From the Pancreatitis Research Laboratory, Department of Visceral
and Transplantation Surgery, University Hospital,
Zürich, 8091, Switzerland, § Institute of
Genomic Medicine, La Jolla, California 92037, ¶ Chirurgie,
Spital Zollikerberg, 8125, Zollikerberg, and
Division of Gastroenterology, Department of Internal Medicine,
University Hospital, Zürich, 8091, Switzerland
Received for publication, November 28, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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A group of 16-kDa proteins, synthesized and
secreted by rat pancreatic acinar cells and composed of pancreatic
stone protein (PSP/reg) and isoforms of
pancreatitis-associated protein (PAP), show structural homologies,
including conserved amino acid sequences, cysteine residues, and highly
sensitive N-terminal trypsin cleavage sites, as well as conserved
functional responses in conditions of pancreatic stress. Trypsin
activation of recombinant stress proteins or counterparts contained in
rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted
in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms
(pancreatic thread protein and pancreatitis-associated thread protein,
respectively) that rapidly polymerize into insoluble sedimenting
structures. Activated thread proteins show long lived resistance to a
wide spectrum of proteases contained in pancreatic juice, including
serine proteases and metalloproteinases. In contrast, PAP II, following
activation with trypsin or pancreatic juice, does not form insoluble
structures and is rapidly digested by pancreatic proteases. Scanning
and transmission electron microscopy indicate that activated thread
proteins polymerize into highly organized fibrillar structures with
helical configurations. Through bundling, branching, and extension
processes, these fibrillar structures form dense matrices that span
large topological surfaces. These findings suggest that
PSP/reg and PAP I and III isoforms consist of a family of
highly regulated soluble secretory stress proteins, which, upon trypsin
activation, convert into a family of insoluble helical thread proteins.
Dense extracellular matrices, composed of helical thread proteins
organized into higher ordered matrix structures, may serve
physiological functions within luminal compartments in the exocrine pancreas.
Pancreatic juice in vertebrates contains a group of 16-kDa
proteins without known enzyme, proenzyme, or inhibitor function in the
digestive process. This group, without defined function, is composed of
the following protein species. Pancreatic stone protein
(PSP/reg)1 is a
16-kDa acidic protein with an isoelectric point in the range of pH
5.5-6. A truncated form of this protein was originally isolated from
calcium carbonate stones surgically removed from the main pancreatic
duct of humans with chronic pancreatitis (1). For several years it was
believed that PSP/reg served as an inhibitor of calcium
carbonate precipitation in pancreatic juice, and it was proposed that
its name should be changed to "lithostathine" (2). However, it was
later shown that PSP/reg has no more crystal inhibitory
activity than several of the pancreatic digestive enzymes (3, 4). Other
studies have demonstrated that the expression of PSP/reg
protein is increased during the regeneration of islets after
nicotinamide treatment and partial pancreatectomy (5, 6). These
observations led to the conclusion that PSP/reg may be a
protein involved in regeneration (7) and furthermore may act as a
growth mediator stimulating the proliferation of Still other investigations sought to implicate PSP/reg in
the digestive process. However, recent studies did not show regulated PSP/reg synthesis and secretion in response to changes in
nutritional substrates in the diet (13).
Pancreatitis-associated protein (PAP) is a 16-kDa basic protein with an
isoelectric point in the range of pH 6.5-7.6. Although most species
contain a single PAP form, rat contains three isoforms, PAP I, PAP II,
and PAP III, transcribed from three separate genes (14-16). PAP levels
increase in pancreatic juice during experimental (17) and clinical (18)
pancreatitis. Although showing an acute phase response under conditions
of pancreatic disease, the function of PAP remains unknown.
PSP/reg and PAP forms, cloned in the rat, mouse, cow, and
man, show similarities in amino acid sequence. At the C terminus there
is a C-type lectin binding sequence, and it has been proposed that this
site might confer bacterial resistance on PAP (14, 19). Recent studies
have demonstrated that PSP/reg and PAP both act as acute
phase reactants in pancreatic juice under a variety of conditions
including acute pancreatitis (20), chronic pancreatitis in male WBN/Kob
rats, and during the post-weaning period (21). Trypsin cleavage of
PSP/reg and PAP has resulted in the appearance of
precipitated proteins believed to represent insoluble thread structures
in humans (22) and cows (23). However, it remains difficult to
understand how precipitation properties could serve useful functions in
pancreatic physiology, and it is not clearly known whether these
precipitated protein forms demonstrate specific or nonspecific structures.
By taking a different approach in this study, we have attempted to
define the functional as well as structural similarities shared between
these molecules as a means to generate clues related to their function.
To this end we have cloned, expressed, and purified recombinant forms
of PSP/reg, PAP I, PAP II, and PAP III in the rat (24, 25).
These purified reagents have allowed us to investigate the structural
and functional properties of these proteins before and after trypsin
cleavage to search for a unifying hypothesis that might explain the
function of these proteins in pancreatic physiology and pathology.
In this paper we have studied the structural/functional consequences of
trypsin activation on these proteins with respect to (i) resistance of
the processed forms to trypsin as well as to the heterogeneous mixture
of proteases in pancreatic juice, (ii) the kinetics of conversion from
soluble to insoluble protein forms, (iii) the kinetics of assembly of
protein subunits into polymerized thread structures, and (iv) the
morphology of polymerized thread structures by scanning, transmission
scanning, and transmission electron microscopy.
Recombinant PSP/reg--
Recombinant PSP/reg was
produced in the baculovirus system and purified as described previously
(3, 24). Monospecific antibodies directed against rat
PSP/reg were generated in rabbits as described earlier
(24).
Construction of Recombinant PAP Isoforms in Pichia pastoris
Vectors--
PAP I cDNA and PAP II cDNA were amplified using
the polymerase chain reaction (PCR) from a rat pancreas cDNA
library using PAP I- and II-specific primers and confirmed by DNA
sequencing. PAP III cDNA was isolated by reverse transcriptase-PCR
using rat ileum mRNA as a template. First, total RNA was extracted
from rat ileum as described (13), and then mRNA was prepared with an Oligotex mRNA minikit (Qiagen). Reverse transcription of 5 µg
of ileal mRNA was performed in a volume of 25 µl at 37 °C for 30 min using 19 units of Moloney murine leukemia virus-reverse transcriptase (Amersham Pharmacia Biotech) and a PAP III-specific primer.
All three PAP isoforms are secretory proteins; therefore, we attempted
to direct the accumulation of recombinant proteins in the media rather
than intracellularly. To ensure that these proteins are secreted by
P. pastoris, the endogenous PAP signal peptides were
substituted by the signal peptide of the
The cDNAs were processed using a two-step PCR amplification
procedure. In a first PCR, an antisense primer was combined with the
matching sense primer (all primer sequences are described in Ref. 25).
Second, the purified PCR products were combined in another PCR with the
same antisense primers and a set of sense primers causing an elongation
of the cDNA. PCR was performed on a PerkinElmer Life Sciences Gene
Amp PCR system 9600 using Taq DNA polymerase (Amersham
Pharmacia Biotech). 30 cycles were run (1 min at 94 °C, 2 min at
55 °C, and 3 min at 72 °C). After each amplification step the
cDNAs were purified by excising them from agarose gels (26). The
recovered cDNAs were purified by phenol extraction and sodium
acetate/ethanol precipitation.
