From the Department of Medical Biochemistry and Microbiology, University of Uppsala, Biomedical Center, Uppsala, Sweden
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ABSTRACT |
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Pre- Bikunin is a 25-kDa serum protein whose polypeptide consists of
two tandemly arranged proteinase inhibitor domains of the Kunitz type
(1). Bikunin is made by hepatocytes and is synthesized as a precursor
also containing Sequence analysis as well as pulse-chase experiments have revealed that
the heavy chains have COOH-terminal propeptides of about 30 kDa
(10-12). These extensions are apparently released by a proteolytic
cleavage between an Asp and a Pro residue (13). The enzyme mediating
this reaction is unknown and in the present study we have defined its
specificity by mutating the amino acid residues near the cleavage site.
It has been demonstrated that the chondroitin sulfate chain of bikunin
is linked to the heavy chains through an ester bond between an internal
GalNAc residue and the Comparison of the amino acid sequences near the site where signal
peptides have been found to be cleaved has yielded a set of rules that
can be used for predicting the cleavage site (15). When these rules are
applied to the sequence deduced from the cDNA of rat H3, they
indicate that the signal peptide ends 12 amino acid residues before the
beginning of the mature polypeptide implying the existence of an
NH2-terminal propeptide (6). Here we confirm this
prediction experimentally, and show that the cleavage, although it
occurs in the ER, is mediated by an enzyme whose sequence specificity
is that of a Golgi proprotein convertase.
Materials--
Tissue culture media, fetal bovine serum,
glutamine, and penicillin/streptomycin were obtained from Statens
Veterinärmedicinska Anstalt (Uppsala, Sweden).
Endo- Production of Specific Antisera--
An antiserum against the
putative NH2-terminal extension of rat H3, PRSPLRLLGKRSC,
was obtained commercially (Medprobe, Oslo, Norway): the corresponding
peptide was conjugated to keyhole limpet hemocyanin with
m-maleimidobenzoyl-N-hydroxysuccinimide ester and
used for the immunization of a rabbit. An antiserum against rat P Construction of Expression Vectors and Mutagenesis--
The
cDNA coding for rat H3 (6) was subcloned into the eukaryotic
expression vector pXM which provides adenovirus late promotor-driven expression of introduced cDNA. Substitutions of amino acid residues were made with the unique site elimination procedure (16) using the
U.S.E. mutagenesis kit (Pharmacia). A cDNA coding for H3 lacking the NH2-terminal propeptide was made by polymerase chain
reaction using a primer corresponding to the 15 last nucleotides in the signal sequence and the 15 first nucleotides in the mature protein. cDNA for H3 lacking the major part of the COOH-terminal propeptide was made by polymerase chain reaction through the introduction of a
stop codon. All constructions were confirmed by DNA sequencing.
Transfection Procedure--
COS-1 cells were grown to
subconfluence (70-80%) in DMEM containing 10% fetal bovine serum, 2 mM glutamine and antibiotics (complete DMEM). After release
with trypsin, the cells were washed with phosphate-buffered saline
supplemented with 10 mM Hepes, pH 8.0, and finally
suspended in phosphate-buffered saline/Hepes at a concentration of
24 × 106 cells/ml. Aliquots of the cell suspension
(0.5 ml) were transferred to electroporation cuvettes (0.5 cm) and 20 µg of plasmid DNA was added together with 50 µg of fish sperm DNA.
After electroporation at 500 microfarads and 0.3 kV with Gene Pulser II
Electroporation system (Bio-Rad), complete DMEM was added and the cells
were immediately plated onto 6-cm Petri dishes. The medium was changed
when the cells had attached after 3-4 h.
