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(Received for publication, May 18,
1995; and in revised form, July 20, 1995) From the
Expression of the pancreatitis-associated protein I (PAP I), an
exocrine pancreatic protein, increases rapidly and strongly in acinar
cells during the acute phase of pancreatitis. This is reminiscent of
the response to stress of acute phase proteins. We have previously
demonstrated that serum factors from rats with acute pancreatitis, but
not from healthy rats, could induce endogenous PAP I gene expression in
the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C.,
Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204,
238-243). In the present work, we have evaluated the influence of
several mediators of inflammation on rat PAP I gene transcription in
these cells. Tumor necrosis factor
The acute phase of pancreatitis is characterized by a pattern of
changes in the expression of secretory proteins(1) . Whereas
expression of most pancreatic enzymes decreases, mRNA levels of the rat
pancreatitis-associated protein (PAP) ( In fact PAP I was not detectable in the pancreas of
healthy animals. It could be evidenced in pancreatic juice 6 h after
induction of an experimental acute pancreatitis, reached a maximum
during the acute phase (12-48 h), and disappeared during
recovery(7) . The rapid and strong induction of the PAPs is
unique among secretory proteins and reminiscent of the response to
stress of acute phase proteins. Recently, we have demonstrated the
presence of factors in serum from rats with acute pancreatitis, but not
from healthy rats, capable of inducing PAP I gene expression in the
pancreatic acinar cell line AR-42J. In addition, the cis-acting element was localized within the 1.2 kilobases
upstream region of the transcription start site(8) . It has
long been known that the changes occurring in the liver and in other
organs during the acute phase response are coordinated by signals
generated at the site of injury, among which several cytokines have
been well characterized, including IL-1, IL-6, TNF
Figure 5:
Identification of two functional IL-6REs
within the PAP I promoter by site-directed mutagenesis. A,
nucleotide substitutions in p-274/+10PAPI-CAT plasmid. B, AR-42J cells were transfected with 20 µg of plasmids
p-274/+10PAPI-CAT, pmut1-274/+10PAPI-CAT,
pmut2-274/+10PAPI-CAT, or
pmut3-274/+10PAPI-CAT. Thirty-six hours after
transfection of AR-42J cells, IL-6 (100 units/ml) and dexamethasone
(100 nM) were added to the culture medium. Cells with no
hormones added were taken as controls. Specific induction by
IL-6/dexamethasone was calculated as the ratios of the values from
induced and control cells. Values represent the means (±
standard error) of six independent transfection
experiments.
Figure 1:
Induction of PAP I
mRNA accumulation by cytokines and dexamethasone treatment. Forty-eight
hours after placing AR-42J cells in culture, cytokines and
dexamethasone, alone or in combination, were added to the culture
medium. After 24 h total RNA was isolated, submitted to electrophoresis
(15 µg/lane) through a formaldehyde-agarose gel, transferred to a
nylon membrane, and hybridized to
Figure 2:
Induction of PAP I/CAT hybrid gene
expression by IL-6 and dexamethasone. AR-42J cells were transfected
with 20 µg of the p-1253/+10PAPI-CAT hybrid gene and
treated with IL-6 (100 units/ml) and dexamethasone (100 nM)
individually or in combination. CAT activities were quantitated using a
phase extraction procedure. CAT activity was normalized for
transfection efficiency, using the ratio of CAT activity to
Figure 3:
Deletion analysis of the rat PAP I
promoter. Numbers in plasmid names refer to the position of
first and last nucleotides of the PAP I gene. Relative CAT activity
(± standard error) in extracts from AR-42J cells transfected
with the corresponding plasmids was measured. CAT activity was
normalized for transfection efficiency, using the ratio of CAT activity
to
A 12-fold
increase in CAT activity was seen upon IL-6/dexamethasone treatment of
cells transfected with constructs containing more than 274 base pairs
of 5`-flanking sequence (Fig. 4). Deletion to position
-180 led to a 3-4-fold drop in induction. Finally, a 2-fold
induction was observed when we transfected with
p-118/+10PAPI-CAT and p-61/+10PAPI-CAT constructs
but not with p+10/-1253PAPI-CAT.
