From the Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, California 94143-0911
Received for publication, September 12, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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In a search for genes encoding the serine
peptidases prostasin and testisin, which are expressed mainly in
prostate and testis, respectively, we identified a related, novel gene.
Sequencing of cDNA allowed us to deduce the full amino acid
sequence of the human gene product, which we term "pancreasin"
because it is transcribed strongly in the pancreas. The idiosyncratic
6-exon organization of the gene is shared by a small group of tryptic
proteases, including prostasin, testisin, and Serine proteases are a fertile family of hydrolases using the
side-chain hydroxyl group of a precisely positioned serine to attack
the carbonyl carbon of a target peptide bond (1). Despite this shared
enzymatic mechanism, serine proteases as a group exhibit a tremendous
range of target specificity. However, some members of the family
recognize and cleave a narrow range of target sequences and are limited
in vivo to hydrolysis of essentially one type of target. An
example is enteropeptidase, which is highly specific for pancreatic
trypsinogens. Some enzymes, like activated pancreatic trypsin itself,
are comparatively omnivorous, hydrolyzing the peptide bond of a broad
range of peptides and proteins at sites containing basic amino acids.
Other serine proteases cleave targets after aromatic, neutral
aliphatic, or acidic residues, but mammalian serine proteases
with tryptic specificity are particularly numerous and
variable in form and function. These include many familiar proteases
with roles in digestion, hemostasis, fibrinolysis, and activation of
complement (2). One of the more intriguing subgroups of tryptic serine
proteases includes prostasin (3-5), testisin (6-8), and This laboratory's interest in Data Base Screening--
Human Amplification and Cloning of Human and Mouse
cDNAs--
Human pancreasin DNA sequence predicted from EST and
genomic sequence was used to design PCR primer pairs, which were used to screen human tissue cDNA preparations for transcripts of the pancreasin gene. Selected amplimers were purified and sequenced by the
general methods described in prior work (10) to confirm the identity of
the fragments. A rapid amplification of cDNA ends (RACE) approach
(15) was used to obtain cDNA encoding additional 3' protein-coding
sequence of pancreasin. This was then used to design PCR primers
(5'-CCCAGCCAGGCCTGAGGACATGAGGCGGCC and
5'-AGGGTATTTGAGAGGGGAGGAAG) bracketing the full protein-coding
sequence. With these primers, a 1046-bp pancreasin cDNA was
amplified from human placental cDNA (Clontech,
Palo Alto, CA), gel-extracted, cloned into pCR2.1 vector (Invitrogen,
Carlsbad, CA), and sequenced. Determination of pancreasin cDNA
sequence permitted establishment of intron-exon splice sites in genomic
DNA encoding the pancreasin gene and generation of specific DNA probes
for blotting studies. Similar approaches were used to obtain cDNA
encoding mouse pancreasin. A PCR primer pair (5'-ATGAGGCAGCCCCACATCGCTGC and 5'-GCGGCCGCCTAGACGATCCTGAGCAGCAGTG) predicted from mouse 5'- and 3'-ESTs was used to amplify a 987-bp cDNA encoding a 328-residue, mouse prepropancreasin coding
sequence, including a carboxyl-terminal extension not predicted by the
human cDNA. This cDNA was obtained from reverse-transcribed
mRNA from the urinary bladder of an adult C57BL/6 mouse.
DNA and Protein Sequence Comparisons--
DNA sequencing was
conducted by University of California at San Francisco's Biomolecular
Resource Center using standard dideoxy techniques. DNA translation,
multiple sequence alignment and dendrograms were generated using
MacVector software (Oxford Molecular, Campbell, CA).
Molecular Modeling--
A homology model of the pancreasin
catalytic domain was constructed in part assisted by an automated
protein modeling tool and server (Swiss PDB Viewer and Swiss-Model,
respectively) (16). Propeptide sequence and the carboxyl-terminal 11 residues were excluded from the model. Coordinates of the
crystal-derived three-dimensional structure of human Gene Structure and Chromosomal Mapping--
Pancreasin cDNA
was used to query GenBankTM genomic sequence databanks to
identify genes with exons matching predicted cDNA sequence.
Intron-exon splice junctions in identified genomic sequence were
established using open reading frames and cDNA alignments by
application of the "5'-GT ... AG-3'" rule for initiating and ending introns, as in prior work from this laboratory (10, 18). Identified genomic sequence was mapped to a specific human chromosomal region through LocusLink (available at www.ncbi.nlm.gov/LocusLink).
