(Received for publication, October 27, 1994)
From the
Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and
rats contain subunits that are type I integral membrane proteins
and
subunits that are disulfide-linked to or noncovalently
associated with membrane-anchored meprin subunits. Both
and
subunits are synthesized with COOH-terminal domains predicted to be
cytoplasmic, transmembrane, and epidermal growth factor-like; these
domains are retained in
subunits but are removed from
during maturation. The present studies establish that an inserted
56-amino acid domain (the ``I'' domain), present in
but
not in
, is necessary and sufficient for COOH-terminal proteolytic
processing of the
subunit. This was demonstrated by expression of
mutant meprin subunits (deletion mutants, chimeric
subunits,
and
mutants containing the I domain) in COS-1 cells. Mutations of
two common processing sites present in the I domain (a dibasic site and
a furin site) did not prevent COOH-terminal proteolytic processing,
indicating that the proteases responsible for cleavage are distinct
from those having these specificities. Deletion of the I domain from
the
subunit resulted in accumulation of unprocessed subunits in a
preGolgi compartment. Furthermore, COOH-terminal proteolytic processing
of wild-type
subunits occurred before acquisition of
endoglycosidase H resistance. Pulse-chase experiments and expression of
an
subunit transcript containing a c-myc epitope tag,
confirmed that proteolytic processing at the COOH terminus occurs in
the endoplasmic reticulum. This work identifies the region of the
subunit that is essential for COOH-terminal processing and demonstrates
that the differential processing of the evolutionarily-related subunits
of meprin A that results in a structurally unique tetrameric protease
begins in the endoplasmic reticulum.
Meprins are plasma membrane metalloendopeptidases of the
``astacin family'' that are expressed at high concentrations
in brush-border membranes of mouse and rat kidney and of mouse, rat,
and human
intestine(1, 2, 3, 4, 5, 6, 7) .
They are glycosylated homo- or heterotetrameric proteins of
and/or
subunits(8) . The mouse
and
subunits
are evolutionarily related (approximately 42% overall sequence
identity), and their cDNAdeduced primary sequences predict a similar
arrangement of functional domains.
Both transcripts encode a
transient NH-terminal signal sequence (S) and a prosequence
(Pro) preceding the catalytic protease domain. Following the protease
domain is a MAM domain, proposed to be an adhesion domain which has
been identified as a component of several cell surface proteins
including meprin, A5 protein, protein tyrosine phosphatase µ, and
enteropeptidase(9, 10) . Near the COOH termini, both
nascent proteins have an epidermal growth factor
(EGF)(
)-like, a hydrophobic putative transmembrane-spanning
(T), and a cytoplasmic (C) domain. Between the MAM and EGF-like domain
is a large domain (182 amino acids for
, 176 amino acids for
) of unknown function, the X domain, which has no known homology
to other proteins. The two transcripts differ in that
encodes a
56-amino acid sequence between the X and EGF-like domains that has no
counterpart in
, indicated above as the inserted (I)
domain(11) .
Previous results indicate that the biosynthetic
maturation of meprin subunits involves NH- and
COOH-terminal proteolytic cleavages and that the mouse kidney
and
subunits are differentially processed in
vivo(11) . During biosynthesis of the
subunit in the
mouse kidney, the signal sequence and the prosequence are removed from
the NH
terminus, whereas the mature
subunit retains
the prosequence and thus has latent proteolytic activity. Differences
in COOH-terminal processing determine the expression of membrane-bound
and secreted forms. The mature
subunit retains all COOH-terminal
domains and is an integral type I membrane protein. The mature
subunit contains neither the COOH-terminal membrane anchor (T) domain
nor the EGF-like domain predicted from the
subunit transcript;
these domains are removed by co- or posttranslational proteolytic
cleavage(s). Thus,
subunits are always membrane-bound, whereas
subunits are either membrane-associated (if complexed with
subunits by disulfide-linkages or adhesion) or secreted into the urine
(as homooligomers). This type of oligomeric organization is unique
among cell surface proteinases.