Figure 4:
Localization of IL-6/dexamethasone
response regions in PAP I promoter. AR-42J cells were transfected with
20 µg of the plasmids described in Fig. 3. Thirty-six hours
after transfection, AR-42J cells were incubated with medium alone
(control) or with IL-6 (100 units/ml) in association with dexamethasone
(100 nM). Specific induction by IL-6/dexamethasone was
calculated as the ratio of the values from induced and control cells.
Values represent the means (± standard error) of four to seven
independent transfection experiments.
Induction of an experimental pancreatitis causes a more than
200-fold increase in PAP I mRNA expression during the acute phase of
pancreatitis(7) . PAP I mRNA accumulation reaches a maximum 6 h
after induction, with a kinetics probably controlled by the cascade of
events taking place during the acute phase. That cascade includes the
activation of monocytes and macrophages and the synthesis and secretion
of inflammatory mediators eventually transported to the target cells.
In support of that hypothesis, we have recently demonstrated the
presence of factors in serum from rats with pancreatitis, but not from
healthy rats, capable to induce PAP I gene expression(8) . The
present work was carried out primarily to localize the cis-regulatory elements in the PAP I gene and to characterize
their response to several acute phase mediators including IL-1, IL-6,
IFN The cytokines tested in this
study had very different effects on PAP I gene expression in AR-42J
cells (Fig. 1). The most striking result was the strong
stimulation of the association IL-6/dexamethasone, and the limited
stimulation by IFN The mechanism by which IL-1
down-regulates the stimulation by IL-6 associated with dexamethasone is
also unknown. IL-1 has already been shown to inhibit IL-6 induction of
the endogenous T kininogen in rat primary hepatocytes(22) , but
IL-1 and IL-6 can also act independently (additive effect) or
synergistically in the regulation of other acute phase genes such as
PAP I gene
expression was significantly induced by IFN We have chosen to address in this study the molecular mechanism of
IL-6 and dexamethasone stimulation of the PAP I promoter. Analysis of
the promoter sequence revealed the presence in two positions of the
potential IL-6 response element of type 2 (CTGGGA), previously
identified in several acute-phase genes (17, 18, 35) and shown to be functional by
mutation analysis(36) . Demonstration that the two IL-6REs
identified in the PAP I promoter were indeed functional was obtained by
mutation and transfection assays (Fig. 5). However, these are
not the only cis-elements involved in the IL-6/dexamethasone
response of PAP I. Another cis-cytokine response element,
localized between -61 and +10, is responsible for a 2-fold
induction. This has been shown previously in other IL-6-activated
cellular genes(37) . For instance, Baumann et al.(38) have demonstrated for several acute phase proteins
that IL-6 acts directly through an IL-6RE but also indirectly by
increasing expression of C/EBPs, which in turn stimulates acute phase
proteins gene expression. Acute phase proteins have been divided
into two subclasses according to their pattern of regulation by
cytokines(17) . The synthesis of class 1 acute phase proteins (e.g. Finally, it is interesting to
note that whereas the PAP I gene is expressed as an acute phase protein
in pancreas, it is constitutively expressed by the epithelial cells of
the intestinal tract(39, 40) . The PAP I promoter is
therefore complex. It confers to the gene the capacity of being
regulated along several pathways, the switch between pathways being
possibly under the control of tissue-specific elements.
Volume 270,
Number 38,
Issue of September 22, pp. 22417-22421, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
IDENTIFICATION OF TWO FUNCTIONAL INTERLEUKIN-6 RESPONSE ELEMENTS IN
THE RAT PAP I PROMOTER REGION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
induced an increase in PAP I
mRNA expression, and interferon caused an even greater increase
in PAP I mRNA level. These stimulations were antagonized by
dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were
ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also
ineffective. IL-6 and dexamethasone together induced a marked
stimulation of PAP I gene transcription, and this effect was slightly
attenuated by IL-1. To analyze the cis-regulatory elements
responsible for the induction of transcription, we fused a 1.2-kilobase
segment of the rat PAP I promoter to the chloramphenicol
acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA
was transfected into AR-42J cells. Addition of IL-6 or dexamethasone
was ineffective, whereas their mixture increased the CAT activity 12
times. Progressive deletions of the PAP I promoter were then fused to
the CAT gene, and the constructs were transfected to AR-42J cells. A
12-fold increase in CAT activity was seen upon IL-6/dexamethasone
treatment with constructs containing more than 274 base pairs upstream
from the cap site. In that region, two sequences are similar to the
canonical IL-6 response element. Site-directed mutagenesis of these
regions strongly decreased induction, showing that they were
functional. PAP I should therefore be classified among acute phase
proteins of class 2, whose expression is increased by IL-6 acting in
combination with glucocorticoids.