mRNA Blotting--
To generate a pancreasin-specific probe,
a 440-bp fragment of pancreasin cDNA was obtained from human
pancreatic cDNA by reverse transcriptase-PCR using the following
primer pair: 5'-GCAAAGACACCGAGTTTGGCTAC and
5'-AGGGTATTTGAGAGGGGAGGAAG. Blots containing purified,
electrophoresed mRNA from a variety of human tissues
(Clontech) were hybridized with the
32P-labeled, 440-bp pancreasin cDNA fragment, then
subjected to autoradiography. The same blots were stripped and probed
with radiolabeled Antibody Generation--
Rabbit polyclonal antisera were raised
against synthetic peptides based on portions of predicted amino acid
sequence corresponding to hypothesized catalytic domain surface loops
(see results under "Molecular Modeling"). Two peptides were
synthesized (CRNTSETSLYQVLLG and CGYQKPTIKNDMLCA) containing residues
78-91 and 202-214, respectively, of prepropancreasin. Both peptides
were conjugated via the amino-terminal cysteines to keyhole limpet
hemocyanin and injected into rabbits. Resulting antisera were screened
and titered by enzyme-linked immunoadsorbent assay. Peptide synthesis,
conjugation, immunizations, bleeding, and titering assays were
conducted by GeneMed Synthesis (South San Francisco, CA). The IgG
fraction of rabbit immunoglobulins was purified from delipidated
antisera on a HiTrap Protein A HP column (Amersham Biosciences,
Piscataway, NJ). Column-bound IgG was eluted using glycine-HCl (0.1 M, pH 2.7).
Cell Culture--
The human pancreatic ductal carcinoma cell
line HPAC (19) was obtained from the American Type Culture Collection
(Manassas, VA) and cultured according to the vendor's recommendations
in medium containing 95% of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium with 1.2 g/liter
NaHCO3, 15-mM HEPES, 2 mg/liter insulin, 5 mg/liter transferrin, 40 µg/liter hydrocortisone, and 10 µg/ml
epidermal growth factor, and 5% fetal bovine serum. Chinese hamster
ovary (CHO) cells were grown in Ham's F-12 medium supplemented with
10% fetal bovine serum.
Immunocytochemical Analysis of Pancreatic Ductal Carcinoma
Cells--
HPAC cells were harvested by trypsinization, washed and
suspended in PBS, and centrifuged onto glass slides. Slides were
air-dried, immersed in methanol followed by acetone (20 min at
Expression of Recombinant Pancreasin in CHO
Cells--
Pancreasin cDNA cloned into pCR 2.1 TOPO T/A vector
served as a template for further constructs. Pancreasin lacks a
traditional nucleotide sequence (20) bracketing the initiator
methionine ATG. Therefore, to boost the prospects of heterologous
expression in CHO cells, the wild-type sequence 5'-GACATGAA was
replaced with optimized sequence
5'-GCCATGGG and incorporated into a PCR forward primer
(5'-ACAACTAATTATTCGAAACGAGGAATTCGCCATGGGGCGGCCGGCGGCGGTGCCG) into
which an EcoRI restriction site also was introduced to
facilitate further cloning. The 3' region of the pancreasin cDNA
was also modified to encode a carboxyl-terminal histidine fusion tag to ease purification. This modification was achieved by replacing the
native stop codon with 9 histidine codons followed by a new stop codon
and an introduced NotI restriction site in a reverse primer
(5'-GTTCGGGCCCAAGCTGGCGGCCGCTCAGTGATGATGGTGATGATGGTGATGATGCTTCTGGCCGCCCAACCTCG). Pancreasin cDNA was amplified by PCR from the pCR2.1-pancreasin template with these modified primers using the following conditions: 95 °C for 10 min, 95 °C for 30 s, 60 °C for 30 s,
and 72 °C 1 min, for 35 cycles. The resulting
His9-tagged pancreasin amplimer was trimmed with
EcoRI and NotI and ligated into similarly
restricted pcDNA3.1 (Invitrogen). In preparation for transfection
with pcDNA3.1-pancreasin, CHO cells were plated in a six-well dish
at a density of 2.5 × 105 cells per well. The cells
were transfected by exposure to 5 µg of plasmid DNA per well plus
LipofectAMINE 2000 (Invitrogen), according to the manufacturer's
protocol. After 48 h of recovery, cells were aliquoted into 10-cm
dishes and cultured for 1 week in the presence of 400 µg/ml G418
(Calbiochem, San Diego, CA) to select for transfected cells. Surviving
colonies were pooled and incubated overnight in 175-cm2
flasks without G418 in the same medium, which was then exchanged for
low protein Opti-MEM I medium (Invitrogen). After 3 days, supernatants
were harvested and used for purification of recombinant, His9-tagged pancreasin.
Purification of Recombinant Pancreasin Expressed by CHO
Cells--
Medium conditioned by pancreasin-transfected CHO cells was
dialyzed against PBS. Imidazole was added to a concentration of 10 mM, and the resulting mixture was shaken overnight at
4 °C with a slurry of nickel-nitrilotriacetic acid-agarose beads
(Qiagen, Valencia, CA). After washing and equilibration with PBS
containing 10 mM imidazole, beads were poured into a column
and washed first with 10 column volumes of PBS containing 10 mM imidazole and 0.3 M NaCl and then
successively with three column volumes of PBS/0.3 M NaCl
containing 30 and 100 mM imidazole, respectively. Residual bound protein was eluted from the beads with PBS/0.3 M NaCl
containing 1 M imidazole. Aliquots of eluted fractions were
assayed for pancreasin immunoreactivity and peptidase activity and
subjected to SDS-PAGE.