The enzymes responsible for
NH- and COOH-terminal proteolytic processing and the
subcellular sites of processing are not yet known. However, there is
evidence that cultured cells could be useful for investigating
COOH-terminal processing events. Expression of rat and human meprin
subunit transcripts in human 293 cells, COS-1 cells, or
Madin-Darby canine kidney cells leads to secretion of the subunit into
the culture medium as a
zymogen(12, 13, 14, 15, 16) .
These and other data indicate that the
subunit is processed at
the COOH terminus in cultured cells as in vivo but that the
NH
-terminal prosequence is not removed in cultured cells as in vivo, and thus these cells produce an inactive meprin
subunit. The amino acid sequence of the mouse
subunit is 86%
identical to the rat subunit and 75% identical to the human sequence,
and the expectation is that mouse subunits are processed as the rat and
human counterparts.
Molecular weight estimates of deglycosylated
meprin subunits from mouse kidney indicated that the processing of
occurs within or very close to the I domain(11) .
Furthermore, this domain contains two sites, a furin-site and a dibasic
amino acid site, that are potential candidates for
processing(17) .
In order to determine the role of the
subunit I domain in processing, chimeric and mutant meprin
subunits were expressed in COS-1 and human 293 cells. In addition,
experiments were designed to determine the subcellular site of
COOH-terminal processing. The studies establish that processing of
subunits occurs early during biosynthesis in a preGolgi
compartment and that the I domain is required and sufficient for
COOH-terminal proteolytic processing.
The amino acid substitutions in the I domain of the mouse
subunit and the deletions of the I domain or portions of the I domain
were generated by site-directed mutagenesis following the method of
Deng and Nickoloff (20) using the Transformer site-directed
mutagenesis kit (Clontech). The mutagenic primers for the base
substitutions were designed to change amino acids
Arg
/Lys
to Gly/Gly (construct
645GG)
and/or Lys
/Arg
to Gly/Gly (constructs
666GG and
645/666GG). The mutagenic primers for the deletion
mutants contained the flanking regions surrounding a deletion of
nucleotides 1897-2064 (construct
628-683,
deletion of nucleotides 1996-2064 (construct
661-683), or deletion of nucleotides 1897-1995
(construct
628-660). All clones were further analyzed
for the desired mutations by DNA sequencing or restriction mapping. The
mutations are shown diagrammatically (see Fig. 2Fig. 3Fig. 4).
Figure 2:
Cell-associated expression of meprin
subunit mutants lacking the inserted domain. Schematic diagrams show
wild-type (wt) and mutant meprin subunits that were
transfected into COS-1 cells. The individual domains are indicated by
the abbreviations: P, protease domain; MAM, adhesion
domain; X, unknown domain; I, inserted domain; E, epidermal growth factor-like domain; T,
transmembrane domain. The
subunit domains are shown in white; shadedareas represent mouse
subunit sequence. All samples were subjected to SDS-PAGE in the
presence of
-mercaptoethanol, transferred to nitrocellulose, and
probed with an anti-meprin
subunit antibody. PanelA, Cellular membranes (cells) and media from
cells transfected with either wild-type meprin
subunit cDNA or
mutant cDNAs; panelB, cellular membrane fractions
before and after treatment with Endo F or Endo H; panelC, membrane fractions isolated from transfected COS-1
cells were treated with sodium carbonate, and supernatant and
precipitated fractions were prepared as described under
``Experimental Procedures.''
Figure 3:
Expression of mutant rat meprin
subunits with inserted mouse
subunit I domain or substituted
COOH-terminal domains. Schematic diagrams show the wild-type (wt) and mutant meprin subunits that were transfected into
COS-1 cells. Rat
subunit domains are shown in gray;
mouse
subunit sequences are shown in white. Tissue
culture media and cellular membrane fractions from COS-1 cells
transfected with the wild-type or mutant
subunits were subjected
to SDS-PAGE in the presence of
-mercaptoethanol. Meprin subunits
were detected with anti-rat meprin
antibodies.