Metabolic Labeling and Pulse-Chase Analysis--
Two days after
the transfection, the cells were rinsed twice with phosphate-buffered
saline and DMEM containing 2 µM methionine and
supplemented with 2 mM glutamine and 1 mg/ml bovine serum albumin was added. After 30 min, new medium containing
[35S]methionine (0.1 mCi/plate) was added and the cells
were labeled for 10 min (pulse period). The medium was then withdrawn
and the cells were incubated for various periods of time in DMEM
containing 2 mM methionine. In some experiments brefeldin A
was present in the medium at a concentration of 10 µg/ml. After the
pulse-chase procedure, the media were collected and the cells rinsed
with ice-cold phosphate-buffered saline. Solubilization and
immunoprecipitation were done as described previously (6). The immune
complexes were analyzed by SDS-PAGE followed by fluorography. The
relative amount of radioactivity in protein bands was determined by
optical densitometry. For radiosequencing, DMEM (supplemented as
described above) lacking valine or leucine and containing 100 µCi/ml
[3H]Val or 100 µCi/ml [3H]Leu was used.
The cells were incubated for 3-5 h and the medium was used for
immunoprecipitations; the localization of the proteins by fluorography
was facilitated by addition of [35S]Met (10 µCi/ml).
NH2-terminal Radiosequencing--
Following
immunoprecipitation and SDS-PAGE, the proteins were transferred to
ProBlottTM membranes (Applied Biosystems). The protein
bands were visualized by autoradiography and those of interest were
excised and analyzed by automated Edman degradation with an ABI 477 sequencer operating in gas-phase mode. The 3H radioactivity
released after each degradation cycle was measured by scintillation counting.
Enzyme Treatments--
Some of the medium samples (500 µl)
were treated with chondroitinase ABC before immunoprecipitation. To
this end, they were supplemented with 50 mM Tris-HCl, pH
8.0, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 mM N-ethylmaleimide, 1 µg/ml pepstatin, and 5 milliunits of chondroitinase ABC and incubated for 14 h at
37 °C. For Endo H treatment, the pellets obtained from the
immunoprecipitation were suspended in 15 µl of 50 mM
sodium acetate, pH 5.6, containing 3 g/liter of SDS. After heating at
95 °C, the supernatants were collected and 10 µl of 50 mM sodium acetate, pH 5.6, containing 0.1 milliunits of
Endo H was added. After 8 h of incubation at 37 °C, sample
buffer was added and the proteins were analyzed by SDS-PAGE followed by fluorography.
Secretion of H3--
The time course for the secretion of H3 from
transfected COS-1 cells was determined by pulse-chase experiments
followed by immunoprecipitation and SDS-PAGE (Fig.
1A). In pulse-labeled cells, the precursor of H3 (with an apparent molecular mass of about 113 kDa)
was detected as a double band (indicated with pH3). After 20 min of
chase, the cleaved form of H3 appeared in the cells (indicated with
H3); upon longer times of chase, it constituted maximally 10-20% of
the total amount in the cell samples. Both uncleaved and cleaved H3
appeared in the medium after a lag period of 20-40 min and attained
maximal levels after 40-80 min. The relative amount of cleaved H3 in
the medium was 45-50%. When a sample from pulse-labeled cells was
pretreated with endoglycosidase H (Endo H), an enzyme removing early
forms of N-linked oligosaccharides (17), the upper band of
pH3 disappeared showing that the heterogeneity was due to incomplete
glycosylation (Fig. 1B, lane 2). The same treatment of a
sample from cells chased for 40 min showed that the cleaved form of H3
was resistant to Endo H (lane 4) indicating that it had
reached the medial Golgi complex. As expected, both cleaved and
uncleaved H3 in the medium were resistant to the enzyme (lane
6).
The observations that the intracellular cleaved form of H3 appeared
only after a distinct lag period and that it was Endo H-resistant
indicated that the cleavage occurred late in the Golgi complex. To
further test this notion, we exploited the effects of the fungal
metabolite brefeldin A on protein secretion. In cells treated with this
compound, protein transport ceases and enzymes normally residing in the
cis, medial, or trans Golgi appear in the ER, where they will modify
the retained secretory proteins (18). However, enzymes residing in the
trans Golgi network or secretory vesicles will not be relocated in the
presence of the drug, and the secretory proteins retained in the ER
will therefore not be modified by these enzymes. As expected, treatment
of the transfected COS-1 cells with brefeldin A prevented secretion of newly synthesized H3 (Fig. 1B, cf. lanes 8 and
10). Furthermore, the retained protein remained uncleaved
(cf. lanes 7 and 9). These results suggest that
the cleavage of the COOH-terminal propeptide normally takes place
beyond the trans Golgi.