)increase
dramatically. Recently, we have described two other PAP-related mRNAs
and named the corresponding proteins PAP II and PAP
III(2, 3) . In consequence, the original PAP became
PAP I. Like PAP I, PAP II and III are induced in pancreas during the
acute phase of pancreatitis. The sequences of the genes encoding rat
PAP I, II, and III have been recently determined (2, 4, 5) . All three genes are organized in
six exons, and similarities observed in their coding sequences extend
to their 5`-flanking regions. In addition, the three genes have been
located to the same position on chromosome 4q33-34(6) ,
suggesting that they derived from the same ancestral gene by gene
duplication., IFN,
leukemia inhibitory factor, IL-11, and oncostatin M. These proteins are
locally produced by the tissue and by circulating mononuclear cells in
response to prototype inflammatory stimuli and can elicit the diverse
biological effects characteristic of the acute phase response.
Interestingly, during the acute phase of pancreatitis, levels of
cytokines are strongly increased in serum(9) . In the current
study, we have evaluated the respective contributions of several
cytokines and of dexamethasone to the transcriptional induction of the
rat PAP I gene in vitro, using a rat pancreatic acinar cell
line.
AR-42J Stimulation
AR-42J pancreatic acinar
cells were obtained from Dr. A. Estival (INSERM U151, Toulouse, France)
and used after 42-49 passages. The cells were routinely
cultivated at 37 °C in a 5% CO
, 95% air atmosphere in
Dulbecco's modified Eagle's medium containing 10% (v/v)
fetal calf serum (Life Technologies, Inc.), 4 mML-glutamine, 50 units/ml penicillin, and 50 mg/ml
streptomycin. The cells were seeded at 3 
10
/100-mm
Petri dish. When cells reached 80-90% confluence (which took
approximately 1 week), they were dissociated with 0.05% trypsin and
0.02% EDTA in Puck's saline A and replated. For the stimulation
experiments, cultures were incubated with either control medium, IL-1,
IL-6, IFN, TNF
, dexamethasone, or a combination thereof.
After 48 h, the medium was removed and replaced with fresh medium
containing the indicated amount of the stimulant. Twenty-four hours
later, total cellular RNA was prepared by the acidic guanidinium
thiocyanate-phenol-chloroform extraction method(10) . Total
cellular RNA (15 µg/lane) was fractionated by electrophoresis on a
1.0% agarose-formaldehyde gel and transferred onto nylon filters
(Hybond). Filters were then hybridized with P-labeled
probes for rat PAP I (7) and
-actin(8) . Filters
were then washed extensively and autoradiographed.CAT Reporter Gene Constructs
All DNA constructs
were generated by polymerase chain reaction using the plasmid P/P as a
template(4) . That plasmid is a pBluescript KS in which was subcloned a 2859-base pair PstI-PstI genomic DNA fragment containing the PAP I
gene, including 1253 nucleotides of the promoter. Accuracy of
polymerase chain reaction was increased by using low dNTP
concentrations(11) , 100 ng of DNA plasmid as template, and
only eight cycles of DNA amplification. Amplification was performed in
1
polymerase chain reaction buffer (50 mM KCl, 10
mM Tris-HCl, pH 8.3, 2 mM MgCl, and 0.01%
gelatin) containing 2 µM dNTP, 1% MeSO, 25
pmol of each primer, and 2.5 units of Taq polymerase in a
final volume of 50 µl. The reaction times were as follows: first
cycle, denaturation at 94 °C for 2 min, annealing at 55 °C for
2 min, and extension at 74 °C for 2 min; for the next 6 cycles,