Figure 4:
Expression of mutant meprin subunits
containing amino acid substitutions or deletions within the inserted
domain. Top, schematic diagrams of the wild-type and mutant
meprin subunits. The wild-type mouse
subunit contains two dibasic
sites within the inserted domain at positions
Arg
/Lys
and
Lys
/Arg
. In the substitution mutants, one
or both dibasic sites were replaced with double glycine residues, as
indicated. The deletion mutants lack either the amino-terminal 33 amino
acids of the I domain (residues 628-660) or the COOH-terminal 23
amino acids of the I domain (amino acids 661-683), as indicated
by the horizontal bars. Samples from cells transfected with wild-type
or mutated meprin
subunit cDNA were subjected to SDS-PAGE in the
presence of
-mercaptoethanol; immunoblots were analyzed using
antimeprin
subunit antibodies.
COOH-terminal deletion
mutants for the meprin subunit were generated by PCR using
mutagenic antisense primers, which changed the codons for Leu
(construct
26), Arg
(construct
76), or Arg
(construct
133) to stop
codons. A diagram for the mutations is shown (see Fig. 5).
Figure 5:
Expression of mutant meprin subunits
lacking portions of the COOH terminus. Top, schematic diagrams
of the wild-type and mutant meprin subunits. Bottom,
concentrated tissue culture media and membrane-enriched fractions from
cells transfected with either vector DNA (control), wild-type
meprin
subunit cDNA, or truncated transcripts were subjected to
SDS-PAGE in the presence or absence of
-mercaptoethanol, followed
by immunoblot analysis using anti-meprin
subunit
antibodies.
To
generate the meprin /
subunit hybrid cDNA encoding mouse
subunit residues Met
-Ser
fused
in-frame to mouse
residues
Pro
-Phe
, a TthIII site
present at position 1832 of the mouse
cDNA was used (see Fig. 2). Full-length mouse meprin
subunit cDNA, obtained
by reverse transcriptase PCR from C3H/HeJ mouse kidney RNA, was cloned
into pcDNAI/Amp and digested with TthIII and EcoRI to
excise nucleotides 1-1832. A TthIII site was introduced
into position 1880 of the meprin
subunit cDNA by PCR; the PCR
product was digested with TthIII and EcoRI and
subsequently ligated into the TthIII-digested pcDNAI/Amp
vector containing the
subunit 3` end.
The chimeric constructs
/
and
/
/
were generated by recombinant PCR
with primers containing overlapping ``5` add on'' sequences
according to the method of Higuchi (21) using mouse
and
rat
cDNA. The
/
construct encodes rat
subunit
amino acids Ala
-Thr
fused in frame to the
mouse
subunit residues Arg
-Gln
.
The
/
/
construct encodes rat
subunit residues
Met
-Thr
, mouse
subunit residues
Arg
-Tyr
, and rat
subunit
residues Val
-Phe
fused in-frame. A
diagram of the constructs is shown in Fig. 3. All PCR reactions
were performed with Pfu DNA polymerase (Stratagene) or Ultma
DNA polymerase (Perkin Elmer) to minimize the possibility of base
misincorporations. The nucleotide numbers and amino acid numbers refer
to the published sequences for mouse
, mouse
, and rat
subunits(2, 3, 4) .
Human 293 cells were transfected with plasmids by the calcium phosphate precipitation procedure(24) . Briefly, 30% confluent cells were cotransfected with 10 µg of expression plasmid and 1 µg of PVA1, a helper plasmid, which enhances expression(25) . The cells were incubated in complete DMEM and grown to 90% confluence prior to exposure to radiolabeled methionine.
To immunoprecipitate the radiolabeled
subunits, cell lysates were mixed with equal volumes of a solution
containing protease inhibitors (1 mM PMSF, 1 mM EDTA,
100 µM 3,4-dichloroisocoumarin, 10 µM E-64)
and incubated with 4 µl of anti-
antiserum or 0.8 µg of
c-myc antibody (Oncogene Science) at 25 °C for 1 h.