The cDNAs were subsequently digested with XhoI and
EcoRI restriction endonucleases (Amersham Pharmacia
Biotech). The digests were purified and subcloned into pBlueScript
(Stratagene) using T4 ligase (Amersham Pharmacia Biotech). TOP F10'
Escherichia coli cells were transformed with the ligated
plasmids by electroporation using a Bio-Rad Pulser II. Purified
plasmids from individual colonies were digested with restriction
enzymes to check for inserts, and some were subsequently sequenced to
confirm the correct orientation of the coding sequence. Plasmids
carrying the correct sequence were then digested with XhoI
and EcoRI restriction enzymes, and the inserts were
subcloned into the Pichia shuttle vector pPIC9 (Invitrogen).
One recombinant vector for each isoform was sequenced again to confirm
the correct insertion and sequence.
Transformation and Selection of High Expression Clones--
The
Pichia strains GS115 and KM71 (Invitrogen) were transformed
with the linearized recombinant pPIC9 vector (SalI
restriction endonuclease) by electroporation according to the
supplier's recommendations. The transformants were plated on
histidine-deficient minimal dextrose (MD) agar plates (1.34% yeast
nitrogen base, 0.00004% biotin and 1% dextrose), and colonies were
analyzed for insert integration by PCR, using the Purification and Analysis of Recombinant Proteins--
High
yield media were collected by centrifugation at 1500 × g for 5 min at 4 °C. The supernatants were centrifuged
again at 5000 × g for 15 min. The supernatants were
diluted 1:3 with chilled MilliQ filtered water (Millipore) and adjusted
to pH 3.6 with HCl. Diluted protein solution (1200-1600 ml) was
applied at a rate of 5 ml/min to a cation exchange column
(SP-Sepharose, Amersham Pharmacia Biotech, 26 × 80 mm) with a bed
volume of 40 ml. The column was washed with 2 volumes of starting
buffer (50 mM MES, 10 mM LiCl, pH 5.3). The
proteins were eluted with a linear LiCl gradient (0-35% elution
buffer: 50 mM MES, 2 M LiCl, pH 6.3), generated
by an AKTA purifier system (Amersham Pharmacia Biotech).
Analysis of Purified Recombinant Proteins by Mass Spectrometry
and N-terminal Sequencing--
To verify the identity of each of the
isoforms, both mass spectrometry (electrospray mass analysis,
PerkinElmer Life Sciences) and N-terminal amino acid sequencing was
performed. The secreted, purified forms of PAP I, II, and III were
desalted by ultrafiltration. To identify the new N terminus of the
trypsin-resistant 14-kDa protein, each isoform was digested with
trypsin as described below. The solutions were centrifuged to pellet
the fibrils and remove the cleaved undecapeptide. For PAP II, the
solution was filtered after digestion with an ultrafiltration device
that retained the C-terminal peptide (10-kDa molecular weight cut-off,
Centricon, Millipore). For mass analysis, aliquots were adsorbed to C18
ZIP tips (Millipore), eluted with 78% methanol, 1% formic acid, and injected into the analyzer.
Production of Antisera against PAP--
One hundred micrograms
of recombinant PAP II were injected in Freund's complete adjuvant into
several subcutaneous deposits in the back of a New Zealand White rabbit
and a guinea pig. After 1 month the animals were boosted with 50 µg
of PAP II in Freund's incomplete adjuvant followed by a similar boost
a month later. Antibody titers were monitored in serum after
venopuncture of the ear vein. Terminal bleeding was performed under
anesthesia (Ketamine/Xylazine) by heart puncture. The antibody directed
against PAP II reacts with PAP II (100%), PAP III (35%), PAP I
(<10%), PATP II (80%), PATP III (<10%), and PATP I (<10%) but
does not react with PSP/reg.
Activation of Pancreatic Juice--
Twenty microliters of rat
pancreatic juice were diluted in 400 µl of Tris-Calcium buffer (10 mM, pH 8.0, 1 mM CaCl2) and
equilibrated at 37 °C. An aliquot was withdrawn, and enteropeptidase
(Worthington) was added at a final concentration of 0.1 unit/ml. The
reaction was continued at 37 °C for 16 h during which several
aliquots were withdrawn as a function of time as indicated in Fig. 1.
The aliquots were transferred into a tube containing 50 µg of FOY 305, a potent trypsin inhibitor, and snap-frozen in liquid nitrogen until all aliquots had been collected. The samples were then rapidly thawed and centrifuged at 10,000 × g for 10 min in a
cooled Beckman centrifuge (TL-100). The supernatant fraction was
transferred to a new tube, and the pellet fraction was washed with 50 µl of Tris-Calcium buffer and centrifuged as described above. The
pellets were dissolved in Tris-Calcium buffer in the original volume of the sample. They were prepared for electrophoresis by adding a 0.5 volume of 3-fold concentrated SDS-sample buffer (150 mM
Tris-HCl, pH 6.8, 3% SDS, 0.015% bromphenol blue, 15% glycerol v/v)
followed by heating for 5 min at 90 °C. To maximize immunoreactivity
of the activated protein forms, Tryptic Activation of PAP, PSP/reg, and Pancreatic Juice for
Morphological Analysis--
Activation of the recombinant proteins was
performed in a volume of 100 or 200 µl in Tris-Calcium buffer.
Proteins (10 or 20 µg) were activated with 0.5-1 µg of trypsin
(Worthington) for 30 min at 37 °C.
SDS-PAGE and Western Blotting--
Standard 15% polyacrylamide
gels were prepared in SDS. Protein samples were heat-denatured at
90 °C in SDS-sample buffer (50 mM Tris-HCl, pH 6.8, 1%
SDS, 0.005% bromphenol blue, and 5% glycerol) in the presence or
absence of Densitometry--
Relative quantities of individual protein
bands were estimated by densitometry. Coomassie Blue-stained gels or
Western blots were scanned with a Scanjet 6300C (Hewlett-Packard). The
files were imported into Adobe Photoshop and quantified using
ImageQuant (Molecular Dynamics) software. Intensities were expressed in
percent of the maximal value as indicated in the figure legends.
Differential Centrifugation of Thread Proteins--
Recombinant
thread proteins were diluted to 10 µg/50 µl in Tris-Calcium buffer
and centrifuged in Microfuge tubes (Beckman) using a table top
ultracentrifuge (Beckman TL 100). Following centrifugation of the
sample at 1000 × g (4 °C, 30 min), the supernatant fractions were transferred to new tubes and centrifuged at 10,000 × g for 30 min at 4 °C. The resulting supernatant
fractions were centrifuged at 100,000 × g for 30 min
at 4 °C. The pellets were washed with Tris-Calcium buffer (described
above) and redissolved in the original volume. Pellet and supernatant
fractions were denatured in the presence of SDS-sample buffer,
including 1% Analysis of Protein Matrices by Scanning Electron Microscopy
(SEM)--
For scanning electron microscopy, pouches of nylon mesh,
~1 cm2, were produced by folding a small piece of nylon
mesh. The edges were heat-sealed, except for a small hole through which
the samples could be introduced. The activation mixtures were
transferred into the pouch, closed with a clamp, and submerged in 50 mM sodium cacodylate, pH 7.5, for 30 min at 4 °C. The
pouch was then transferred to 2% glutaraldehyde in 100 mM
sodium cacodylate, pH 7.5. After fixation at 4 °C overnight, the
pouches were opened and processed for SEM analysis using standard
procedures. The samples were viewed on a JEOL (JSM-25S II) scanning
electron microscope. Photographs were taken via an attached computer
using the software DISS (Digital Image Scanning System, Prophysics, Switzerland).
