated in parallel without endoglycosidases using an identical
buffer. The samples were then subjected to SDS-PAGE followed by
immunoblotting. C, cell lysates; S, supernatants.
B, immunofluorescence localization of SEP,
SEP
, and MTP in CHO cells. Under permeabilization
conditions, CHO/SEP, CHO/SEP
, and CHO/MTP cells
exhibited strong intracellular staining, whereas CHO-K1 cells, the
parental CHO cell line, exhibited no staining.
cells with antibodies that recognize the common
C-terminal ectodomain of SEP. After permeabilization, both cells showed
strong intracellular staining. This intracellular staining pattern is
indistinguishable from the results observed when we stained the
microsomal triglyceride transfer protein, an ER resident protein, using
a polyclonal antibody against human MTP (8). These intracellular
staining patterns were typical ER patterns (Fig. 2B). Taken
together, these data demonstrated that the membrane-bound SEP and
SEP
are localized in the endoplasmic reticulum.
proteins were
subjected to endo-
-N-acetylglucosaminidase D digestion. Retention refers to the protein never being exported out of the ER. In
the retrieval mechanism, however, proteins escape from the ER to the
cis-Golgi where the oligosaccharide is converted from
Man8GlcNAc2 to
Man5GlcNAc2 by mannosidase-I and then retrieved to the ER (11). Although Endo H digests both ER and
cis-Golgi types of glycosylation, Endo D uniquely hydrolyzes
only N-linked oligosaccharides of the
Man5GlcNAc2 (12, 13). As shown in Fig.
2C, both SEP and SEP
were resistant to Endo
D. In addition, it has been accepted that the incubation of cells with
the microtubular inhibitor nocodazole alters the distribution of ER
proteins by recycling from the intermediate compartment (14). We
observed that the distribution of both SEP and SEP
were
not altered by this treatment (data not shown). Therefore, these
results suggest that these proteins localized in the ER by a retention mechanism.
Determines Its ER
Localization--
To analyze the features of the membrane-bound
SEP/SEP
responsible for ER retention, we substituted the
cytoplasmic transmembrane and luminal domains of this protein with the
corresponding domains of bECE-1b (Fig.
3A). The bECE-1b is another
member of this metalloprotease family, and this protein is normally
transported to the cell surface (2). Because no differences were
observed in the localization and/or retention of the membrane-bound
SEP/SEP
, only the findings with SEP
are
presented.
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Fig. 3.
The luminal domain of
SEP protein is a determinant for ER
retention. A, a schematic diagram of
the wild-type SEP
and wild-type bECE-1b proteins and of
SEP
-bECE-1b chimeras. The cytoplasmic tail and luminal
domain of the SEP
sequence are indicated as open
bars, and the transmembrane helix is indicated by a closed
bar. The sequence derived from the cytoplasmic tail and the
luminal domain of bECE-1b is shown as dotted bars, and its
transmembrane helix is shown as a striped bar. B,
immunofluorescence microscopy of CHO cells transiently transfected with
plasmids encoding wild-type SEP
, S/S/E, E/E/S chimeras
or wild-type bECE-1b. Permeabilized cells were stained with a
polyclonal antibody directed against mouse SEP or bovine ECE-1
C-terminal peptide followed by fluorescein-conjugated secondary
antibody. C, glycosidase digestion of SEP
,
S/S/E, E/E/S, and bECE-1b. Cell lysates from cells expressing these
constructs were digested with Endo H (H) and PNGaseF
(F) as described under "Experimental Procedures."
Control samples (
) were incubated in parallel without
endoglycosidases using an identical buffer. The samples were then
subjected to SDS-PAGE followed by immunoblotting.
cytoplasmic,
SEP
transmembrane, and bECE-1b luminal, Fig.