Concentrated media were mixed with equal volumes of lysis buffer and
then treated as cell lysates. Then, 30 µl of protein A-Sepharose
(1:1 suspension) was added to the mixture. After a 1-h incubation, the
protein-A Sepharose complex was precipitated by centrifugation and
washed twice with 1 ml of lysis buffer (at half the concentration given
above), and once with 20 mM Tris-HCl, pH 7.5, containing 137
mM NaCl, 0.1% Tween-20. Finally, the immunoprecipitates were
prepared for SDS-PAGE by suspending and boiling in 50 µl of SDS
sample buffer. Generally, 25% of the sample obtained from one 100-mm
plate was applied to each lane for SDS-PAGE.
When mouse meprin subunits were expressed in COS-1
cells, most of the immunoreactive material was found in the tissue
culture medium as secreted protein with a subunit mass of about 95 kDa (Fig. 1, topgel, lane3).
The secreted protein was catalytically inactive, but could be activated
with trypsin treatment (data not shown). A small fraction of the
expressed meprin
subunits was found associated with cellular
membranes (Fig. 1, topgel, lane4); the cell-associated meprin consisted of several bands
with apparent molecular masses of 86-102 kDa, with the 86 kDa
band being the predominant form. The supernatant fractions of cell
homogenates did not contain any immunoreactive material (data not
shown). Under nonreducing conditions, the secreted and the
cell-associated meprin subunits were oligomeric with apparent molecular
weights larger than 180,000 (Fig. 1, top gel, lanes 5 and 6). The cell-associated meprin subunits were
sensitive to Endo F and Endo H, indicating that complex glycosylation
had not yet occurred (Fig. 1, bottomgel).
Treatment with either of the endoglycosidases produced two bands with
molecular masses of approximately 81 and 65 kDa (Fig. 1, bottomgel, lanes1-3), with
the 65 kDa band being the predominant form. In contrast, the secreted
meprin subunits in the culture medium contained some carbohydrate
chains that are resistant to Endo H treatment (Fig. 1, bottomgel, lanes4-6). Endo
F treatment decreased the molecular mass of the secreted meprin
subunit from approximately 95 to 67 kDa; Endo H treatment decreased the
molecular mass to approximately 79 kDa. Thus, in transfected COS-1
cells, the mature mouse
subunit is primarily a secreted protein.
A small amount of meprin subunits remains associated with the
endoplasmic reticulum (ER), indicated by the Endo H sensitivity. There
are no detectable amounts expressed on the cell surface because no Endo
H-resistant forms of meprin were detected associated with cells. These
results are generally consistent with those obtained for the
transfected human and rat meprin
subunits in COS-1
cells(12, 13, 15) . Furthermore, the
molecular mass of the predominant cell-associated deglycosylated mouse
subunit indicated that COOH-terminal processing occurs in the ER.
Figure 1:
Immunoblot analysis of the mouse meprin
subunit expressed in COS-1 cells. COS-1 cells were transfected
with wild-type meprin
subunit cDNA as described under
``Experimental Procedures.'' Concentrated tissue culture
media (medium) and membrane-enriched fractions (cells) were subjected to SDS-PAGE and immunoblotting using
anti-meprin
subunit antibodies. Topgel,
SDS-PAGE in the presence or absence of
-mercaptoethanol. Lanes1 and 2 contained control samples transfected
with vector DNA only; lanes3-6 contained
samples after transfection with meprin
subunit cDNA. Bottomgel, SDS-PAGE in the presence of
-mercaptoethanol
and immunoblot analysis of membrane-enriched fractions or media
samples. Samples were incubated with SDS and
-mercaptoethanol in
the presence or absence of Endo F or with Endo H. The relative
positions of the molecular mass markers (in kDa) are
shown.