For scanning transmission electron microscopy (STEM, Philips CM 120)
analysis, a small amount of fibrillar material was removed from the
surface of the pouch and plated with gold. To exclude gold-derived
artifacts, the following pilot study was performed. After
glutaraldehyde fixation, the pouch (see above) was immersed in 2%
OsO4, 0.1 M sodium cacodylate buffer for 3 h. The pouch was rinsed and processed for SEM and STEM. In the absence
of gold plating, the quality of resolution of the protein matrix was
inferior compared with the gold-plated sample. However, it was
concluded that the structure of the unplated sample was comparable to
the structure of the gold-plated sample.
Analysis of Protein Matrices by Transmission Electron
Microscopy--
To prepare thin sections of fibrils for
examination in the electron microscope, the activation mixtures were
initially embedded in Epon. However, this approach required extensive
centrifugation after each medium change and a polymerization step at
60 °C. The resulting preparations appeared amorphous due to the loss
of the fibrillar architecture. To circumvent this problem, we omitted centrifugation and used an alternative procedure that did not require
polymerization at 60 °C. The digests were mixed with 0.1 volume of
25% glutaraldehyde and left at 4 °C overnight. Then OsO4 was added to a final concentration of 2 mM, and fixation was continued at room temperature for
another hour. The fibrils demonstrated a black appearance and settled
to the bottom of the vessel. Unicryl (British Biocell Int., Cardiff,
UK), an embedding medium that polymerizes at low temperatures under UV
light, was used according to the manufacturer's recommendation. Thin
sections were cut with a diamond knife and examined in an electron
microscope (Philips 400). As a negative control for this procedure,
buffer was substituted for the activation mixture and processed as
described above. Although some amorphous material was generated during
OsO4 fixation, the fibrillar structures were not observed
in the control.
For negative contrast staining of fibrils, a small drop of activated
sample was placed on the surface of a grid, dried, exposed to
phosphotungstic acid (27), and examined in the electron microscope.
Computer Programs for Sequence Analysis--
The multiple
sequence alignment was created by "PileUp," an algorithm for
progressive, pairwise sequence alignments by Feng and Doolittle (28).
The same program produces a dendrogram that depicts the clustering
relationships. The determination of sequence similarity and identity
was performed by pairwise analysis using the program "Gap." Gap and
PileUp are part of the Wisconsin package version 9, supplied by
Genetics Computer Group (Madison, WI). GCG is run on a UNIX system
(Silicon Graphics) maintained by the Rechenzentrum der
Universität Zürich.
Conservation of the N-terminal and C-terminal Peptides in a Family
of 16-kDa Secretory Proteins--
Fig. 1
shows the sequence alignment of the N-terminal and C-terminal regions
of three isoforms of PAP with PSP/reg in the rat. The mature
secretory proteins are defined by two peptide domains separated by a
highly conserved trypsin cleavage site. In three of the four secretory
stress proteins (PSP/reg, PAP I, and PAP III), the
Arg11-Ile12 bond represents the most sensitive
cleavage site for trypsin. Upon trypsin cleavage the N-terminal
undecapeptide is separated from the C-terminal peptide, which varies
from 138 residues in PAP to 133 residues in PSP/reg.
In addition to similarities in size, these proteins show similarities
in sequence and protein domains. PSP/reg shows 43% identity and 54% similarity with PAP I, 45% identity and 52% similarity with
PAP II, and 50% identity and 57% similarity with PAP III. PSP/reg is five amino acids shorter than the three PAP
isoforms. The conservation of six cysteine residues suggests that three disulfide bonds are conserved. At the C terminus there is a conserved sequence indicating a C-type lectin domain (Fig. 1). Although the
function of this signature sequence has not been elucidated, CTLs have
been found in a variety of proteins that demonstrate diverse functions
in different cellular and extracellular compartments (29). However, the
conserved N-terminal trypsin cleavage site is not observed in other
proteins bearing CTLs.
The secreted and proteolytic processed forms of recombinant
PSP/reg and recombinant PAP shown in Fig. 1 were analyzed by
mass spectrometry and N-terminal sequencing. The secreted forms of PAP
I, II, and III all conformed with the expected amino acid sequence
(first 10 amino acids determined), starting with a glutamine residue in
each case. The processed forms of PAP also conformed with the expected
sequence (first 5-7 amino acids determined) starting with an
isoleucine (PATP I, III) or a threonine (PATP II) residue. Mass
analysis yielded the following measurements: PAP I, 16623.6 (theoretical 16623.6); PATP I, 15414.0 (15414.2); PAP II, 16404.2 (16403.2); PATP II, 15203.9 (15203.9); PAP III, 16247.8 (16248.0); and
PATP III, 15021.5 (15019.7). Mass determinations indicated that
PSP/reg and each of the PAP isoforms were not modified by
glycosylation or other posttranslational mechanisms.
Trypsin Activation of Rat Pancreatic Juice Generates
Protease-resistant Products with Low Solubility--
By utilizing
purified recombinant PSP/reg and PAP III, we have recently
demonstrated that trypsin activation leads to the formation of 14-kDa
trypsin-resistant products (25). In order to determine if the 14-kDa
products are resistant to the wide spectrum of proteases observed in
activated pancreatic juice, we studied the identity and longevity of
these products generated from rat pancreatic juice containing high
levels of PSP/reg (320 µg/ml) and PAP (116 µg/ml). Fig.
2 shows the Coomassie Blue and immunostained products that appear in soluble and insoluble fractions of pancreatic juice, diluted into 20 volumes of buffer and activated with enterokinase at 37 °C, over a period of 960 min (16 h).
At the beginning of the experiment (0-, 0.5-, and 1.0-min time points),
the major digestive enzymes and zymogens present in pancreatic juice
may be easily identified (30, 31). These include amylase (major band at
55 kDa), procarboxypeptidase A and B isoforms at 40-45 kDa, and serine
protease zymogens between 21 and 27 kDa. At later time points these
bands disappear in response to the degradative effects of activated
proteases. Despite the progressive degradation of the digestive enzymes
and zymogens, a prominent Coomassie Blue-stained band appears at 14 kDa
in the pellet fraction at 10 min and remains largely intact throughout the duration of the experiment.