3A) in which the cytoplasmic tail and transmembrane domain
of SEP
were fused to the luminal domain of bECE-1b. When
the subcellular localization of the chimeric protein was analyzed in
transfected CHO cells unexpectedly, the S/S/E chimera was expressed on
the cell surface as detected by immunofluorescence in permeabilized cells (Fig. 3B). This surface labeling in permeabilized
cells was also observed in the wild-type bECE-1b, suggesting that like bECE-1b this chimera exits from the ER very efficiently, which results
in high concentrations of the chimera at the cell surface. Nontransfected control cells did not react with the anti-ECE-1 polyclonal antibody (data not shown), consistent with the evidence that
CHO cells have little or no endogenous activity of ECE-1 (2).
Therefore, it appears that in the absence of the SEP
luminal domain, the cytoplasmic SEP
tail in combination
with its transmembrane domain is not sufficient to achieve retention.
is
sufficient for the correct targeting of the protein. A chimeric
construct in which both the cytoplasmic tail and transmembrane domain
of SEP
were replaced by those of bECE-1b (E/E/S, ECE-1b
cytoplasmic, ECE-1b transmembrane, and SEP
luminal, Fig.
3A) was thus created. Immunofluorescence analysis of
transient transfectants expressing E/E/S chimera showed the same
distribution as the wild-type SEP
. This chimeric protein
behaved similarly to wild-type SEP
as indicated by its
typical internal staining pattern and its lack of cell surface staining
(Fig. 3B). These data indicate that the SEP
luminal domain, independent of the transmembrane domain, determines the
localization of SEP
protein in the ER.
stable transfectant cells, we
provide a biochemical analysis of SEP
trafficking and
turnover by pulse-chase experiments. The CHO/SEP
cells
were pulse-labeled with 35S-amino acids for 30 min and
followed by chase periods at designated times. At specific time
intervals, cell extracts were prepared and immunoprecipitated with an
anti-SEP antibody. Before analysis by SDS-PAGE, half of the samples
were treated with Endo H. During all the chase periods, the proteins
remained fully sensitive to Endo H, suggesting that it never acquired
any Golgi enzyme modifications (Fig.
4A). In addition, as shown in
Fig. 4B, the membrane-bound SEP
was retained
in the ER with a half-life of more than 2 h. Therefore, it appears
that the retention of membrane-bound SEP in the ER is not because of
gross misfolding. Our previous findings also support this conclusion.
We have reported that both the membrane-bound and the soluble forms of
SEP have an activity to hydrolyze big ET-1 (4).
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Fig. 4.
Biosynthesis and Endo H sensitivity of
SEP . A, CHO cells
stably expressing SEP
were labeled for 30 min with
35S label and then chased in the presence of complete
medium for 0, 1, 2, and 4 h. Thereafter, half of the
SEP
protein was digested with Endo H before analysis on
SDS-PAGE. C, cell lysate. B, the relative amount
of immunoprecipitate at each time point was calculated as a percentage
of the amount labeled at 0 h. The quantitative data shown
represent the mean of results from three independent experiments.
polypeptides predicted from the cDNA are identical except for the
23 amino acids unique to SEP, we hypothesized that the structural
determinants, which cause the release of the soluble form of SEP, must
be embedded within these 23-amino acid insertion. To test this
hypothesis, we made a construct (E/E/
/E) in which these 23 amino
acids (residues 41-63 of SEP) were inserted in frame between the
transmembrane and extracellular domains of bECE-1b. When cells
expressing this construct were analyzed by immunoblotting, the chimeric
protein was secreted efficiently as in the case of wild-type SEP (Fig.
5A), thus demonstrating that
the insertion domain of SEP is necessary and sufficient for the
cleavage process.
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Fig. 5.
The structural determinant of SEP cleavage is
embedded within the insertion region. A, immunoblot
analysis of supernatant and cell lysate from CHO/SEP and
CHO/(E/E/ /E) chimera. CHO cells were transiently transfected with an
expression construct containing SEP or E/E/
/E chimera. Cell lysates
(C) and supernatants (S) from each cell were
separated on 7.5% SDS-PAGE under reduced conditions, blotted, and
detected using anti-C-terminal peptide antisera against SEP or ECE-1.