The COOH-terminal Propeptide Remains Associated with H3 after
Cleavage--
In the experiments described in Fig. 1A, a
polypeptide of 33 kDa coprecipitated with H3 (lane 9,
denoted C). To determine if this was the COOH-terminal
propeptide, we analyzed the polypeptide by radiosequencing. To this
end, cells expressing H3 precursor were labeled with
[3H]Val and the heavy chain was isolated from the medium
by immunoprecipitation. Following SDS-PAGE and transfer to a membrane,
the 33-kDa polypeptide was located by fluorography and subjected to
Edman degradation. The first elevated radioactivity value was obtained
in cycle 7 (Fig. 2), in agreement with
the sequence of the COOH-terminal propeptide, shown as one-letter
symbols above the bars.
The reason for the coprecipitation of the COOH-terminal propeptide and
H3 could be either that the propeptide was attached to the heavy chain
or that the antiserum contained antibodies against the propeptide. The
latter alternative does not seem to be the case since we found that the
propeptide expressed alone in COS-1 cells was not recognized by the
antiserum (result not shown). To test whether the COOH-terminal
propeptide was actually attached to H3, we ascertained the
sedimentation behavior of the propeptide in the presence or absence of
the heavy chain. To this end, COS-1 cells expressing either the H3
precursor or only the propeptide (with a Myc tag) were labeled with
[35S]Met. Samples of the media were then analyzed by
velocity centrifugation followed by immunoprecipitation and SDS-PAGE.
This experiment showed that the COOH-terminal propeptide formed from
the H3 precursor by intracellular proteolytic cleavage sedimented at
the same rate as cleaved and uncleaved H3 (Fig.
3, upper panel) whereas the COOH-terminal propeptide expressed alone sedimented more slowly (lower panel). The simplest interpretation of these results
is that the propeptide remains attached to H3 after cleavage.
The COOH-terminal Propeptide Is Needed for Coupling of H3 to
Bikunin--
The observation that there is an association between the
COOH-terminal propeptide and the heavy chain suggested to us that the
propeptide might have a role in the coupling of H3 to bikunin. To test
this idea, we exploited our previous finding that coexpression of H3
and the bikunin precursor in COS-1 cells leads to coupling of the two
polypeptides (6). The result of such an experiment is shown in Fig.
4 in which the cell medium of the
transfected cells was analyzed with antibodies against bikunin
(lane 1) or H3 (lane 2). The resulting protein
complex, which has an apparent molecular mass of 180 kDa upon SDS-PAGE,
is indicated with an arrow. To ascertain whether the
COOH-terminal propeptide of H3 is necessary for the coupling reaction,
we coexpressed bikunin and H3 lacking the propeptide; secretion of the
truncated heavy chain was normal but no 180-kDa complex was formed
(lane 3).
The analysis of the medium from cells coexpressing bikunin and H3 with
antibodies against bikunin showed, as earlier reported (6, 19), that
both bikunin precursor with and without chondroitin sulfate as well as
mature bikunin were secreted; the respective protein bands are
indicated with 1, 2, and 3 (Fig. 4, lane 1). When antibodies
against H3 were used, there was a weak band at the position of the
COOH-terminal propeptide (lane 2, indicated with
C). The relative amount of this band was 2-3 times lower than that of the band appearing when H3 was expressed alone (Fig. 1A, lane 9), consistent with our conclusion that the
propeptide is released upon coupling; the occurrence of the propeptide
in this sample can be accounted for by the presence of cleaved but uncomplexed heavy chain (indicated with H3). Furthermore, when the
immunoprecipitation was done with antibodies against bikunin, there was
even less radioactive material at the position of the propeptide
(lane 1) and pretreatment with chondroitinase ABC reduced this amount to less than 1% of the radioactivity in the 180-kDa band
(not shown).