In order to determine the composition of this 14-kDa protease-resistant
band, we used polyclonal antibodies individually directed against
PSP/reg and PAP to determine the representation and
longevity of PTP and PATP isoforms in this region of the gel. The
antibody directed against PSP recognizes both PSP and PTP. The antibody directed against PAP II reacts with PAP II (100%), PAP III (35%), PAP
I (<10%), PATP II (80%), PATP III (<10%), and PATP I (<10%) but
does not react with PSP/reg.
Between 1 and 10 min PSP/reg disappears from the supernatant
fraction, and PTP appears in the pellet fraction. However, PTP appears
in the supernatant fraction before it appears in the pellet fraction,
suggesting that soluble PTP requires time to polymerize into insoluble
PTP complexes. Similar findings are observed in the conversion of PAP
to PATP, with two exceptions. First, whereas conversion of
PSP/reg occurs between 1 and 10 min, the conversion of PAP
occurs more slowly, largely between 10 and 60 min. Second, the PAP/PATP
signal is less intense, due to the lower concentration of PAP isoforms
and possibly compounded by a lower binding of the antibody to its
target protein(s).
These findings indicate that trypsin activation leads to the rapid
conversion of PSP/reg and PAP forms to trypsin- and
protease-resistant forms of PTP and PATP, respectively. The longevity
of PTP and PATP forms in the presence of fully activated pancreatic
juice containing numerous protease forms is impressive and suggests that the two groups of thread proteins have evolved with similar properties of protease resistance that may be related to the ultimate function of these proteins in pancreatic physiology and pathology.
To gain more insight into the temporal aspects of activation and
polymerization of thread proteins, the gels and blots were analyzed by
densitometry. Fig. 3A shows
the relative abundance of Coomassie Blue-stained products in the pellet
fraction as a function of time. Fig. 3B shows the kinetic
data for conversion of PSP/reg to PTP as well as the
conversion of PTP from a soluble form to an insoluble form. The data
demonstrate that proteolysis precedes the polymerization process by
several minutes and identifies an intermediate state between soluble
precursor and insoluble product. Furthermore, the data suggest that PTP
occurs in a soluble form before its polymerization into insoluble
thread structures. Fig. 3C shows kinetic data for the
conversion of PAP to PATP and similarly identifies an intermediate
state represented by soluble PATP prior to its polymerization into
insoluble thread structures.
In the presence of fully activated pancreatic juice at 37 °C the
half-life is 100-150 min for the PATP band, 800 min for the PTP band,
and 400 min for the Coomassie Blue band. Thus, the activation of
secretory stress proteins into insoluble thread proteins leads to
strong resistance to the degradative effects of pancreatic proteases.
Kinetics of Proteolytic Conversion of Recombinant PAP Isoforms and
Assembly of Products into Insoluble Thread Complexes--
Because our
PAP antibody could not track individual PAP/PATP forms and furthermore
recognized PAP II with greater affinity than PAP I or PAP III, we
studied the conversion of purified recombinant PAP forms to PATP forms
under three conditions as follows: (i) trypsin activation, (ii)
activation in the presence of enteropeptidase-treated pancreatic juice,
and (iii) trypsin activation followed by the addition of pancreatic
juice after the formation of protein fibrils. Fig.
4 shows the results of trypsin activation
on PAP I, PAP II, and PAP III and monitors proteolytic processing to
smaller thread proteins in soluble and insoluble fractions. These
kinetic studies demonstrate that PAP I and PAP III isoforms are
rapidly, i.e. within 1 min, cleaved from 16-kDa precursors
to 14-kDa products. 14-kDa products initially appear as soluble
intermediate products but rapidly polymerize into insoluble sedimenting
structures. The 50% threshold for formation of insoluble products was
3 and 0.8 min for PATP I and PATP III (Fig. 4, A and
C), respectively. In contrast, although PAP II was rapidly
processed to 15- and 14-kDa forms by trypsin (PAP II contains an
additional trypsin cleavage site at
Lys5-Ala6), these products remained soluble
throughout the duration of the 16-h incubation period.
During proteolytic processing intermediate sized peptides (15 kDa)
appeared transiently in the incubation mixtures containing PAP
isoforms. The intermediates are consistent with proteolytic processing
at lysine residues contained within the N-terminal undecapeptide
(cf. Fig. 1). These intermediate forms are most prominent in
the case of PAP II, which demonstrates an Arg-Thr trypsin
cleavage site. This finding is consistent with Arg-Iso being a more
active trypsin cleavage site than Arg-Thr, which is observed in PAP
isoforms I and III.
Fig. 4, D-F, shows the survival of PATP isoforms after
16 h of incubation in the presence of (i) trypsin, (ii) activated
pancreatic, and (iii) trypsin-activated PAP followed by addition of
activated pancreatic juice (with low endogenous PSP/reg and
PAP levels). These studies demonstrate that PATP I and PATP III
isoforms show complete resistance to trypsin and partial resistance to
the mixture of proteases contained in pancreatic juice. These proteases
include serine proteases (trypsin, chymotrypsin, and elastase) and
metalloproteinases (carboxypeptidases A and B). Thread proteins exposed
to pancreatic proteases, after fibril formation had been completed,
appeared to show greater resistance to pancreatic proteases (45 and
54% survival after 16 h of incubation for PATP I and PATP III,
respectively) than those formed in the presence of pancreatic proteases
(4 and 27% survival after 16 h of incubation for PATP I and PATP
III, respectively). PATP III showed greater resistance to pancreatic proteases than PATP I. In contrast, although PATP II showed trypsin resistance, it was completely digested within 60 min of addition of
activated pancreatic juice.
Secretory Thread Proteins Show Differential Sedimentation
Properties--
In order to compare the physical properties of
insoluble thread proteins, we activated purified recombinant forms of
PSP/reg, PAP I, PAP II, and PAP III. Trypsin cleavage
converted these recombinant forms into PTP, PATP I, II, and III,
respectively. In previous studies we had observed that PTP, PATP I, and
PATP III sediment under conditions of centrifugation at 10,000 × g for 10 min at 4 °C. In contrast, PATP II could not be
sedimented under these conditions.
To explore in greater detail the sedimentation properties of these four
activated proteins, we studied their sedimentation at varying
conditions of centrifugation. Fig.
5A shows the results of
sedimentation of trypsin-activated proteins (10 µg of protein/50 µl) at 1,000, 10,000, and 100,000 × g for 30 min at
4 °C. Pellet fractions from the three sedimentation conditions and
the final supernatant fraction were submitted to SDS-PAGE for each of
the recombinant forms.
The data indicate that 67% of PATP I is sedimented at 10,000 × g, an additional 14% is sedimented at 100,000 × g, and 11% remains in the final supernatant fraction. Under
these conditions PATP III shows greater insolubility with 87%
sedimented at 10,000 × g and minimal amounts observed
in the 100,000 × g fraction and the final supernatant
fraction. PTP appears to sediment over a wider range of g
forces, demonstrating 25% in the 1,000 × g fraction, 52% in the 10,000 × g fraction, 23% in the
100,000 × g fraction, and negligible amounts in the
final supernatant fraction. In contrast, PATP II appears to be largely
soluble under these conditions: 14% sedimented up to 100,000 × g and 86% was observed in the final supernatant fraction.