To
determine whether the I domain of is necessary for COOH-terminal
processing, two mutant meprin subunits lacking this domain were
expressed in COS-1 cells (Fig. 2). In construct
628-683, the I domain of
was deleted by
site-directed mutagenesis. In construct
/
, the
subunit
domains COOH-terminal to the X domain were replaced with the
corresponding domains of the
subunit. Analysis of the culture
media and membrane fractions by immunoblotting showed that, in contrast
to the wild-type
subunit, neither of the mutant meprin subunits
were secreted into the culture medium, they were found exclusively in
membrane fractions (Fig. 2, panelA). In
addition, the molecular masses of the cell-associated mutant subunits
were slightly larger than the molecular masses of the secreted
wild-type
subunit (approximately 105 kDa for the
/
chimeric subunit and 102 kDa for the
628-683 mutant,
compared with 95 kDa for the wild-type secreted
subunit). The
larger molecular masses of the mutant subunits compared with the
wild-type subunit provide further evidence that these mutants are not
proteolytically processed at the COOH terminus but retain the EGF-like
and transmembrane domains. To test whether the mutant proteins are
expressed on the cell surface or retained intracellularly, the isolated
membranes were subjected to endoglycosidase digestion (Fig. 2, panelB). The
628-683 mutant was Endo
H-sensitive, and thus was retained in a preGolgi compartment. In
contrast, the
/
chimeric subunit was resistant to Endo H
digestion, indicating that this subunit was present in a postGolgi
compartment, most likely the cell surface. The results of transfecting
the
/
chimeric subunit indicate that the COOH terminus of the
subunit, but not the
subunit, contains the information for
COOH-terminal processing. The
628-683 mutant was not
processed and not able to leave the ER, indicating the importance of
the I domain for processing and secretion.
To further test whether
the COOH-terminal removal of the membrane anchor of occurs in the
ER, carbonate treatment of isolated membranes from transfected COS-1
cells was performed. Carbonate treatment releases lumenal proteins and
peripheral membrane proteins from membrane vesicles, while
transmembrane proteins remain associated with the membrane vesicles and
thus precipitate during centrifugation (27) . Carbonate
treatment of membranes from cells transfected with wild-type
subunits solubilized at least 50% of meprin
subunit protein but
failed to solubilize any meprin protein from cells transfected with the
628-683 mutant (Fig. 2, panelC). Thus, ER-associated wild-type meprin
subunits
are lumenal/peripheral ER-associated proteins that lack the
COOH-terminal membrane-anchoring domain, while the
628-683 mutant is membrane-anchored. These data provide
further evidence that COOH-terminal processing of the membrane
anchoring domain occurs early in meprin biosynthesis, before the
acquisition of complex carbohydrate modifications, and that the I
domain is essential for proteolytic processing.
To test whether the
I domain is sufficient to determine meprin subunit processing, the
subunit I domain was inserted in-frame into the rat
subunit
sequence. For this experiment the rat
subunit, which is more than
90% identical to the mouse subunit, was used (rather than the mouse
subunit) because of the availability of a specific and sensitive
antiserum to the rat
protein. The insertion of the I domain was
achieved in two steps; in the first step, the COOH-terminal domains of
(EGF-like, transmembrane, and cytoplasmic) were replaced with the
subunit COOH-terminal domains (I, EGF-like, transmembrane, and
cytoplasmic) (construct
/
). In the second step, the EGF-like,
transmembrane, and cytoplasmic domains of the
/
construct
were replaced with those of
(construct
/
/
). After
expression in COS-1 cells, wild-type rat
subunits were
predominantly membrane-bound as previously reported for rat and human
subunits. In contrast, both mutants
/
and
/
/
were predominantly secreted (Fig. 3). Thus,
insertion of the
subunit I domain into the
subunit sequence
is sufficient to direct COOH-terminal proteolytic processing and
secretion of normally membrane-bound
subunits.