Specificity of Cleavage at COOH Terminus--
The amino acid
sequence close to the cleavage site of the COOH-terminal propeptide of
H3 is shown in Fig. 5A. Amino
acid residues conserved in this protein in man, mouse, and rat (13) are
in bold. As a first step in the identification of the enzyme cleaving the COOH-terminal propeptide, we determined which of the conserved residues are essential for cleavage. To this end they were mutated as
shown in Fig. 5B. The corresponding polypeptides were then expressed in COS-1 cells and the degree of cleavage assessed by SDS-PAGE (Fig. 5C). This analysis showed that Asp at
P3 and P1 as well as Pro at P1' are
absolutely required (<1% cleaved). Substitution of Gln for His at
P2' and Tyr for Phe at P3' reduced cleavage approximately 3-fold whereas substitution of Val-Val for Ile-Ile at
P4' and P5' was without effect. Interestingly,
mutation of Val to Ala at P4 resulted in a significantly
higher cleavage (lane 2).
Detection of NH2-terminal Propeptide--
As mentioned in
the Introduction, analysis of the cDNA of H3 suggests that the
signal peptidase cleaves 12 amino acid residues before the beginning of
the mature polypeptide implying the existence of an
NH2-terminal propeptide. To experimentally test this
hypothesis, we wanted to block the release of the putative propeptide
and then determine the NH2-terminal sequence of the
secreted proprotein. With the assumption that the two basic amino acid
residues preceding the NH2 terminus of mature H3 (Fig.
6A, right-hand arrow) are essential for cleavage, we mutated these to Asn and Ser. The modified protein was then expressed in COS-1 cells which were labeled with [3H]Leu. The protein was isolated from the cell medium by
immunoprecipitation followed by SDS-PAGE, subjected to Edman
degradation, and the radioactivity released in each cycle was measured
(Fig. 6B, closed bars). Elevated values were obtained at
cycles 6, 8, and 9. This result is consistent with the cleavage of the
signal peptide occurring between amino acid residues 21 and 22 (left-hand arrow in A); the corresponding
sequence is shown above the bars. When the same experiment was done
with cells labeled with [3H]Val (open bars),
no significant increase of radioactivity was obtained, consistent with
the absence of Val in the NH2 terminus of the
propeptide.
As a control, wt H3 produced from COS-1 cells was also analyzed by
radiosequencing. Labeling with [3H]Leu yielded elevated
radioactivity values in cycles 2, 6, 8, and 9 (Fig. 6C, closed
bars). This result can be explained by the occurrence of similar
amounts of H3 molecules with and without the NH2-terminal
propeptide; the corresponding sequences are shown above the bars in
plain and bold letters, respectively. Consistent with this conclusion,
wt H3 labeled with [3H]Val yielded increased values at
cycles 6 and 7 (open bars). A quantitative analysis of the
data in Fig. 6C indicates that 35-40% of H3 was cleaved
(average of two
experiments).2
Specificity of Cleavage at NH2 Terminus--
Comparison of
the amino acid sequences of H3 from man, mouse (13), and rat (6) shows
that the basic amino acid residues near the cleavage site of the
NH2-terminal propeptide (see Fig. 6A), at
P1, P2, and P6, are conserved. As
described above, simultaneous substitution of those at P1
and P2 against non-basic ones abolishes cleavage (Fig.
6B). To assess the relative importance of each of the
conserved basic residues, we mutated them one at a time (Fig.