In order to investigate whether PATP II enters into insoluble
aggregates at acidic or alkaline pH, we suspended trypsin-cleaved PATP
II at different pH values, and we monitored aggregation with SDS-PAGE
after centrifugation at 10,000 × g for 10 min. Fig.
5B demonstrates the absence of sedimenting complexes at all
pH values tested under these conditions.
These data confirm the insoluble nature of PTP, PATP I, and PATP III
and the soluble nature of PATP II. Questions now arise whether these
insoluble complexes are specific or nonspecific and whether they serve
functions important in pancreatic physiology.
Trypsin Activation of Purified Recombinant Secretory Stress
Proteins Generate Matrices of Highly Organized Fibrils--
To
characterize the morphological structures of the insoluble thread
proteins (PTP, PATP I, and PATP III), the recombinant precursors were
activated with trypsin and analyzed by a variety of electron microscopy
methods. Previous studies that attempted to elucidate the structures of
PTP-derived fibrils were hampered by fibril fragmentation and the
collapse of fibrils when they were placed onto a surface and dried
down. Collapse of fibrils resulted in a nonspecific amorphous
appearance of the matrix complex without distinguishable markings.
To circumvent the collapse of the three-dimensional architecture, we
constructed small pouches of nylon mesh, which were then filled with
fibril-containing solutions. The pouch could then be transferred into
the various washing and fixation solutions without the need for
attachment to a glass support. Fig. 6
shows fibrils generated in this manner and examined in the scanning electron microscope. With the exception of PATP II, which did not form
visible fibrils, all other forms produced a matrix of highly organized
fibrils.
Fig. 6 shows scanning electron micrographs of matrices formed from PATP
I (A and D), PATP III (B and
E), and PTP (C and F). Low power
micrographs (A-C) indicate the appearance of tight matrices covering the nylon mesh in the pouch. Matrices appear to attach to the
nylon mesh. At higher power differences were observed in matrices
formed from activation of the three recombinant proteins. PATP I
(D) shows tight bundling of filaments into larger diameter fibers that contribute to the meshwork. PATP III (E) shows
loose bundling of filaments in a nodular distribution particularly near the edges. PTP (F) shows more delicate filaments comprising
the meshwork and less bundling. The inset in Fig.
5B shows that individual fibrils appear to "grow out" of
the densely polymerized matrix. Extensive branching appears in each of
the observed matrices.
These studies demonstrate that insoluble thread proteins form dense,
highly organized three-dimensional matrices. Through bundling,
branching, and extension processes, these fibrillar threads achieve
higher ordered matrix structures that span large topological surfaces.
High Resolution Analysis of Fibrillar Structures Using Transmission
Electron Microscopy and Scanning Transmission Electron
Microscopy--
In order to determine the fine structure of individual
filaments, we examined the morphological appearance of matrices using a
variety of techniques that employ transmission EM and scanning transmission EM (Fig. 7,
A-E).
In Fig. 7A, a PTP matrix analyzed by scanning transmission
electron microscopy is shown to examine the surface and branching properties. To exclude artifacts caused by gold plating, we explored whether the size and surface features of filaments were altered by gold
spraying methods. Osmium-fixed samples were examined (i) without prior
gold treatment, (ii) with lightly sprayed gold treatment (shown in
A), and (iii) with the standard gold treatment. The morphology of the filaments showed little or no change as a function of
gold treatment. However, the visualization of the filaments was
improved with gold spraying.
Fig. 7, B (PTP) and C (PATP III), shows filaments
that were negatively stained with phosphotungstic acid. This procedure, which causes extensive fragmentation of filaments, compares favorably with negatively stained micrographs that appear in the literature (22,
23).
To examine the higher ordered structure of polymerized thread proteins,
fibrils were fixed in glutaraldehyde and osmium, embedded in low
temperature Unicryl, sectioned, and examined with transmission EM.
Under these conditions filaments (Fig. 7, D, PTP, and
E, PATP III) appeared to be composed of subunits assembled
into helical fibrillar structures. Favorable sections in this
micrograph also suggested the presence of cavities within PATP III
filamentous structures.
The data indicate that, regardless of the method employed or the
protein species examined (PTP, PATP I, and PATP III), filaments exhibit
a consistent diameter of ~15-20 nm.
Morphological Structure of Filaments and Matrices Generated from
Activated Pancreatic Juice--
In order to determine if matrices and
filaments formed from pancreatic juice mimic those observed from
activated recombinant stress proteins, we activated rat pancreatic
juice and prepared it for examination by scanning EM using the nylon
pouch method described above.
We chose samples of pancreatic juice obtained from male WBN/Kob rats,
which contain high levels of PSP/reg (150 µg/ml) and PAP
(50 µg/ml) and spontaneously develop chronic pancreatitis. Fig.
8, A and C, shows
low power and high power micrographs, respectively, of matrices
generated by trypsin activation of juice from these animals. The
morphological appearance of the filaments and three-dimensional architecture of the matrix appear to be intermediate between those observed for pure PTP and pure PATP I in Fig. 6.
Fig. 8, B and D, shows low power and high power
micrographs, respectively, of matrices generated by trypsin activation
of pancreatic juice from normal Wistar rats (PSP/reg, 14 µg/ml; PAP, 1.4 µg/ml). The morphological appearance of the
filaments and three-dimensional architecture of the matrix appear to be
intermediate between those observed for pure PTP and pure PATP III in
Fig. 6. We also noted that the amount of matrix covering the nylon mesh
pouch was considerably less in the sample shown in B than that of A and correlated with the levels of secretory stress
proteins in the two pancreatic juice samples.
These data indicate conclusively that trypsin activation of pure
pancreatic juice can generate insoluble matrices of filamentous thread
proteins that have the capacity to attach to and cover large surface
areas in a manner similar to that observed for matrices generated from
purified recombinant secretory stress proteins.
This study investigates a group of 16-kDa proteins present in rat
pancreatic juice without known enzymatic or inhibitory function in the
digestive process. In addition to the similarity in size between
PSP/reg and PAP isoforms, a comparison of amino acid
sequences reveals significant structural homologies, including
conservation in the position of six cysteine residues, which implies
the conservation of three disulfide bonds. The conserved structural
features of PSP/reg and PAP, including the locations of
tryptophan residues, suggest that the members of this group of
secretory proteins demonstrate highly conserved three-dimensional
structures.2 In the rat all
three of the PAP genes and the PSP/reg gene colocalize to
the same segment on chromosome 4 (32), observations that suggest a
common ancestral gene. Taken together, the conserved structural
features in this group of genes suggest that they form a gene family
generated through gene duplication processes.
The structural similarities in these genes are augmented by functional
homologies. First, each of these proteins is synthesized and secreted
by pancreatic acinar cells. Second, their synthesis and secretion is
increased in response to conditions of pancreatic stress as follows.
During experimental acute pancreatitis, PAP (33, 34) is increased in
pancreatic juice and PSP/reg mRNA is increased in
pancreatic tissue (35). During chronic pancreatitis, both in man and in
an animal model, the male WBN/Kob rat, PAP and PSP/reg
levels are increased in pancreatic tissue (21, 36-38) and juice (21).