Immunoblotting--
Aliquots of cell supernatants or were
subjected to reducing, gradient SDS-PAGE, and transferred to
polyvinylidene difluoride membrane (Immun-Blot, Bio-Rad, Hercules, CA),
which was washed and hybridized for 1 h with rabbit
anti-pancreasin IgG diluted 1:1000 in Tris-HCl (50 mM, pH
7.5) containing 0.5 M NaCl, 0.1% Tween 20, and 5% nonfat
milk. The blots were washed, incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa
Cruz, CA) diluted 1:2500 in the same buffer, and washed again.
Immunoreactive bands were visualized by enhanced chemiluminescence
using Lumiglo reagents (Cell Signaling Technology, Beverly, MA) and
Hyperfilm (Amersham Biosciences, Arlington Heights, IL).
Peptidase Activity of Recombinant Pancreasin--
Highly
immunoreactive, purified fractions eluted from the nickel bead column
were assayed for amidolytic activity using tryptic, chymotryptic, and
elastolytic peptidyl 4-nitroanilide (NA) substrates. These included
benzoyl-L-Arg-NA, tosyl-Gly-L-Pro-Arg-NA,
tosyl-Gly-L-Pro-Lys-NA, D-Pro-L-Phe-Arg-NA,
benzoyl-L-Lys-Gly-Arg-NA,
succinyl-L-Ala-Ala-Pro-Phe-NA, succinyl-L-Phe-Pro-Phe-NA, and succinyl-Ala-Ala-Pro-Val-NA.
All peptidyl NA substrates were obtained from Sigma, except for
benzoyl-L-Lys-Gly-Arg-NA, which was prepared as described
previously (21). All assays were conducted at 37 °C in Tris-HCl (20 mM, pH 7.0) containing 2-mM substrate.
Appearance of free nitroaniline was monitored at 405 nm using a
kinetic, 96-well microplate spectrophotometer (Thermomax, Molecular
Devices, Sunnyvale, CA) at 37 °C. For comparison, pancreatic trypsin
(bovine TPCK (L-1-tosylamido-2-phenylethyl chloromethyl
ketone)-treated; Sigma) was assayed in similar fashion. Susceptibility
to potential inactivators, including soybean trypsin inhibitor,
aprotinin, benzamidine, p-amidinophenylmethanesulfonyl fluoride, tosyl-L-Lys-chloromethylketone,
D-Phe-L-Pro-Arg-chloromethylketone, leupeptin,
E-64, pepstatin, and EDTA, was assessed using
tosyl-Gly-L-Pro-Arg-NA as above after 30 min of
preincubation of pancreasin with protease inhibitor.
cDNA and Deduced Amino Acid Sequence of
Prepropancreasin--
Screening of human and rodent EST databases in
GenBankTM revealed several cDNAs encoding fragments of
human (e.g. AI272325, AA321681, and AA368960) and murine
(e.g. AI070303, BB627930, and BB11542) homologues of
prostasin and
The primary structure of pancreasin's prosequence and proximal
catalytic domain, including Arg-34 and idiosyncratic Met-35, is
supported by the sequences predicted for chimpanzee and mouse pancreasin (Fig. 2), as deduced from
genomic DNA and cDNA, respectively. Furthermore, rat EST BF551850,
which encodes the amino terminus of putative rat pancreasin and is 77%
identical (49 of 64 amino acid residues) to human pancreasin in the
region of overlap, also contains these features. This suggests that
mammalian pancreasins are activated by tryptic hydrolysis at Arg-34 and
that Met-35, although apparently unique among serine proteases, is a
conserved and possibly essential feature of pancreasins. As expected of a catalytically competent serine protease, the pancreasin catalytic domain possesses all three of the essential "catalytic triad" residues (using standard chymotrypsinogen numbering: His-57, Asp-102, and Ser-195) conserved in all serine proteases, as well as an aspartate
at the base of the primary specificity pocket in position 189, found in
all proteases of tryptic specificity (Fig. 2). This is consistent with
pancreasin's observed cleavage site preference for substrates with P1
arginine (see below). Because pancreasin is predicted to be activated
by tryptic hydrolysis at Arg-34 and is itself a tryptic enzyme, it may
catalyze its own activation. This possibility is consistent with the
finding of active pancreasin in medium conditioned by transfected CHO
cells. However, the actual site of activation (intracellular
versus extracellular) and the mechanism (autoactivation
versus other) remain to be established. Pancreasin may be
secreted initially as a zymogen, where it also could be activated in
the pancreatic ductal lumen by trypsin or glandular kallikrein. The
predicted site of propeptide hydrolysis at Arg-34 is not preceded by
the series of aspartate residues required for recognition by
enteropeptidase, which, therefore, is unlikely to activate
propancreasin. The secreted material would be in a position to interact
with other proteins in pancreatic secretions and with potential protein
targets on the apical surface of epithelial cells lining the pancreatic
ducts.