7A) and determined the degree
of cleavage. For these experiments the immunoprecipitation was first
done with antibodies against the NH2-terminal propeptide
and subsequently with antibodies against the whole protein (denoted I
and II, respectively); the amount of H3 obtained in the second
extraction relative to the amount obtained with both antibodies was
taken as a measure of the degree of cleavage. These experiments showed
that 33-37% of wt H3 was cleaved (lanes 1 and
2), which agrees with the result obtained by amino acid
sequencing (Fig. 6C). The proportion of H3 lacking the
COOH-terminal extension was the same in the samples obtained with the
two different antibodies, implying that the NH2-terminal propeptide does not affect the subsequent cleavage of the COOH-terminal propeptide. Substitution of one of the basic amino acids, reduced the
cleavage to 7-15% (Fig. 7B, lanes 3-8). With the same
type of analysis in which the residues at P1 and
P2 had both been mutated, no cleavage was detected (<1%).
When this mutant was coexpressed with bikunin, the same amount of the
180 kDa complex was detected in the medium as with the wild type
protein (not shown). Analysis of wt H3 from cells labeled for 5 min
yielded essentially the same degree of cleavage as for the secreted
form (not shown) implying that processing of the
NH2-terminal propeptide occurred in the ER.
Many secretory proteins are synthesized as precursors that are
proteolytically cleaved during their transport to the cell surface.
Normally this cleavage occurs just before the proteins are released: in
the trans Golgi network or in the secretory vesicles (20). In several
cases the cleavage has been shown to make the proteins biologically
active suggesting that an early activation would be harmful to the cell
(21). The cleavage of proproteins typically occurs next to two basic
residues and recently, a family of enzymes was identified that mediates
this reaction (22).
In this study we have used the heavy chain of rat P We have earlier shown that coexpression of bikunin and heavy chain in
COS-1 cells leads to coupling of the two polypeptides via the
chondroitin sulfate chain of bikunin (6). Using this system we now show
that no complex is formed when the COOH-terminal propeptide is deleted
(Fig. 4). Since H3 expressed without the COOH-terminal propeptide is
secreted at the same rate as the normal protein, its inability to
become linked does not seem to be due to misfolding. Our finding that
the propeptide remains bound to H3 after cleavage is consistent with
the idea that it mediates the coupling, as shown schematically in Fig.
8. The mechanism for this reaction is
presently unknown. Part of the amino acid sequence of the COOH-terminal
propeptide shows similarity with multicopper oxidases (13, 6). Further
mutational analysis should reveal if the corresponding amino acid
residues are essential for the coupling reaction.
-inhibitor is a serum protein consisting
of two polypeptides named bikunin and heavy chain 3 (H3). Both
polypeptides are synthesized in hepatocytes and while passing through
the Golgi complex, bikunin, which carries a chondroitin sulfate chain,
becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain.
Using COS-1 cells transfected with rat H3, we found that in the absence
of bikunin, the cleaved propeptide remained bound to the heavy chain
and that H3 lacking the propeptide sequence did not become linked to
coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed
that part of the molecules had a 12-amino acid residue long
NH2-terminal propeptide. Cleavage of this propeptide,
which occurred in the endoplasmic reticulum, was found to require basic
amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in
transit. Deletion of the NH2-terminal propeptide or
blocking of its release affected neither transport nor coupling of the
heavy chain to bikunin.
INTRODUCTION
Top
Abstract
Introduction
References
1-microglobulin (2, 3). Late during its
intracellular transport, the bikunin precursor acquires a chondroitin
sulfate chain and shortly afterward,
1-microglobulin is
released by proteolytic cleavage (4). Just before this cleavage, part
of the bikunin precursor molecules becomes covalently linked via the
chondroitin sulfate chain to one or two polypeptides of about 80 kDa,
named the heavy chains. In this fashion, pre-
-inhibitor (P
I)1 is formed from one
bikunin and one heavy chain and inter-
-inhibitor (I
I) from one
bikunin and two heavy chains. The polypeptide compositions of these
proteins have been shown to differ between species. I
I and P
I
from man and rat contain H1 and H2, and H3, respectively (5, 6); the
corresponding bovine proteins contain H2 and H3, and H2, respectively
(7). The physiological function of P
I and I
I is not clear but
in vitro experiments have shown that the two proteins are
required for the formation of the hyaluronan-containing matrix that
surrounds certain cells (6, 8). Other results suggest that I
I might
have a role in inflammation (9).