denaturation at 94 °C for 10 s, annealing at 55 °C for 2 min,
and extension at 74 °C for 2 min; for the last cycle, denaturation
at 94 °C for 10 s, annealing at 55 °C for 2 min, and extension
at 74 °C for 10 min. The product was blunt-ended with Klenow
polymerase, kinased, and ligated into the SalI site of the
promoterless vector pCAT-Basic (Promega) to generate the plasmids
p-1253/+10PAPI-CAT, p-926/+10PAPI-CAT,
p-685/+10PAPI-CAT, p-444/+10PAPI-CAT,
p-379/+10PAPI-CAT, p-317/+10PAPI-CAT,
p-274/+10PAPI-CAT, p-180/+10PAPI-CAT,
p-118/+10PAPI-CAT, p-61/+10PAPI-CAT, and
p+10/-1253PAPI-CAT. Mutant plasmids were also constructed, to
monitor the function of two sequences in the PAP I promoter
corresponding to potential IL-6 response elements. Plasmids
pmut1-274/+10PAPI-CAT,
pmut2-274/+10PAPI-CAT, and
pmut3-274/+10PAPI-CAT were generated, in which IL-6RE-1,
IL-6RE-2 or both were modified, respectively (Fig. 5). The
following oligonucleotides were used:
5`-CCTTGTGTCGTTAGAAACAGAGTATCTGGAAAAGGGTGTGGAGGGTTCAAAC-3`,
5`-CCTTGTGTTTCCCAGAACAGAGTATAGTAGGCAGGGTGTGGAGGGTTCAAAC-3`, and
5`-CCTTGTGTCGTTAGAAACAGAGTATAGTAGGCAGGGTGTGGAGGGTTCAAAC-3`, the
underlined sequences corresponding to modified regions. The
oligonucleotide used in opposite orientation was
5`-TGGATGGTTTGTGAGGACAGA-3` in all cases. Numbers in plasmid names
refer to the positions of first and last nucleotides of the insert in
the PAP I gene. Plasmid DNA was purified with the Qiagen plasmid Kit
(Diagen, Hilden, Germany) and the DNA concentration measured
spectrophotometrically. Sequences were verified by the chain
termination method using the T7 sequencing kit (Pharmacia Biotech
Inc.). Plasmids were also checked for purity, concentration,
supercoiling, and restriction pattern by agarose gel electrophoresis.
Cell Transfection and CAT Assays
Fifty to 60%
confluent AR-42J (100-mm Petri dish) were transfected using the calcium
phosphate DNA coprecipitation method(12) . The transfection
mixture contained 20 µg of test plasmid and 4 µg of
pCMV/-gal (Promega). Twelve hours after the addition of the DNA,
cells were subjected to 20% (v/v) glycerol for 2 min, and the cells
were washed with serum-free medium and transferred to serum-containing
medium. Thirty-six hours later each dish was treated with a different
stimulant for 24 h. Each construct was monitored in triplicate. In all
cases, at least two separate plasmid preparations were tested in the
transfection experiments. Cells were then harvested, and extracts were
prepared. CAT activity was determined using a phase extraction
procedure(13) .
Effect of IL-1, IL-6, IFN
AR-42J is a pancreatic
tumor cell line derived from an azaserine tumor of the rat exocrine
pancreas(14) , which has retained most characteristics of the
acinar cells. In these cells, the basal level of the PAP I transcript
is extremely low, as in normal pancreas(4) . We have monitored
PAP I mRNA expression following treatment with several cytokines and
dexamethasone. As shown in Fig. 1, treatments with IL-1 (50 and
500 units/ml), IL-6 (100 and 1000 units/ml), or dexamethasone (10 and
100 nM) alone were ineffective. Treatment with TNF, TNF
, and
Dexamethasone on AR-42J PAP I mRNA Levels (500,
1000, and 5000 units/ml) or IFN (100, 500, and 1000 units/ml)
induced weak PAP I mRNA expression by comparison with untreated cells.
However, dexamethasone clearly inhibited their stimulatory effects.
Combinations of IL-1 with IL-6 or dexamethasone were also ineffective.