Two consensus N-linked glycosylation sites,
which lie in the catalytic domain (see Figs. 1 and 2), predict that the
mature, active enzyme is glycosylated. Results of SDS-PAGE and
immunoblotting of recombinant pancreasin support this prediction, as
discussed below. The pancreasin cDNA open reading frame predicts 11 cysteines in the preproenzyme, one of which is in the signal peptide
and therefore will be unavailable to participate in disulfide linkages in the mature protein. Based on alignments with trypsin and other serine proteases with Cys-Cys pairings established in crystal-derived tertiary structures, each pancreasin Cys can be paired with another. Thus, no unpaired cysteines are expected to be available to form intermolecular disulfide linkages. By analogy to chymotrypsinogen, Cys-26 of the propeptide is linked to Cys-144 in the catalytic domain.
This predicts that the propeptide of pancreasin, like that of mature,
active chymotrypsin, remains attached to the catalytic domain after
hydrolysis of Arg-34. Predicted specific Cys-Cys pairings for
pancreasin are shown in Fig. 1C.
The prepropancreasin open reading frame ends in a stop codon in a
position that is 11 residues beyond that of the corresponding carboxyl-terminal region of soluble Relationship to Other Serine Proteases--
Searches of protein
sequence databases with full-length human pancreasin deduced as the
query sequence reveal homology with several published and partly
characterized proteins, the most closely related of which are the
Xenopus epidermis-specific protease xepsin (26) and
embryonic serine protease-1 (27), mammalian prostasins (4),
Xenopus channel-activating protease (24), mouse distal
intestinal serine protease (DISP) (25), and mammalian Molecular Model--
The homology model constructed from human
Location and Organization of the Pancreasin
Gene--
Interrogations of GenBankTM with query sequences
based on pancreasin cDNA identified highly homologous sequences in
a 40.2-kb cosmid clone of human genomic DNA (accession number
AC004036). This clone is part of a contig localizing to chromosome 16p,
the same chromosomal arm containing Tissue Expression of Pancreasin mRNA--
As shown in Fig.
6, hybridization of a pancreasin-specific
probe with blotted mRNA from multiple tissues reveals a strong signal from the pancreas but not from any other tissue surveyed. However, more sensitive reverse transcriptase-PCR-based screening identifies the predicted 440-bp transcript in several other tissues, of
which lung and placenta are strong (Fig.
7). The same PCR primers also yield
pancreasin-derived amplimers from HPAC pancreatic carcinoma cells (Fig. 7). However, cDNA from the MRC5 line of human
fibroblasts does not yield the amplimer (not shown), reinforcing the
selectivity of pancreasin expression predicted by the mRNA blotting
and reverse transcriptase-PCR findings.
Expression of Pancreasin Protein in Pancreatic Carcinoma
Cells--
As shown in Fig. 8,
antibodies raised against synthetic pancreasin peptides reveal
predominantly cytoplasmic reactivity in HPAC cells subjected to
immunocytochemical analysis. Consistent with predictions from
hydropathy analysis showing that the mature protein is not
membrane-anchored, there is no strong staining of the cell surface.
Some of the cells exhibit eccentric, perinuclear immunoreactivity,
consistent with the presence of pancreasin in the Golgi apparatus or
endoplasmic reticulum. Thus, pancreasin transcripts are translated into
protein in a cell line derived from the tissue in which pancreasin
expression is highest, based on the survey of tissue mRNA levels
shown in Fig. 6.
Properties of Recombinant Human Pancreasin--
As shown by the
immunoblot in Fig. 9, CHO cells
transfected with His9-tagged human pancreasin
express a 40- to 41-kDa protein that binds strongly to our polyclonal
anti-serum raised against synthetic pancreasin peptides. This
immunoreactive band was detected in pancreasin-transfected cells but
not in control cells transfected with empty vector (not shown),
suggesting that CHO cells do not natively express detectable amounts of
pancreasin. Immunoreactive pancreasin is detected primarily in
conditioned medium rather than in cell extracts, suggesting that
recombinant human pancreasin is secreted by transfected CHO cells and
not stored. The ~9-kDa reduction in size of the recombinant
pancreasin band achieved by incubation with peptide
N-glycosidase F indicates that pancreasin is
N-glycosylated, likely at both of the predicted sites given the magnitude of the size reduction. The size of native pancreasin (without the His9 tag) should be slightly smaller than that
of the recombinant enzyme, assuming equivalent levels of
glycosylation.
Substrate preferences of pancreasin in comparison with trypsin are
shown in Fig. 10A. Based on
hydrolysis of a sampling of peptidyl-NAs, pancreasin is more selective
than trypsin in the types of tryptic substrates it hydrolyzes. However,
like trypsin, it has no chymotryptic or elastolytic activity. The best
substrate is tosyl-Gly-L-Pro-Arg-NA, which is also a good
substrate for human
As shown in Fig. 10B, recombinant human pancreasin rather
remarkably resists inactivation by large molecular mass inhibitors such
as aprotinin and soybean trypsin inhibitor, which effectively inhibit
pancreatic trypsin. Although broad resistance to proteinaceous inhibitors is rare among characterized mammalian serine proteases, it
is a feature of
In conclusion, our data reveal the gene, cDNA, predicted protein
structure, and activity profile of a novel, secreted serine protease
expressed in several tissues but most strongly in the pancreas.
Characterization of recombinant pancreasin reveals an active,
inhibitor-resistant peptidase with a preference for hydrolysis of
peptide substrates after arginine residues.