-carbon of the COOH-terminal amino acid (5,
14). The mechanism for the formation of this bond is unknown but in this paper we present evidence that the COOH-terminal propeptide is
required for the coupling reaction.
EXPERIMENTAL PROCEDURES
-N-acetylglucosaminidase H (Endo H) and fish sperm
DNA were from Boehringer-Mannheim. Brefeldin A, chondroitinase ABC, and
antibodies against the c-Myc epitope were from Sigma. The expression
vector pXM was from Genetics Institute Inc. (Cambridge, MA), pSecTag
from Invitrogen (Leek, The Netherlands), oligonucleotides from DNA
Technology (Aarhus, Denmark), restriction endonucleases from Amersham,
and Pfu polymerase from Stratagene. Tran35S-label (>1000
Ci/mmol) was from ICN and [3H]leucin (52 Ci/mmol) and
[3H]valine (44 Ci/mmol) from Amersham. cDNA for rat
1-microglobulin-bikunin was a gift from B. Åkerström (Lund University, Sweden).
I
was obtained by injecting the protein intramuscularly into a rabbit;
P
I was purified from rat plasma as described previously (6).
Antibodies specific for H3 were obtained from this antiserum by
affinity chromatography (6). The specificities of the different antisera were examined by immunoblotting of purified protein and plasma samples.
RESULTS
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Fig. 1.
Time course for secretion of H3 in COS-1
cells. A, the cells were transfected with cDNA for
H3, labeled with [35S]Met for 10 min, and chased for the
times indicated. H3 was then immunoprecipitated from cell lysates and
media, and detected by SDS-PAGE followed by fluorography; pH3,
H3, and C indicate the H3 precursor, mature H3, and a
coprecipitated polypeptide (presumably the COOH-terminal propeptide),
respectively. The position of 14C-labeled reference
proteins are shown with their molecular masses in kDa. B,
some of the samples were treated with Endo H before electrophoresis;
c is cell lysate and m is medium. Shown are also
the effects of brefeldin A (BFA) on secretion and
cleavage.
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Fig. 2.
Identification of polypeptide coprecipitating
with H3. Cells expressing H3 were labeled with
[3H]Val and the protein was extracted from the medium by
immunoprecipitation followed by SDS-PAGE and fluorography. The
coprecipitated 33-kDa polypeptide, indicated with C in Fig.
1A, was excised from the gel and subjected to Edman
degradation. The radioactivity recovered in each cycle is shown as
bars. The amino acid sequence of the COOH-terminal
propeptide is shown above the bars. Because of
spill over in the sequential extractions, the radioactivity of a
negative cycle following a positive one is elevated.
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Fig. 3.
Binding of COOH-terminal propeptide to H3
detected by velocity centrifugation. Cells expressing the H3
precursor or the COOH-terminal propeptide were labeled for 4 h
with [35S]Met. Samples of the media were layered on
sucrose gradients (5-25% w/w) which were centrifuged at 100,000 × g for 20 h and fractions were collected from the
bottom. The recombinant polypeptides were isolated by
immunoprecipitation and detected by SDS-PAGE followed by fluorography.
A and B show the result obtained with cells
expressing H3 and the COOH-terminal propeptide, respectively. The
precursor of H3, cleaved H3, and the COOH-terminal propeptide are
indicated with pH3, H3, and C, respectively. Note
that the propeptide is in the same fraction as H3 in A but
has sedimented more slowly in B.
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Fig. 4.