The I domain of
contains two dibasic sites, amino acids
Arg
/Lys
and Lys
/Arg
that could serve as processing sites by enzymes with specificity
for dibasic motifs. Amino acids Lys
/Arg
are
also part of the consensus sequence for cleavage by furin-like
processing enzymes (RX(K/R)R)(17) . To test whether
the furin-like consensus sequence or the other dibasic site are
cleavage sites in
, the dibasic sites were replaced by glycine
residues, and the resulting mutant proteins were expressed in COS-1
cells and analyzed for secretion and cellular localization (Fig. 4, leftgel). Eliminating either or both
of the two dibasic sites did not prevent secretion of the meprin
subunit into the culture medium, indicating that neither of these sites
is essential for processing.
In an attempt to map the cleavage site
to a smaller region within the inserted domain, portions of the
inserted domain were deleted (constructs 628-660 and
661-683), and the resulting mutants were analyzed (Fig. 4, rightgel). Both mutants were
processed and secreted into the culture medium. The apparent molecular
mass of
628-660 seemed slightly larger than the
molecular mass of the wild-type or
661-683 protein;
however, after deglycosylation with Endo F, all three proteins had
indistinguishable molecular masses (data not shown). The mutant
661-683 was secreted in amounts comparable with
wild-type
subunits, while the
628-660 construct
was secreted at somewhat lower levels. These results indicate that the
entire 56-amino acid sequence of the I domain is not required for
processing and that, if the processing site is located within the
inserted domain, more than one peptide bond can be cleaved by the
processing machinery.
To determine whether the COOH-terminal domains
in are important during meprin biosynthesis for either
dimerization or transport of meprins through the secretory pathway,
several COOH-terminal deletions were constructed and expressed in COS-1
cells (Fig. 5). Up to 133 amino acids, including the I domain,
were deleted from the
subunit COOH terminus. All truncated
proteins were secreted, and none of the deletions affected the level of
protein biosynthesis or the ability of subunits to dimerize.
Pulse-chase experiments in 293 cells showed that mouse meprin
subunits were present in two major forms (95 and 86 kDa) after 10 min
of exposure to [
S]methionine and that with time,
the 95-kDa form was converted to the 86-kDa form (Fig. 6). By 30
min, most of the radiolabeled subunits were of the 86-kDa form, and a
small amount of this form persisted in cells up to 6 h. The
cell-associated forms of the
subunit were Endo H-sensitive for
all time points (data not shown). Immunoprecipitable radiolabeled
subunits were detected in culture media by 1 h; secreted forms of the
subunit were Endo H-resistant as shown in Fig. 1. Thus, the
pulse-chase experiments indicated that two major glycosylated forms of
are produced early in biosynthesis (about 15-30 min), and
that there is a conversion from the larger to the smaller form before
the meprin
subunits acquire complex glycosylation.
Figure 6:
Pulse-chase radiolabeling of transfected
293 cells expressing wild-type mouse meprin subunits. Cells were
radiolabeled with [
S]methionine for 10 min and
then incubated in nonradioactive methionine-containing medium for the
times shown. At each time point, cells were harvested and lysed, and
the cells and medium were prepared as described under
``Experimental Procedures''. Meprin
subunits were
immunoprecipitated with an anti-meprin
subunit antibody. The
proteins were subjected to SDS-PAGE in the presence of
-mercaptoethanol and visualized by
fluorography.
In order to
determine more definitely whether the processing observed in
pulse-chase experiments was due to COOH-terminal proteolytic
processing, a rat meprin cDNA with a c-myc epitope at
the COOH terminus of the subunit was transfected into 293 cells. The
disappearance of the c-myc tag was studied by radiolabeling
the transfected cells with [
S]methionine (Fig. 7). A monoclonal antibody against the c-myc epitope only immunoprecipitated the largest form of radiolabeled
subunits. The anti-
meprin subunit antibody
immunoprecipitated several smaller forms of the subunit in addition to
the COOH-terminally unprocessed form. Furthermore, all forms associated
with cells were Endo H-sensitive, indicating that they exist in the ER.
The results of this experiment confirm that the COOH terminus of the
meprin
subunit is proteolytically processed in the ER.