B, amino acid sequence of the insertion region of SEP and
cleavage phenotype of wild-type and mutant SEP forms. The numbering
refers to the predicted amino acid residue starting from the
N-terminal methionine of mouse SEP. The mutants are named by the
position of their deletions as shown at the left margin of the figure.
Dash lines indicate deleted regions of the SEP sequence.
Wild-type and mutant SEP cDNAs were transiently transfected into
CHO cells and supernatant, and cell lysates were separated on 7.5%
SDS-PAGE and subjected to immunoblotting. Symbols refer to
the proportion of mutant SEP secreted. The proportion secreted was
<10% (
), 10-40% (+), 50-80% (++), or >90% (+++) of total SEP
relative to wild-type SEP secretion.
62-63) or mutated to
Ser62-Ser63 (KR-SS) or
Asn62-Gly63 (KR-NG), but all these mutant SEP
forms were cleaved as efficiently as wild-type SEP in CHO cells (Fig.
5B).
58-63) completely prevented SEP cleavage as did the deletion of
the C-terminal 7, 8, 9, 17, 19, and 21 amino acids (Fig.
5B). In contrast, the deletion of 14 amino acids at the
N-terminal of the SEP insertion (
41-54) did not have any effect,
and only after we deleted 17 amino acids (
41-57) was SEP secretion
abolished. Therefore, the C-terminal of this insertion seems
dispensable for the secretion of this protein. To further define the
important amino acids in the C-terminal of the SEP insertion, we kept
Arg58 in the C-terminal deletion mutant (
59-63) and
observed that this mutant was 50% cleaved compared with the wild-type
form. The lengthening of the C terminus with Thr59
(
60-63) did not change the cleavage efficiency, but the extension with two valine residues (amino acids 60 and 61) (
62-63) increased the cleavage to a level similar to that of the wild-type form. To
identify the minimal requirement of the N-terminal residues, we
extended the
41-57 mutant with Glu57 (
41-56)
producing a significant amount of protein in the medium (approximately
50% compared with the wild-type). When we lengthened
41-57 mutant
until Trp55 (
41-54), this mutant plasmid (
41-54)
was 100% cleaved. These results indicate that a stretch of
seven amino acids (WDERTVV) in the C-terminal part of the SEP insertion
is the minimal structural determinant of the SEP cleavage.
(Fig. 4A), we
hypothesized that soluble SEP is cleaved in the ER and then transported
to the downstream secretory pathway until it is secreted. For this
purpose, we used a fusion protein secECE-1 in which the signal peptide
of human placental alkaline phosphatase (PLAP) is joined in frame to
the luminal domain of bECE-1b as a tool to compare the intracellular
trafficking and turnover of the protein with SEP (19). The signal
peptide of placental alkaline phosphatase has been shown to be cleaved
by a signal peptidase in the ER (20).
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Fig. 6.
Posttranslational modification of secECE-1,
SEP, and bECE-1b and ER localization of membrane-bound
E/E/ /E. Equivalent numbers of CHO cells
stably expressing secECE-1 (A), SEP (B), or
bECE-1a (C) were pulse-labeled with
[35S]methionine for 30 min and then chased for a
designated time in complete medium. SecECE-1 and SEP proteins were
immunoprecipitated both from the supernatants (S) and cell
lysates (C), whereas bECE-1b protein was immunoprecipitated
only from cell lysates. Before analysis on SDS-PAGE, half of the
samples were treated with Endo H. An autoradiograph from a
representative experiment is shown. D, immunofluorescence
microscopy of transiently transfected CHO cells expressing E/E/
/E
chimera. Fixed and permeabilized cells were stained with a bovine ECE-1
antibody.
, which lacks the insertion domain.
This protein never received Golgi enzyme modifications (Fig.