During maximal caerulein stimulation and supramaximal caerulein-induced
pancreatitis, PAP and PSP/reg levels are increased in
pancreatic tissue (34) and
juice.3 During the
post-weaning period of pancreatic organ growth, PSP/reg levels are increased in pancreatic juice, and PAP levels show transient
increases in tissue (39). During pancreatic regeneration following
partial pancreatectomy, PSP/reg levels increase in
pancreatic tissue (5, 40) (PAP levels have not been measured in this experimental protocol). The similarity in tissue response and secretion
between PSP/reg and PAP isoforms under conditions of stress
due to (i) post-weaning glandular growth including experimental pancreatic regeneration, (ii) hormone stimulation, and (iii)
experimental and clinical disease suggests homologies of function for
this family of proteins.
The wide divergence of proposed functions for this group of secretory
proteins, summarized in the Introduction, suggests that the primary
function of this gene family remains to be elucidated. In this study we
have focused on an important structural feature, the conserved trypsin
cleavage site (Arg11-Ile12 in
PSP/reg, PAP I, and PAP III isoforms and
Arg11-Thr12 in PAP II). This cleavage site is
conserved in all proteins related to this gene family, both within a
single species as well as across species lines. These proteins include
human, rat, and mouse PSP/reg and PAP, bovine pancreatic
thread protein, the trypsin cleavage product isolated from bovine
pancreatic homogenates treated with trypsin, and PSP/reg
protein isolated from pancreatic tissue in the hamster. This list may
be extended to include islet neogenesis-associated protein (41) and
islet neogenesis-associated protein-related protein (42). These
proteins all share the C-type lectin binding domain, conserved amino
acid sequences, including six cysteine residues, and a conserved
trypsin cleavage site at the N terminus. In their unactivated state
PSP/reg and PAP isoforms remain soluble during their
secretory passage through the pancreatic duct and their delivery to the
intestinal milieu. Cleavage at the conserved Arg11-Ile12 site leads to the removal of an
N-terminal undecapeptide from the C-terminal polypeptide. This
proteolytic processing feature has not been observed in any other
proteins bearing a C-type lectin domain.
By using rat PSP/reg and PAP activated by trypsin, we have
demonstrated in this study that the corresponding C-terminal peptides or thread proteins (pancreatic thread protein and
pancreatitis-associated thread protein, respectively) remain largely
resistant to proteases contained in pancreatic juice, including serine
proteases (trypsin, chymotrypsin, and elastase) and metalloproteases
(carboxypeptidases A and B). Despite the presence of numerous potential
cleavage sites within their amino acid sequence, thread proteins show
impressive protease resistance and survive the proteolytic degradation
of the proteases themselves. Longstanding protease resistance implies that thread proteins undergo a significant change in conformational structure following activation and enter into tightly folded structures relatively impervious to the effects of exogenous proteases.
When pancreatic juice containing high levels of PSP/reg and
PAP was activated, PTP and PATP survived through the 16-h time period
(960 min). By utilizing antibodies to track the conversion of stress
proteins to thread proteins, the half-lives of PATP forms and PTP were
100-150 and 800 min, respectively. Our recombinant studies indicated
that PATP I and PATP III were entirely resistant to trypsin and
substantially resistant to the mixture of pancreatic proteases after
fibril formation. PATP III was resistant to pancreatic proteases both
during and after fibril formation. In contrast, although PATP II was
resistant to trypsin, it was completely degraded by pancreatic
proteases within 60 min of adding pancreatic juice.
In these activation studies we also measured the kinetics of conversion
of soluble intermediate proteins into insoluble sedimenting forms.
These studies demonstrate that PTP, PATP I, and PATP III rapidly appear
in the pellet fraction as insoluble thread proteins. The 50% threshold
for conversion of soluble thread proteins to insoluble thread forms
were 0.8, 3, and 5 min for PAP III, PTP, and PAP I, respectively. PATP
II did not appear in an insoluble fraction. These findings suggest
that, with the exception of PAP II, polymerization of soluble thread
proteins (monomers) into insoluble forms (polymerized fibrils) is a
specific assembly process that is dependent on time and protein concentration.
We have also studied the solubility characteristics of each of the rat
pancreatic secretory thread proteins in detail and further confirmed
that they fall into two groups. PATP I, PATP III, and PTP sediment at
low speed centrifugation, whereas PATP II remains largely in the
soluble fraction at either low speed or high speed centrifugation.
Changes in incubation temperature had little effect on the physical
characteristics of sedimentation for any of these isoforms. We conclude
that PAP II is a nonsedimenting secretory stress protein.
Among pancreatic secretory stress proteins, PAP II shows a number of
characteristics that distinguish it from the other forms, including (i)
rapid digestion in the presence of pancreatic proteases, (ii) inability
to polymerize into fibrils that may be isolated by low speed or high
speed centrifugation, and (iii) undetectable fibrils analyzed by
scanning and transmission electron microscopy. A dendrogram depicting
the sequence relationship between PSP/reg and PAP isoforms
of human, bovine, and rodent origin clearly demonstrates that PAP II is
separated in a different evolutionary cluster distinct from either
rodent PAP I and III isoforms or human PAP and bovine PAP. PAP II may
serve as a facilitator or inhibitor in the coordinated assembly of
insoluble thread structures during pancreatic stress reactions.
Alternatively, PAP II may represent a nonfunctional secretory stress
protein that has lost the critical sequences required for
polymerization into fibrillar thread structures.
In humans and cows with chronic calcific pancreatitis, PTP has been
identified as a major organic component of calcified stones. In both
species thread proteins were isolated and shown to be fibrillar by
negative staining in the electron microscope (22, 23). Purified
PSP/lithostathine has been shown to form similar fibrils after
tryptic cleavage in vitro (43), and prolonged incubation of
PSP, purified by column chromatography, suggested that activation may
occur by autoproteolysis. The studies reported here further extend the
existence of fibrillar thread structures among pancreatic secretory
proteins to include not only PTP but also PATP I and PATP III isoforms.
This group of insoluble thread proteins appears to be generated from a
family of pancreatic secretory stress proteins following trypsin activation.
The assembly of soluble thread proteins into insoluble fibrillar
matrices by all of the 16-kDa proteins (PTP, PATP I, and PATP III),
save one (PATP II), further substantiates the functional similarities
in this family of secretory stress proteins. Analysis of the morphology
of these fibrillar matrices by scanning and transmission electron
microscopy, under a variety of staining and embedding methods, reveals
highly organized structural features associated with extensively
polymerized structures. Although features differ somewhat among
individual thread matrices, there is a strong tendency for polymerized
protein threads to bundle together. Bundling properties appear to be
most developed (strong curvilinear bundles) in the case of PATP I
filaments, less developed, and nodular, in the case of PATP III, and
least developed (feathery matrix) in the case of PTP. Extensive
branching of filaments in all three species leads to dense fibrillar
matrices covering large topological surfaces.