-tryptase. Like the
other genes, the pancreasin gene resides on chromosome 16p. Pancreasin
cDNA predicts a 290-residue, N-glycosylated, serine
peptidase with a typical signal peptide, a 12-residue activation
peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic
domain. Unlike prostasin and other close relatives, human pancreasin
and a nearly identical chimpanzee homologue lack a carboxyl-terminal
membrane anchor, although this is present in 328-residue mouse
pancreasin, the cDNA of which we also cloned and sequenced. In
marked contrast to prostasin, which is 43% identical in the catalytic
domain, human pancreasin is transcribed strongly in pancreas (and in
the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at
all in kidney and prostate. Antibodies raised against pancreasin detect
cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and
secreted as an active tryptic peptidase. Pancreasin's preferences for
hydrolysis of extended peptide substrates feature a strong preference
for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by
benzamidine and leupeptin but resists several classic inhibitors of
trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the
pancreas with novel physical and enzymatic properties. These studies
provide a rationale for exploring the natural targets and roles of this enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-tryptase
(9, 10). These enzymes are tryptic in specificity (i.e.
prefer arginines and lysines in target peptides) and are synthesized
with a distinctive carboxyl-terminal peptide or glycosylphosphatidyl
inositol membrane anchor. Subsequently, they may be released from their
anchor and secreted. The genes of these three enzymes share an
idiosyncratic organization of introns and exons and reside on the short
arm of chromosome 16 (5, 10, 11). However, they differ widely in
dominant tissue pattern of expression: i.e. kidney and
prostate (prostasin) (3, 4, 12), eosinophils, testicular germ cells and
sperm (testisin) (6, 8, 13), and airway and gut mast cells
(
-tryptase) (9, 10). The functions of these proteases are being
actively investigated. In the case of prostasin, one likely role that
has emerged is regulation of transmembrane ion flux via epithelial sodium channels (14). This non-classic regulatory role for one member
of the prostasin subgroup of tryptic mammalian serine proteases hints
that we can expect unconventional roles for other members of the subgroup.
-tryptase and prostasin (10) led us
to seek genes and transcripts encoding related enzymes in the human
genome. As detailed below, our search identified a new family member,
which we term "pancreasin" because it appears to be predominantly
transcribed by pancreatic tissue as well as by a cell line derived from
pancreatic ductal epithelium. The pancreasin gene shares the
idiosyncratic gene structure of human prostasin, testisin, and
-tryptase and resides like the others on chromosome 16p.
Furthermore, recombinant expression reveals that it is a catalytically
competent, tryptic peptidase, and proteinase. However, its substrate
preferences and inhibitor profile are unique and, unlike its closest
relatives, it is synthesized and secreted without a membrane anchor.
The distinct patterns of expression, catalytic, and structural features
predict that pancreasin's functions are distinct from those of its
closest known relatives.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-tryptase and prostasin
cDNA sequence and Basic Local Alignment Search Tool (available at
www.ncbi.nlm.nih.gov) algorithms were used to query human expressed
sequence tag (EST)1 and
genomic sequence databases in GenBankTM. Iterative searches
using identified individual human EST sequences were used to confirm
and extend sequence derived from a given "hit" and to arrive at a
consensus sequence. Predicted human cDNAs corresponding to a novel
-tryptase/prostasin homologue identified in this manner were used to
interrogate non-human EST databases to identify murine homologues.
II-tryptase
(Protein Data Bank number 1AOL) (17), which is pancreasin's closest
relative for which diffraction data are available, served as template
for the model, which was optimized by idealizing bond geometry and
removing unfavorable contacts.
-actin to control for differences in mRNA loading.
20 °C for each solvent), rinsed with PBS, then incubated for
1 h with blocking solution containing 5% nonfat dry milk, 3%
normal goat serum, 0.1% Triton X-100, and 1% glycine in PBS. Blocked
slides were incubated overnight at 4 °C with various dilutions of
rabbit preimmune IgG or anti-pancreasin IgG, washed with PBS containing
0.05% Tween-20, incubated for 1 h with fluorescein-conjugated
goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA), and washed
again with PBS/Tween 20. Slides were coverslipped in the presence of
Vectashield medium (Vector Laboratories) and imaged by fluorescence microscopy.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-tryptase that have not been described previously.
Human EST-based primers were used to obtain more complete protein
coding sequence of a cDNA encoding a novel protein, which we term
pancreasin based on the features of the predicted
translation product, as described below. Comparisons with partial
sequence AX001350 predict that the pancreasin transcript contains a
5'-untranslated region (UTR) of at least 198 nucleotides. 3'-RACE
reveals a 3'-UTR of 571 nucleotides containing a polyadenylation signal
and poly(A) tail (Fig. 1).