4A). A strikingly different result was observed for the
membrane-bound form of wild-type bECE-1b. It has been shown that the
bECE-1b protein was transported to the down secretory pathway after
synthesis and finally resided in the plasma membrane (2). At 0 h
of chase (30 min of pulse), we already observed the Endo H resistant
band in the membrane-bound bECE-1b, and after 1 h, a significant
amount of protein was exposed to the Golgi or plasma membrane as
observed by its resistance to Endo H. Meanwhile, the protein retained
in the ER (Endo H sensitive, lower band) was gradually
exported to the plasma membrane and disappeared from the ER within
4 h (Fig. 6C). These findings show that the trafficking
of membrane-bound SEP is distinct from that of membrane-bound bECE-1b
and behaved similarly to membrane-bound secECE-1. Furthermore, when we
immunostained the E/E/
/E chimera (Fig. 5A) in CHO cells,
we observed a typical ER-staining pattern (Fig. 6D). This
suggests that to be secreted, this chimera protein is initially
targeted to the ER from which proteolytic cleavage occurs and then
transported to the down secretory pathway. Taken together, these data
strongly indicate that SEP is cleaved in the ER and subsequently is
secreted rapidly into the medium.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. These findings indicate that the sites where SEP
functions as a metalloprotease are determined, at least in part, by an
alternative splicing mechanism.
viral promoter in our expression construct, we did not observe any
leakage of the SEP protein in the downstream secretory pathway. This
implies that this localization mechanism could not be saturated by
overexpression, suggesting the retention mechanism (27). Moreover, our
experiments with Endo D (specific for
Man5GlcNAc2 trimming) and nocodazole (data not
shown) also provide further evidence. Nevertheless, the idea that SEP
proteins localize in the ER by a retention mechanism remains a working hypothesis because the available results could not exclude a rapid recycling from the intermediate compartments.
KR mutant protein was completely
secreted when we transfected an expression construct of this mutant
into CHO cells (Fig. 5B) as well as human embryonic kidney
293 cells (data not shown). Although we cannot exclude the possibility
that the presence of SEP proteins in the supernatant is because of
overexpression, it is worth noting that the expression level of
SEP/
KR was not different as compared with the wild-type SEP. In
addition, if overexpression is the reason, we should observe leakage
from the ER in other mutants. In fact, we failed to detect SEP
secretion in the
41-57 and in the
58-63 mutants (Fig.
5B). Accordingly, based on the results presented in this
paper, we conclude that SEP was not cleaved at the dibasic residues. We
were also able to exclude the possibility of autoproteolytic cleavage,
as in the case of profurin cleavage (28) by finding that the inactive SEP mutant in which the glutamic acid residue in the zinc binding consensus sequence was replaced with glycine, was completely secreted (data not shown). Therefore, we assumed that SEP might be cleaved by
the membrane secretase in a fashion similar to that of some membrane
proteins, such as pro-TGF-
(30), angiotensin-converting enzyme (31), kit ligand (32), or colony-stimulating factor-1 (33).
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ACKNOWLEDGEMENTS |
---|
We thank Toshio Terashima in the Department of Anatomy for providing open access to the facilities. We also thank Catherine Lynn T. Silao for critical reading of the manuscript and Tetsuaki Hirase for technical assistance.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Division of Cardiovascular and Respiratory Medicine, Dept. of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe 6500017, Japan. Tel.: 81-78-382-5846; Fax: 81-78-382-5859; E-mail: emoto@med.kobe-u.ac.jp.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M101703200
2 S. B. Raharjo, N. Emoto, M. Yokoyama, and M. Matsuo, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
NEP, Neutral
endopeptidase;
ECE, endothelin-converting enzyme;
bECE-1b, bovine
endothelin-converting enzyme-1b;
SEP, soluble secreted endopeptidase;
ET, endothelin;
BAC, bacterial artificial chromosome;
PCR, polymerase
chain reaction;
CHO, Chinese hamster ovary;
MTP, microsomal
triglyceride transfer protein;
PAGE, polyacrylamide gel
electrophoresis;
Endo H, endo--N-acetylglucosaminidase H;
PNGaseF, peptide-N-glycosidase F;
Endo D, endo-
-N-acetylglucosaminidase D;
TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone;
ER, endoplasmic reticulum.
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