High resolution analysis of fibrillar matrices by transmission EM
suggests the presence of subunit structures apparently assembled into a
helical fibrillar structure. Certain favorable sections point to the
presence of a cavity within these filamentous structures. However,
further structural studies will be required to determine the precise
subunit structures of these filaments and the nature of cavities,
should they exist. Our investigations to date with purified recombinant
proteins have been confined to the assembly of homologous filaments.
Polymerization of heterologous mixtures of thread proteins forms the
basis for a separate study.
We have demonstrated that samples of pancreatic juice, upon trypsin
activation, also form dense fibrillar matrices similar to those
observed with purified recombinant proteins. Both normal pancreatic
juice (Wistar rat), containing small concentrations of stress proteins
(14 µg/ml PSP/reg and 1.4 µg/ml PAP), and pathological juice (male WBN/Kob rats), containing elevated concentrations of stress
proteins (150 µg/ml PSP/reg and 50 µg/ml PAP), form dense fibrillar matrices. However, the extent of matrix formation appears to correlate with the amount of stress proteins observed in
samples of juice.
Based on the findings presented here we propose that PSP/reg
and PAP form a family of secretory stress proteins which, upon trypsin
or "trypsin-like" activation, convert to a family of insoluble thread proteins. We further propose that the activated thread proteins
represent the active form of these molecules whereas the secretory
proforms represent inactive precursors. In order to begin to unify the
terminology in this complex area, we suggest that PSP/reg
may refer to "pancreatic stress protein" as well as "pancreatic
stone protein."
Concerning their function, this family of secretory stress proteins may
serve to form dense extracellular fibrillar complexes that attach to
and span large topological surfaces during conditions of luminal
stress. For example, these dense fibrillar structures may provide a
luminal matrix from which the repair or regeneration of ductal
structures may be orchestrated under conditions of stress. Since islet,
acinar, and ductal cells are all derived from proliferating duct cells
(44), it is possible that neogenesis of exocrine and endocrine tissue
occurs as part of the more general process of ductal proliferation
during pancreatic development or regeneration after partial surgical
ablation of the gland. The mitogenic activity reported for
PSP/reg (8) would be consistent with this notion. Further
work is clearly needed to determine the role of luminal matrices during
pancreatic development, growth, and repair and to determine the
location of the activating enzyme, whether due to a soluble activity in
pancreatic juice or a surface activator associated with ductal cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells. Tissue
culture studies implied a mitogenic activity of PSP/reg on
the growth of various cell types (8, 9), and application of
PSP/reg was observed to partially ameliorate diabetes in NOD mice (10). Recently, a receptor was cloned from regenerating islets
that binds PSP/reg and causes an increase in proliferation of cells transfected with a vector containing the receptor cDNA (11). Reg II (PAP I) appears to be involved in regeneration of motor
neurons by acting as a Schwann cell mitogen (12).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mating factor. The latter
contains a Kex2 signal cleavage site consisting of a Lys-Arg followed
by a Glu. Since all PAP isoforms start with a Glu, the PCR and
subcloning strategy was designed to produce a cDNA with the Lys-Arg
site followed by the first amino acid of the PAP isoforms (Glu). At the
5'-end of the cDNAs an XhoI site was included that
facilitates ligation of the cDNA with the pBlueScript and recreates
the correct reading frame of the
-mating factor signal peptide in
pPIC9 (viz. manual of the Pichia Expression Kit, Invitrogen).
-factor primer and
the 3' AOX1 (alcohol oxidase) primer. Positive clones were selected and
tested with respect to their expression levels. By using a shaking
incubator at 280 rpm, colonies were grown at 29 °C in baffeled
flasks, each containing 80 ml of buffered minimal glycerol (BMG) medium
(100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen
base, 0.00004% biotin, and 1% glycerol). Culture growth was monitored
by measurement of the absorbance (600 nm); when an
A600 between 2 and 6 was reached, the cells were
harvested by centrifugation. To induce expression of the recombinant
protein, the cells were resuspended in 12 ml of BMM medium (BMG medium,
containing 0.5% methanol instead of glycerol) and cultured for 6 days.
The protein secretion was monitored in 24-h intervals by
electrophoretic analysis of the supernatant (Coomassie-stained SDS-15%
polyacrylamide gels). The most productive clones were selected and
cultured in increased culture volumes. The highest yields were found in
5-day-old cultures.
-mercaptoethanol was omitted from the buffer solutions.
-mercaptoethanol as indicated above. Proteins resolved in
gels were stained with Coomassie Brilliant Blue (0.1% in 30%
methanol, 10% acetic acid, Bio-Rad). For Western blot analysis, the
proteins were transferred to polyvinylidene difluoride membranes
(Bio-Rad) (24) on a semidry blotting apparatus (Amersham Pharmacia
Biotech). The membranes were blocked with 1% bovine serum albumin in
Tris-buffered saline (20 mM Tris, pH 7.5, 150 mM NaCl). Guinea pig anti-PAP II, diluted 1:3000 (25), and
phosphatase-coupled anti-guinea pig IgG, diluted 1:10,000 (Sigma), were
used to detect PAP and PATP. For the detection of PSP/reg
and PTP, chemiluminescence (ECLplus, Amersham Pharmacia Biotech) was
employed. The primary rabbit anti-PSP/reg antibody was
diluted 1:20,000 in Tris-buffered saline containing 1% bovine serum
albumin. The membranes were washed in Tris-buffered saline containing
0.05% Tween 20. The secondary antibody, peroxidase-coupled goat
anti-rabbit IgG (Sigma) was diluted 1:25,000 in the same buffer.
Development and detection followed the manufacturer's recommendations.
-mercaptoethanol, for 5 min at 90 °C and submitted
to SDS-PAGE.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conservation of the N-terminal trypsin
cleavage site in a family of secretory stress proteins. The amino
acid sequences of the mature (secreted) rat proteins
PSP/reg, PAP I, II, and III were aligned. The N termini of
the four sequences each contain a propeptide (Pro,
solid line) of 11 amino acids ending with the highly
conserved trypsin cleavage site (Try). This cleavage site is
conserved in 16 PSP/reg-related proteins from 6 different
species (human, bovine, pig, rat, mouse, and hamster). All but one of
the activation peptides contain 11 amino acid residues. Position 12 marks the N termini of the activated proteins (dashed line).
The complete sequence of the stress proteins is 144 (PSP) to 149 (PAP
isoforms) amino acids in length. The position of the putative cleavage
sites for the 15-kDa intermediate PAP isoforms (lysines
underlined) is marked at the top ( ). The
C-terminal alignment, separated by two dots, includes the
C-type lectin domain signature (underlined residues).
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Fig. 2.
Trypsin activation of pancreatic juice
generates 14-kDa products that are protease-resistant and sediment
under conditions of low speed centrifugation. Pure pancreatic
juice from a rat with high levels of PSP/reg and PAP was
activated with enteropeptidase. Aliquots were withdrawn at the
indicated time points (min), and reactions were stopped by the addition
of FOY-305, a protease inhibitor. The samples were centrifuged, and the
supernatant fractions were transferred to a fresh tube. The pellet
(P) and supernatant (S) fractions were analyzed
separately by SDS-polyacrylamide gel electrophoresis. The top
panel of the figure shows the Coomassie Blue-stained gel.