The cDNA encodes a predicted 290-amino acid preproprotease and is
highly similar to the sequence of a predicted gene product termed
marapsin (GenBankTM AJ306593). The deduced amino acid
sequence of prepropancreasin/marapsin begins with a typical 22-amino
acid signal peptide, which predicts that the nascent protein is
directed initially to the endoplasmic reticulum and, like most serine
proteases, is secreted outside of the cell. Indeed, as discussed below,
the behavior of recombinant prepropancreasin expressed in CHO cells
supports this prediction. Immediately following the signal peptide is a
12-residue pro- (activation) peptide ending in Arg, followed by a
256-amino acid serine protease catalytic domain. The amino-terminal
residue of the predicted mature protease after propeptide hydrolysis
at Arg-34 is methionine, which appears to be unique among
trypsin family serine proteases, the great majority of which have an
isoleucine at this position. The amino-terminal residue dives into the
hydrophobic interior of mature proteases, allowing the positively
charged
-amino moiety to form a salt bridge with the negatively
charged carboxylate side chain of a highly conserved aspartate (residue 228 of prepropancreasin), thereby bringing the residues involved in
catalysis into productive alignment. Studies of trypsin involving mutation of the residue equivalent to pancreasin's Met-35 suggest that
activity of the mature enzyme is preserved with a variety of amino
acids containing aliphatic side chains (i.e. isoleucine, valine, and alanine) at that position, although methionine itself was
not examined (22). Our finding that recombinant pancreasin is
catalytically active as a tryptic protease indicates that the amino-terminal methionine side chain is tolerated in the binding pocket, although perhaps with assistance from structural accommodations peculiar to pancreasin. Methionine's presence in this critical position could render pancreasin susceptible to oxidative modification to the sulfoxide, especially in the zymogen form, in which the Met-35
side chain should be more exposed at the protein surface than in the
enzyme's mature, active conformation.
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Fig. 1.
Human pancreasin predicted primary structure
and post-translational processing. A, pancreasin cDNA
and deduced amino acid sequence, beginning with the predicted initiator
methionine and ending with the poly(A) tail. Predicted signal and pro
(activation) peptides are italicized and
underlined, respectively. The "catalytic triad" residues
common to all active serine proteases are boxed. The 3'-UTR
continues after the stop codon until the polyadenylation site, which is
preceded by a conventional polyadenylation signal
(underlined). B, compares results of Janin
hydrophobicity analysis (as implemented in MacVector) of the
290-residue human prepropancreasin amino acid sequence with that of the
predicted 328-residue mouse enzyme. Although both proteases contain an
amino-terminal hydrophobic sequence typical of a signal peptide, only
the substantially longer mouse protein contains a carboxyl-terminal
hydrophobic sequence (arrow). The mouse pancreasin
carboxyl-terminal hydrophobic sequence is similar to that found in
prostasin and other close relatives. C, the predicted
structure of mature, processed human pancreasin. The cDNA sequence
predicts that pancreasin is translated initially as a single-chain,
290-amino acid precursor with a signal peptide (Pre),
propeptide (Pro), and catalytic domain. We predict that the
signal peptide is removed co-translationally in the endoplasmic
reticulum, leaving a proenzyme, which is activated subsequently by
hydrolysis of the propeptide segment at Arg-34, leaving a 256-residue catalytic domain (heavy
chain) that remains attached to the propeptide segment (light chain)
via a disulfide linkage involving Cys-26 and Cys-144, as shown. The
locations of other Cys-Cys pairs are shown, by analogy to pairings
involving homologous cysteines in trypsin. Consensus
N-linked glycosylation sites are found in two positions,
Asn-55 and Asn-79. Also shown are positions of the "catalytic
triad" residues found in all active serine proteases (His-75,
Asp-124, and Ser-229, corresponding to His-57, Asp-102, and Ser-195
using standard chymotrypsinogen numbering) and specificity-determining
Asp-223, which is characteristic of trypsin-family serine proteases
with specificity for lysine and arginine in peptide and protein
targets.
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Fig. 2.
Comparison of primary structure of pancreasin
and homologues. This figure aligns the deduced amino acid sequence
of human, chimpanzee, and mouse pancreasin and three of their closest
relatives: Xenopus epidermal protease xepsin (AB018694) and
Xenopus embryonic serine protease (ESP)-1, along with human
-tryptase. Chimpanzee pancreasin cDNA and amino acid sequence
was deduced from GenBankTM-deposited draft genomic sequence
(AC097329.1) and is 98% identical in amino acid sequence to the human
preproenzyme. Mouse pancreasin amino acid sequence was deduced from
cloned and sequenced cDNA and is 80% identical to human pancreasin
in overlapping catalytic domain. Amino acids identical in all six
proteases are marked with an asterisk (*); residues that are
similar but not identical in the proteases are marked with a
period (.). The predicted amino terminus of the mature
catalytic domain, after tryptic hydrolysis at an arginine residue
conserved in all of these proteases, is marked with a plus
sign (+). Predicted N-glycosylation sites in
pancreasins are underlined. Residues constituting the
"catalytic triad" common to all serine proteases are in
boldface. The key aspartate conferring tryptic specificity
is underlined and in boldface. Note that mouse
pancreasin, as well as the two frog proteases and
-tryptase, contain
hydrophobic, carboxyl-terminal extensions compared with the primate
pancreasins, which therefore appear to differ from the other enzymes in
not being synthesized in a membrane-anchored form. Xepsin's
carboxyl-terminal extension has been truncated for clarity.