Figure 7:
Immunoprecipitation of transfected meprin
subunits containing a c-myc epitope tag at the COOH
terminus, with anti-meprin
subunit antibodies or anti-c-myc antibodies. Human 293 cells were transfected with expression
vector (control) or expression vector containing the rat
meprin
cDNA tagged with a c-myc epitope at the COOH
terminus (c-myc). The cells were radiolabeled with
0.1 mCi/ml [
S]methionine for 1 h. Radiolabeled
meprin
subunits were then immunoprecipitated from cell lysates,
subjected to SDS-PAGE, and visualized by fluorography. Samples in lanes1-3, 6, and 7 were
immunoprecipitated with anti-meprin
subunit antibodies. Samples
in lanes4 and 5 were immunoprecipitated
with anti-c-myc antibodies. For lanes2 and 3, immunoprecipitated cellular proteins were treated with Endo
H and F, respectively, prior to
electrophoresis.
It is clear from the results presented herein that several
important events in the maturation of meprin subunits occur in the ER
(shown diagramatically in Fig. 8). The experiments indicate that
subunits are translated, dimerize, form intersubunit disulfide bridges,
and are glycosylated (high mannose), and, if the I domain is present,
the domains COOH-terminal to the X domain are proteolytically removed
in the ER. Once all these events occur, the subunits appear to exit the
ER, obtain complex sugars (probably in the Golgi apparatus), and are
secreted from cells through the constitutive pathway. This was the
scenario for most of the transcripts expressed herein in the cultured
cells, including wild-type subunits (Fig. 8, panelA),
subunits truncated down to the X domain,
subunits with the dibasic sites of the I domain mutated to GG,
subunits with the COOH-terminal or NH
-terminal halves of
the I domain deleted, and
subunits with the I domain inserted
between the X and EGF-like domains. There was very little evidence of
monomeric or nonglycosylated forms of these proteins in the cells,
indicating that dimerization and high-mannose glycosylation take place
immediately after translation. This could be important for the
stabilization of the subunits. Many proteins are degraded in the ER if
not properly assembled, and the oligomerization and glycosylation of
the subunits might protect the subunits from destruction in the ER, as
was shown for proteins such as the T cell receptor subunits, class II
major histocompatibility complex molecules, and the transferrin
receptor(28, 29, 30) . In addition, there was
no evidence for complex glycosylated meprin
subunits in the
transfected cells, which indicates that once the subunits leave the ER
they are secreted rapidly, without much delay in other subcellular
compartments.
Figure 8:
Diagrammatic representation of the
biosynthesis of meprin subunits. Meprin subunits transfected into COS-1
cells were either secreted (A), transported to the cell
surface as membrane-bound proteins (B), or retained in the ER
as membrane-bound proteins (C). The subunit sequences
are indicated by openstructures;
subunit
sequences are indicated by shadedstructures. The I
domain is indicated in panelA as a loop between the EGF-like domain (oval) and the main body of
the subunits which includes the X, MAM, and protease domains (rectangles); the I domain is not present in transcripts of panelB and C. Core N-linked glycosylation is indicated for all nascent proteins
by Y-shaped structures perpendicular to the subunits. Complex
glycosylation is indicated by circles on the Y-shaped
structures.
A different final localization was observed for
wild-type subunits (Fig. 8B) and the
/
chimera without the I domain. These subunits also dimerize, leave the
ER, and enter the Golgi where they obtain complex glycosylation. Thus,
they probably follow the constitutive pathway to the cell surface, as
the secreted forms. However, the lack of the I domain in these subunits
prevents their COOH-terminal processing, and as a consequence they
remain membrane-bound after they reach the cell surface.