Requirement of COOH-terminal propeptide of H3
for coupling to bikunin. COS-1 cells were transfected with
cDNA for the bikunin precursor (Bk) and H3 with or
without its COOH-terminal propeptide (H3 and
H3 CPP, respectively). The cells were then labeled for
1 h with [35S]Met and antibodies against bikunin
(lane 1) or H3 (lanes 2 and 3) were
added to samples of the media. The immunoprecipitates were analyzed by
SDS-PAGE followed by fluorography. The brackets numbered
1-3 show the bikunin precursor with and without chondroitin
sulfate and mature bikunin, respectively. The precursor of H3, cleaved
H3, and the COOH-terminal propeptide are indicated with pH3,
H3, and C, respectively. The arrow indicates
the complex formed between the bikunin precursor and H3 upon
coexpression (lanes 1 and 2). When H3 lacking the
COOH-terminal propeptide was coexpressed with bikunin, no such complex
was formed (lane 3).
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Fig. 5.
Specificity of cleavage of COOH-terminal
propeptide. A, the amino acid sequence around the
cleavage site for the COOH-terminal propeptide of H3 with the conserved
residues in bold. B, the conserved residues were mutated as
indicated, the new amino acid residues being chosen based on the most
frequently occurring mutations accepted by natural selection (29).
C, COS-1 cells were transfected with the corresponding
cDNAs and then labeled for 20 min with [35S]Met
followed by a 90-min chase. H3 was extracted from the media by
immunoprecipitation and analyzed by SDS-PAGE followed by fluorography;
the uncleaved and mature form of H3 are indicated with pH3
and H3, respectively. The degree of cleavage for each
mutated H3 is shown to the right in B: ++, +,
and represent a processing efficiency of 45-55, 8-18, and
<1%, respectively. Each mutant was analyzed in at least three
independent experiments.
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Fig. 6.
Radiosequence analysis of secreted H3.
A, H3 has been proposed to be synthesized with a 12-amino
acid residues long NH2-terminal propeptide; the
right and left-hand arrows indicate the
NH2 terminus of the mature heavy chain and the proposed
cleavage site of the signal peptide, respectively. B, the
existence of the NH2-terminal propeptide was tested by
radiosequence analysis of H3 in which the formation of the mature form
was blocked by mutating the amino acid residues at P1 and
P2. The altered protein was expressed in COS-1 cells and
labeled with either [3H]Leu or [3H]Val.
Following immunoprecipitation and SDS-PAGE, the labeled protein was
subjected to Edman degradation. The radioactivity measured at each
cycle is plotted for [3H]Leu (closed bars) and
[3H]Val (open bars). The amino acid sequence
of the proposed NH2-terminal propeptide is shown
above each bar. C, wild type H3 was analyzed as
described in B; the amino acid sequences of the
NH2-terminal propeptide and of the mature heavy chain are
shown above each bar in plain and
bold letters, respectively.
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Fig. 7.
Specificity of cleavage of
NH2-terminal propeptide. A, the basic amino
acid residues near the cleavage site of the NH2-terminal
propeptide (see Fig. 6A) were mutated as shown.
B, the resulting proteins were expressed in COS-1 cells
which were labeled with [35S]Met. The uncleaved form of
H3 was then extracted from the medium by antibodies against the
propeptide and analyzed by SDS-PAGE followed by fluorography (denoted
I). The remaining cleaved form was subsequently extracted
with antibodies against the whole protein (denoted II).
Lanes 9 and 10 show H3 lacking the
NH2-terminal propeptide. The degree of cleavage as
described in Fig. 5B is shown to the right
in A.
DISCUSSION
I, heavy chain 3 (H3), as a model for the proteolytic processing of the heavy chains of
the bikunin-containing proteins. The cleavage sites for the
COOH-terminal propeptides of these proteins differ from those of other
proproteins in that they lack adjacent basic amino acid residues (23).
Using site-directed mutagenesis we have now shown that five of the
conserved residues flanking this site in H3 are essential for cleavage;
to our knowledge there is no known proteinase that recognizes the
corresponding sequence. We also found that the COOH-terminal propeptide
(in the absence of bikunin) remains attached to the heavy chain. The
conserved amino acid residues near the cleavage site that we found not
to be required for cleavage, P4, P4', and
P5', might mediate this interaction. The degree to which
the heavy chains are cleaved during secretion varies greatly between
different cell types. Thus, experiments with human hepatocytes have
shown that H3 and H2 are completely cleaved in these cells whereas in
the human hepatoma cell line Hep G2, they are partially or not
processed at all, respectively (12); these observations indicate that the cleavage is not autocatalytic.