By contrast, combination of IL-6 with dexamethasone induced a strong
stimulation of PAP I gene transcription and subsequent mRNA
accumulation. Surprisingly, when IL-1 was added together with IL-6 and
dexamethasone, the induction was partially inhibited. The extremely low
basal level of PAP I expression in AR-42J cells and the strong signal
observed in Northern blots upon stimulation suggest that PAP I gene
expression in these cells is primarily controlled by transcriptional
regulation. Differences in
-actin mRNA concentrations were mainly
due to differences in total RNA on the filters, estimated from
methylene blue coloration.
P-labeled cDNAs specific
for the PAP I and
-actin mRNA. Results for IL-1, IL-6, and
dexamethasone are given in panelA. Results for
IFN, TNF
, and dexamethasone are given in panelB; results with dexamethasone and the combination
IL-6/dexamethasone, from an experiment run in parallel, are given as
controls. Concentration of cytokines and dexamethasone are provided in
the corresponding tables. Lanes7 and 13 in panelA and lane9 in panelB refer to experiments where no cytokines or
dexamethasone were added.
Analysis by Progessive Deletion of Sequences Required for
IL-6/Dexamethasone Induction of PAP I Gene
The presence of a
functional promoter and tissue-specific elements in the 5`-flanking
region of the rat PAP I gene was tested by transient expression assays.
A 1253-base pair fragment containing the PAP I 5` region was fused to
the bacterial gene coding for chloramphenicol acetyltransferase (CAT)
whose expression can be easily monitored in transfected cells.
Transfection experiments showed that p-1253/+10PAPI-CAT was
able to promote basal transcription in AR-42J cells(8) . Fig. 2shows the comparison of CAT activity in extracts from
AR-42J cells transfected with the above constructs, then stimulated
with IL-6, dexamethasone, or IL-6 and dexamethasone. Treatments with
IL-6/dexamethasone increased CAT activity 12 times, whereas IL-6 or
dexamethasone alone were ineffective. In order to identify the regions
necessary for IL-6/dexamethasone induction, progressive deletions of
the PAP I promoter were performed, and the resulting constructs were
transfected into AR-42J (see ``Experimental Procedures''). As
shown on Fig. 3, deletions in the 5` to 3` direction resulted in
a stepwise decrease of CAT gene expression in the AR-42J cell line.
Deletion down to position -926 did not alter significantly the
expression of the reporter gene. Deletion to nucleotide -685
resulted in about 30% decrease in expression. Progressive deletion of
the next 368 nucleotide (to position -317) did not alter CAT
activity further. An additional deletion to nucleotide -274
caused a decrease to 40% of the control. Extending deletion to
nucleotide -180 caused a decrease to 20% of the control, and a
further deletion of 62 base pairs (to position -118) resulted in
a reduction of the CAT activity to about 10% of control. Finally,
deletion down to nucleotide -61 further reduced activity about 3
times, although it remained slightly above background.
-galactosidase activity. Values represent the means of six
independent transfection experiments (± standard error). In each
experiment, CAT activities were expressed relative to the level of CAT
activity in untreated control cells, which was assigned a value of
1.0.
-galactosidase activity. Values represent the means of six to
nine independent transfection experiments. Values were expressed as
percentage of the p-1253/+10PAPI-CAT
activity.
Mutation Analysis Reveals Two Functional IL-6 Response
Elements (IL-6RE) in the PAP I Promoter Region
Computer-assisted
search for sequences similar to previously described IL-6RE showed two
potential regions within the PAP I promoter, at positions -266 to
-260 (in antisense orientation) and -249 to -243 (in
sense orientation). To analyze these sequences, site-directed
mutagenesis of the regions from -266 to -260 and -249
to -243 was conducted within the CAT construct
p-274/+10PAPI-CAT and the influence of the mutations on the
amplitude of CAT induction by IL-6/dexamethasone was monitored. With
mutant pmut1-274/+10PAPI-CAT, in which the sequence from
-266 to -260 was modified, induction was reduced to 22% of
control (Fig. 5). Similarly, replacement of the sequence from
-249 to -243 (mutant pmut2-274/+10PAPI-CAT)
reduced the induction to 35% of control. Induction with mutant
pmut3-274/+10PAPI-CAT, which carried both mutations, was
further reduced compared to pmut1-274/+10PAPI-CAT or
pmut2-274/+10PAPI-CAT constructs. Hence, hexanucleotides
TTCCCAG and CTGGAAA are actually involved in the response of the
promoter to IL-6/dexamethasone. However, stimulation could not be
completely abolished by mutation of the two IL-6REs. The remaining
induction (about 2 times) suggests that additional active cis-regulatory elements are present within the
-274/+10 region of the PAP I promoter.