A third
pathway was observed only for subunits with the I domain deleted (Fig. 8C). These subunits accumulate as unproteolyzed
high mannose precursors in the ER without being further transported to
the Golgi. Thus, the I domain is essential for processing. The data
further indicate that the COOH-terminal domains of
contain
information that leads to retention in the ER. It is clear that the I
domain in itself is not essential for intracellular transport because
COOH-terminally truncated subunits lacking the I domain are secreted
from cells. Rather, it is more likely that the deletion of the I domain
causes the accumulation in the ER by preventing the proteolytic removal
of an ER retention determinant COOH-terminal to the I domain. The
retained
subunit with a deleted I domain differs from the
/
chimeric subunit, which is not retained, only in that the
COOH-terminal EGF, T, and C domains are derived from
instead of
; thus these COOH-terminal domains determine retention or movement
out of the ER. It is probable that determining factors for retention
lie in the T or C domains of
. The transmembrane and cytoplasmic
domains of
and
share no significant homology, in contrast
to the rest of the subunit protein. There is a growing number of
membrane proteins, in which ER retention signals have been identified
in the transmembrane or cytoplasmic domains(31, 32) .
These signals are distinct from the tetrapeptide KDEL-like sequences at
the COOH termini of lumenal ER proteins, which determine retention
through a receptor-mediated retrieval pathway(33) . The T
domain of
contains a motif of repeating glycines, also found in
class II major histocompatibility complex molecules and glycophorin,
which have been proposed to form a surface that can mediate
interactions with other transmembrane
helices(29, 34) . These repeating glycines are not
present in the
T domain. The retention of unprocessed
subunits ensures that
subunits only exit the ER after
COOH-terminal processing, and may be an example of the quality control
function of this subcellular compartment(35) .
The results
herein demonstrate that the I domain is essential for processing.
However, the attributes of this domain that are required for processing
are not yet defined. It cannot be completely ruled out that the I
domain determines processing at a nearby sequence in through
conformational effects. However, the observation that the I domain can
function as a processing domain in the context of a different protein
(the
subunit) makes it more likely that the processing site(s)
lies within the I domain itself. This is also consistent with previous
calculations of the molecular size of processed deglycosylated
subunits from mouse kidney, which indicated that the mature
subunit ends in the NH
-terminal part of the I
domain(11) . The simple possibilities of the furin site or
dibasic site, or both sites being essential sites for cleavage were
eliminated by the site-directed mutagenesis experiments. Deletion of
the NH
- or COOH-terminal halves of the I domain also did
not prevent processing of the subunits, as they were still
predominantly secreted. Thus, more than one cleavage site can be used
by the processing enzyme(s). It is possible that the processing site is
not a specific recognition sequence but that processing occurs at an
exposed region that is vulnerable to proteolysis. The I domain might be
a region of poorly defined secondary or tertiary structure such as a
surface exposed loop, which is susceptible to cleavage by resident ER
proteases that are known to degrade misfolded proteins.
Dimerization is clearly not dependent on the T domain, as with some other proteins such as class II major histocompatibility complex molecules, glycophorin, and hemagglutinin, nor does it depend on the EGF-like or C domains because deletion of all of these still results in dimers(29, 34, 36) . Perhaps the MAM or X domains are important for alignment of the subunits and intersubunit disulfide bridge formation.
It is well established that the
proteolytic system in the ER is responsible for the total degradation
of many misfolded or incompletely assembled proteins, as well as
resident ER
proteins(28, 37, 38, 39, 40) .
There is also some indication that processing for antigen presentation
can occur in the ER(41) . Known instances of limited
proteolysis in the ER, as seen with the meprin subunit, are rare,
with the exception of signal peptide cleavage by signal
peptidase(42, 43) . The specific limited protein
processing described in the present work may constitute a new function
for ER proteases. Autocatalytic processing of some proteases, e.g. kexin, occurs in the ER(44) , but it is unlikely that the
processing of meprin
is due to self-cleavage because the subunits
are synthesized as inactive zymogens in COS-1 and 293 cells. Proteases
that have been implicated in ER degradation are cysteine proteases, e.g. ER-60 and ER-p72(39, 45, 46) ,
and a serine protease(37) . Meprins provide a good system for
investigating and identifying the ER proteases involved in the limited
proteolysis function of this subcellular compartment.