-tryptases (23). However, this
carboxyl-terminal extension is much shorter and less hydrophobic (see
Fig. 1B) than the putative membrane-anchoring
carboxyl-terminal domains of otherwise similar channel-activating
protease (24), prostasin (4), testisin (6),
-tryptase (10), and
distal intestinal serine protease (25). Hydropathy analysis (Fig.
1B) suggests that the pancreasin carboxyl-terminal extension
is too short and hydrophilic to form a membrane-spanning helix. Our
finding of secretion of pancreasin from CHO cells further supports the predicted lack of a membrane-anchoring segment in the human enzyme. Interestingly, the predicted carboxyl-terminal extension of mouse prostasin (see Fig. 2) is 35 amino acid residues longer than that of the human enzyme. Hydropathy analysis (Fig.
1B) predicts that the mouse extension does form a
transmembrane anchor. Similarly, the amino acid sequence deduced from a
set of overlapping rat ESTs (AI070303, AI716503, and AI575237) encoding
the carboxyl-terminal portion of putative rat pancreasin is 78%
identical (87 of 111 of overlapping amino acid residues) to human
pancreasin in this region. Nonetheless, like the mouse sequence, it
contains a hydrophobic open reading frame that extends 35 amino acid
residues beyond that of the human sequence. Thus, pancreasins in
rodents may contain a membrane anchor, even if primate pancreasins do not. The presence of a membrane anchor could greatly influence protease
function by limiting the spectrum of targets to proteins that are in
the plasma membrane or directly in contact with it. Without an anchor,
human pancreasin may diffuse away from its cell of origin to reach more
remote targets. In this regard it should be noted that some proteases,
e.g. prostasin, thought to be synthesized initially with a
membrane anchor subsequently might be solubilized by cleavage of the
anchor at the membrane surface (4).
-tryptases (9,
10). As shown in Fig. 3, dendrograms
prepared from alignments of pancreasin catalytic domain alone (to avoid the biasing effects of comparing proteases with and without available preprosequence and carboxyl-terminal extension) continue to reveal strong homology with xepsin and embryonic serine protease, which are of
unknown function in frogs. However, the most closely related catalytic
domain is that of brain-specific protease-2, a partially sequenced
serine protease of unknown function from rat hippocampus (28). Rat
brain-specific protease-2 is highly similar to the predicted protein
product of an uncharacterized human gene (SP001LA, predicted from
genomic sequence; GenBankTM accession number AC003965),
which is distinct from the pancreasin gene, but which also maps to
chromosome 16. Furthermore, as noted, the partial sequence of a much
more closely related rat gene product is predicted from EST libraries.
Thus, pancreasin and brain-specific protease genes are not orthologous.
However, it is possible that xepsin or embryonic serine protease-1 is
pancreasin's orthologue in frogs. Despite a number of shared features
(including gene structure, as discussed below), the
-tryptases,
DISP, channel-activating proteases, prostasins, and testisins are less
closely related to pancreasin and therefore are less likely to serve
similar functions.
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Fig. 3.
Dendrogram of pancreasins and related
proteases. The amino acid sequence of the catalytic domains of
human, chimpanzee, and mouse prostasin and its closest relatives were
subjected to tree analysis using the unweighted pair group with
arithmetic mean multiple sequence alignment algorithm in MacVector 7.1. Prepropeptides and carboxyl-terminal extensions were excluded from the
alignment to limit distortions in phylogenetic distance created by
variations in length of sequence on either side of the catalytic
domain. The length of each branch of the tree is proportional to the
fraction of mismatched amino acids in pairs of aligned sequences. The
pancreasin branch is depicted in heavy black lines. The
nearest relatives to pancreasins are rat brain-specific protease (BSP)
2 (an uncharacterized, partially sequenced gene product of unknown
function), two additional Xenopus proteases, xepsin, and
embryonic serine protease (ESP)-1. The "tryptase" group (which
includes mouse distal intestinal protease, DISP, as well as -,
-,
and
-tryptases) forms a separate branch, as does the prostasins and
testisins. The sequence of human pancreatic trypsin is included as an
outlier from this otherwise fairly closely related collection of
proteases. Pancreasins are unlikely to be orthologues of rat BSP-2
because: (a) the magnitude of mismatch is too great,
(b) ESTs suggest the existence of rat proteins even more
closely matched to Pancreasin, and (c)
GenBankTM-deposited human genomic sequence contains a gene
that is a better candidate as a BSP-2 orthologue. The
GenBankTM accession numbers of the sequences used to
generate this tree are as follows: human pancreasin (AY030095), chimp
pancreasin (AC097329), mouse pancreasin (BB627930 and BB11542), rat
BSP-2 (AJ005642), frog xepsin (AB018694), frog embryonic serine
protease, ESP-1 (AB038496), human
-tryptase (AF191031), mouse
-tryptase (AF175760), mouse DISP (AJ243866), human
-tryptase
(M30038), human
I-tryptase (M33491), mouse tryptase-1/MCP-6
(M57626), human prostasin (L41351), mouse prostasin (BC003851), frog
channel-activating protease, CAP (AF029404), human testisin (AF058300),
mouse testisin (AY005145), and human trypsin (M22612).