, TNF
, and dexamethasone. or TNF
, compared to the absence of effects
of IL-1 or IL-6. Another intriguing finding was the inhibition by IL-1
of IL-6/dexamethasone stimulation. However, a growing number of reports
show that expression of acute phase protein genes is not always
mediated by single cytokines but by combinations of several cytokines (15, 16, 17) or by cytokines in association
with cofactors such as glucocorticoids(16) . It was also shown
that one cytokine may modulate the effect of other
cytokines(17, 18) . These findings suggest that
specific responses of a cell to various inflammatory stimuli are
mediated by specific combinations of cytokines and/or glucocorticoids.
Although an important regulatory function during the acute phase
reaction has been attributed to glucocorticoids and IL-6(17) ,
at the utilized doses, dexamethasone and IL-6 alone were unable to
induce PAP I gene expression in AR-42J cells ( Fig. 1and Fig. 2). Then, two mechanisms may account for the synergy
between IL-6 and dexamethasone. First, glucocorticoid and IL-6 response
elements might be localized in close vicinity on the PAP I promoter. In
that instance, interaction of the nuclear factors binding the two
transcription activator elements might enhance individual responses
that would be otherwise too weak to be observed. Such a situation was
reported for the -acid glycoprotein(19) .
Second, dexamethasone could stimulate IL-6 receptor synthesis in AR-42J
cells, resulting in an increased number of IL-6 receptors at the cell
surface. This was already observed in hepatic
cells(20, 21) . That mechanism might apply to the PAP
I gene since, in the hepatic carcinoma cell line HepG2 transfected with
p-1253/+10PAPI-CAT, CAT activity could be strongly induced
(25-fold) by IL-6 alone (data not shown). Hence, the IL-6 enhancer
element of the PAP I promoter does not require glucocorticoids to be
active. More studies are, however, necessary to understand the
synergistic effect of IL-6 and dexamethasone on the PAP I gene
induction in AR-42J cells.-acid glycoprotein, haptoglobin, hemopexin, complement
C
, and serum amyloid A(17) . Therefore, relative
positions of the different enhancer sequences are likely to influence
the effect of cytokines acting in combination. A mechanism involving
interaction of IL-1 with expression of the IL-6 receptor, as suggested
for dexamethasone, cannot be ruled out, although such regulation has
never been reported in other systems. However, inhibition by
interaction of IL-1 with the IL-6 receptor is unlikely because the two
cytokines have their own specific membrane receptors. or TNF
, although
100-fold less than with IL-6 and dexamethasone. Other genes induced by
TNF are also induced by
IFN(23, 24, 25) . This may be due to the
ability of TNF
and IFN to activate the same transcription
factors, such as interferon regulatory factors 1 and
2(26, 27) . A similar PAP I mRNA induction was
obtained with 100, 500, or 1000 units/ml IFN
, but an inhibitory
effect was observed when we incubated the cells in presence of more
than 500 units/ml TNF
(Fig. 1), suggesting a toxic effect
of this cytokine. Addition of dexamethasone to these cytokines
inhibited induction, as already reported for other
genes(28, 29, 30, 31, 32, 33, 34) .
Again, the opposite effect of dexamethasone on IL-6 and TNF or
IFN underscores that understanding the mechanism of effector
action requires a detailed topological analysis of promoter sequences.
-acid glycoprotein, C-reactive protein,
haptoglobin, and serum amyloid A) is induced by IL-1 or combinations of
IL-1 and IL-6, whereas the genes for class 2 acute phase proteins (e.g. -macroglobulin,
-antichymotrypsin, and fibrinogen) are mainly
regulated by IL-6 and glucocorticoids. PAP I is therefore an additional
member of the second group of acute phase proteins, with the original
feature of being a secretory protein.
)
We are grateful to R. Grimaud and P. Garrido for
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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