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Fig. 8.
Model of intracellular assembly of
pre- -inhibitor. During, or immediately
after synthesis of the heavy chain, an NH2-terminal
propeptide is released. Bikunin (Bk) is synthesized as a
precursor also containing
1-microglobulin
(
1µ). As this precursor passes through the Golgi
complex, it acquires a chondroitin sulfate chain (CS). In
the Golgi complex, a COOH-terminal propeptide of H3 is cleaved off but
remains associated with the mature protein until the bikunin precursor
becomes linked to the heavy chain through the chondroitin sulfate
chain. Finally
1-microglobulin is released by a
proteolytic cleavage.
On the basis of sequence analysis, all heavy chains of the bikunin-containing proteins have been proposed to have NH2-terminal propeptides (11, 12). In this study of rat H3 we have obtained experimental evidence for the existence of such a propeptide. Furthermore, we have confirmed an earlier observation indicating that the cleavage occurs in the ER (12). Using mutational analysis we now show that cleavage of the NH2-terminal propeptide requires basic amino acid residues at P1, P2, and P6 (Fig. 7). These characteristics agree with the substrate specificity of the Golgi enzyme furin, a ubiquitously occurring proprotein convertase (24). This enzyme is synthesized in an essentially inactive form and becomes fully active only when it has left the ER. However, the newly synthesized form does display some (autocatalytic) activity in the ER (21), which could be sufficient for mediating the cleavage of pro-H3. It is interesting to note that there are basic amino acid residues at all the proposed cleavage sites of the heavy chains, suggesting that they are all cleaved by the same enzyme. In contrast to the previously studied liver cells (12), COS-1 cells cleave the NH2-terminal propeptide in the ER only partially. This fact made it possible to observe that there was little additional processing in the Golgi complex, indicating that the NH2-terminal propeptide is hidden in the mature polypeptide.
For many proteins, the propeptides have been found to be required for
proper folding (25). This does not appear to be the case for the
NH2-terminal propeptide of H3, as judged by the fact that
when H3 was expressed without the NH2-terminal propeptide, it was secreted normally and could be coupled to bikunin. However, absence of the NH2-terminal propeptide during synthesis
might affect other properties of the protein adversely, perhaps those associated with its function in the formation of the
hyaluronan-containing matrix that surrounds various cell types
(26-28).
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ACKNOWLEDGEMENT |
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We thank Markku Salmivirta for critical comments on the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Erik, Karin, and Gösta Selanders Foundation, Polysackaridforskning AB, Uppsala, and the Swedish Natural Science Council.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: Box 575, S-751 23
Uppsala, Sweden. Tel.: 46-18-471-4350; Fax: 46-18-471-4975; E-mail:
Thuveson{at}medkem.uu.se.
2 In a previous study we reported that the cleavage of the NH2-terminal propeptide of H3 is complete in COS-1 cells (6). This conclusion was based on the observation that when the 2 basic amino acid residues next to the cleavage site were mutated to Asn and Ser, there was a distinct shift in the electrophoretic mobility of the protein band upon SDS-PAGE (6). We later realized, however, that the cause of the mobility shift was an inadvertent introduction of a site for N-linked glycosylation.
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ABBREVIATIONS |
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The abbreviations used are:
PI, pre-
-inhibitor;
I
I, inter-
-inhibitor;
H3, heavy chain 3;
ER, endoplasmic reticulum;
Endo H, endo-
-N
-acetylglucosaminidase H;
PAGE, polyacrylamide gel electrophoresis;
wt, wild type;
DMEM, Dulbecco's modified Eagle's medium.
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REFERENCES |
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