M, marker proteins with the sizes indicated in kDa. The
middle panel (PSP) shows an immunoblot of the
same gel analyzed for the proteolytic conversion of PSP/reg
to PTP (arrowheads indicate their position). The
bottom panel shows an immunoblot analyzed for the
proteolytic conversion of PAP to PATP (arrowheads).
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Fig. 3.
Kinetics of tryptic conversion of secretory
stress proteins to soluble and insoluble thread proteins in pancreatic
juice. The gel and immunoblots shown in Fig. 2 were analyzed by
densitometry and plotted as a function of time. A, relative
abundance of Coomassie Blue-stained 14-kDa bands (CBB) in
the pellet fraction ( ) at each time point. Maximum
density was arbitrarily set at 100%. B, relative abundance
of PSP/reg (
) and PTP in the soluble
(
) and PTP in the insoluble (
) fractions.
The sum of soluble and insoluble PTP at 10 min was set as 100%.
C, PAP (
) and PATP (
) in the
soluble and PATP in the insoluble (
) fractions were
determined from the PAP immunoblot as described for B. The
PAP value at 1 min was set at 100%; for PATP, the combined soluble and
insoluble fraction at 10 min was set as 100%. Note that the antibodies
do not recognize the unprocessed and processed forms with equal
sensitivity. The 15-kDa band (see Fig. 4) was not quantified. Similar
activation kinetics were observed for trypsin-activated pancreatic
juice.
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Fig. 4.
Kinetics of tryptic conversion of recombinant
PAP isoforms to soluble and insoluble thread proteins. Top
panels, Coomassie Blue-stained gels of PAP I, II, and III
activated with trypsin. Samples were obtained at the indicated times
(min) and separated into pellet (P) and supernatant
(S) fractions by centrifugation at 10,000 × g for 10 min at 4 °C. Molecular weight markers on the
left at 14 and 18 kDa. Bottom panels, densitometric
analysis of these gels is shown in A (PAP I), B
(PAP II), and C (PAP III) as a function of time through 960 min and monitors 16-kDa soluble precursors ( ), soluble
intermediate products at 15 kDa (
), and 14 kDa (
)
soluble and insoluble sedimenting products (
).
D (PAP I), E (PAP II), and F (PAP III)
show bar graphs indicating the percent survival of PATP isoforms after
16 h of incubation. PAP was incubated with trypsin (stippled
bar); PAP isoforms were added to pancreatic juice activated by
enteropeptidase (lightly hatched bar), and PATP (trypsin
activated) was added to pancreatic juice activated by enteropeptidase
(darkly hatched bar). PATPs survival in pancreatic juice
(with low levels of PSP/reg and PAP) are taken from a
separate set of experiments.
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Fig. 5.
Members of the family of secretory thread
proteins show differential sedimentation. A, pancreatic
thread proteins were centrifuged at increasing g force as
follows: initially at 1,000 × g for 30 min at 4 °C,
then the supernatant fractions were transferred and centrifuged at
10,000 × g for 30 min at 4 °C, and finally at
100,000 × g for 30 min at 4 °C. The supernatant and
the pellet fractions were dissolved in sample buffer, and proteins were
resolved by SDS-PAGE. The bands were analyzed by densitometry and
expressed as percent of the total (pellet fractions plus the
supernatant fraction). The top panel shows the section of
the gel used for densitometric analysis. B, pH-independent
solubility of PATP II. PAP II was digested with trypsin, acidified to
pH 3, and neutralized by buffer exchange (AN). Aliquots were
then mixed with buffers of various pH and centrifuged. Proteins in the
pellet and supernatant were resolved by SDS-PAGE. M, markers
at 14 and 18 kDa.
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Fig. 6.
Trypsin activation of purified recombinant
secretory stress proteins generates a matrix of highly organized
fibrils. Scanning electron microscopy of PTP and PATP generated
in vitro. The micrographs demonstrate fibrous networks
following activation of PAP I, PAP III and PSP/reg with
trypsin. A and D show the matrix obtained with
PATP I (A, bar 70 µm; D, 3 µm).
B and E show the matrix obtained with PATP III
(B, 20 µm; E, 7 µm). C and
F show the matrix obtained with PTP (C, 40 µm;
F, 4 µm). Inset in B gives a higher
magnification micrograph showing individual fibrils that emerge from
the dense matrix. A and C, part of the plastic
mesh of the pouch can be seen.
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Fig. 7.
High resolution structural analysis of
activated secretory stress proteins by transmission electron
microscopy. A, scanning transmission electron
micrograph of the PTP matrix (bar, 100 nm). B and
C, negative staining of PTP (B) and PATP III
(C), original magnification × 71,000, bar
200 nm; D and E, thin section of PTP
(D) and PATP III (E) matrix embedded in low
temperature Unicryl (original magnification × 180,000, bar 70 nm). Structural analysis by each of these methods
indicate filamentous structures with diameters approximating 20 nm.
High resolution analysis of PATP III in D and E
suggests that protein subunits are organized in helical configurations.
Favorable sections in this panel suggest the presence of cavities
within these filamentous structures (arrowheads).
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Fig. 8.
Trypsin activation of 14-kDa proteins in
pancreatic juice generates a matrix of highly organized fibrils.
Pancreatic juice was incubated with trypsin and then transferred to a
plastic pouch for fixation and drying. A and C,
pancreatic juice from a WBN/Kob rat with high levels of
PSP/reg (150 µg/ml) and PAP (50 µg/ml); bars
70 and 3 µm, respectively. B and D, fibrils
generated in pancreatic juice from a Wistar rat with lower levels of
PSP/reg (14 µg/ml) and very low levels of PAP (1.4 µg/ml); bars 70 and 3 µm, respectively. A much smaller
area of the pouch was covered with fibrillar material when pancreatic
juice from Wistar rats was activated, and these fibrils are decorated
with protein droplets in contrast to the "pure" threads from the
WBN/Kob rat.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Höchli, R. Keller, H.-P. Gautschi, and Prof. Th. Bächi from the EM-Zentrallabor, University of Zürich, for excellent technical support and help in sample processing.
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FOOTNOTES |
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* This work was supported by the Amélie Waring Stiftung, the Hartmann Müller-Stiftung, and the Swiss National Science Foundation Grant 32-52661.97.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:
Pankreatitis-Forschungslabor, DL36, Sternwartstrasse 14, 8091 Zürich, Switzerland. Tel.: 411 255 2071; Fax: 411 255 4393;
E-mail: rgf@chi.usz.ch.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M010717200
2 The published sequence of rat PAP II contains an error: residue 79 of the mature secreted protein is glycine rather than tryptophan and should read WIGLH. This was verified by DNA sequencing and confirmed by mass spectroscopy analysis.
3 D. Bimmler, M. Scheisser, A. Lüssi, and R. Graf, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PSP/reg, pancreatic stone protein, regenerating protein; CTL, C-type lectin domain (Ca2+-dependent carbohydrate-recognition domain); PAP, pancreatitis-associated protein; PATP, pancreatitis-associated thread protein; PTP, pancreatic thread protein; SEM, scanning electron microscope; STEM, scanning/transmission electron microscope; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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