II-tryptase as a starting point is shown in Fig.
4. This model predicts that the topography of charged and uncharged amino acids in the vicinity of the
substrate binding and catalytic sites is unique compared with that of
tryptase and other serine proteases. Because binding of potential
substrates and inhibitors involves contacts with amino acid side chains
in this region, the differences between pancreasin and other proteases
predict differences in substrate specificity and inhibitor
susceptibility. On the other hand, the position of pancreasin Asp-223
in the model predicts that the enzyme's primary specificity
(i.e. its preference for the P1 residue on the
amino-terminal side of the scissile bond) is for basic residues, as was
found to be true of the recombinant enzyme (see below). The model also
shows that both consensus N-linked carbohydrate attachment
sites lie fairly close to the binding site of the P' (carboxyl-terminal) side of peptide substrates. Therefore, attached sugars may narrow the spectrum of potential substrates and inhibitors by impeding access to the active site. Due to a modest excess of amino
acids with acidic side chains over those with basic side chains, the
pancreasin catalytic domain is predicted to be acidic with a net charge
at pH 7 of approximately
3, not counting any additional negative
charge contributed by N-linked carbohydrates. However, if
the complete propeptide (which contains four basic residues) remains
attached to the mature enzyme, the net charge of the two-chain complex
is +1, and thus is slightly basic. Although the mature enzyme is not
predicted to be strongly cationic, the model suggests that there are
patches of positive charge that could bind to polyanions such as
heparin and related glycosaminoglycans and proteoglycans.
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Fig. 4.
Homology model of pancreasin. A
model of the predicted pancreasin catalytic domain was generated
starting from the crystallographically derived structure of human
II-tryptase. The propeptide and the carboxyl-terminal 11 residues of
pancreasin were omitted because they have no counterpart in the
tryptase structure. In the model shown, the catalytic triad residues
(His-75, Asp-124, Ser-229) in the active site are red, basic
residues (lysine and arginine) are blue, acidic residues
(aspartate and glutamate) are green, and predicted
N-linked carbohydrate (CHO) attachment sites (Asn-55 and
Asn-89) are cyan. Note that sugars (not shown) attached to
Asn-55 and Asn-89 could influence access and binding of substrates in
the active site. The ribbon structure shows side chains of
the catalytic triad residues and putative N-linked
asparagines. "Front" views show the active site face one, with the
extended substrate-binding site oriented roughly vertically. "Side"
views depict the active site binding cleft in profile, with the
carbohydrate attachment sites to the left. The distribution
of surface side chains of basic and acidic residues suggests patches of
positive charge, despite an overall excess of residues with acidic side
chains.
-tryptase (TPS1),
-tryptases (e.g. TPSB1),
-tryptase
(TPSG1), and testisin (PRSS21) genes (7, 9, 10,
18, 29). The pancreasin gene appears to reside on the centromeric side
of the tryptase locus and on the telomeric side of the testisin locus
(i.e. between the two), although there are persistent
ambiguities in mapping data in this region. The predicted organization
of the pancreasin gene is shown in Fig. 5
and compared with that of related proteases. Its most distinctive feature is distribution of the "prepro" coding segments among three
different exons, including exon 2, which is only 27 bp. This pattern,
including the phase and placement of the first intron and the small
size of the second exon, is described only in genes encoding
pancreasin's close relatives, such as prostasin (5),
-tryptase
(10), testisin (7), and DISP (25). Pancreasin's first, third, and
fifth introns are large compared with those of its relatives, due, in
part, to insertion of Alu repetitive sequences not present
in the other genes (Fig. 5).
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Fig. 5.
Organization of human pancreasin and related
genes. Exons of human pancreasin, prostasin, testisin,
-tryptase, and
/
-tryptase genes are numbered and
indicated by boxes. Untranslated regions, prepropeptides,
catalytic domains, and carboxyl-terminal membrane anchors are
designated by open, downward hatched,
black, and upward hatched boxes, respectively.
Lines represent introns and flanking regions. The locations
of codons encoding the His (H), Asp (D), and Ser
(S) catalytic triad residues are indicated. The phase of
each intron (0, I, or II) is shown.
Note the similarity in phase and placement of introns in pancreasin,
prostasin, testisin, and
-tryptase genes, each of which contains a
preprosequence divided among three exons, which is a distinctive
feature of this group of protease genes, but not in genes encoding
/
-tryptases. Pancreasin's first, third, and fifth introns are
large compared with the other genes. The third and fifth introns
contain an Alu-type repetitive element not present in the
corresponding intron in the other genes. The prostasin, testisin, and
-tryptase genes each contain an extended 3'-open reading frame
encoding a putative transmembrane segment and small cytoplasmic tail.
This transmembrane segment is not present in human pancreasin or
/
-tryptases. These findings suggest close evolutionary
relationships among these genes.
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Fig. 6.
Blotting of tissue mRNA.
Electrophoresed poly(A) mRNA from multiple human tissues was
transferred to nitrocellulose, hybridized at high stringency with a
radiolabeled probe prepared from a 440-bp portion of pancreasin
cDNA, then subjected to autoradiography (upper panels).
After stripping, the blot was hybridized with a radiolabeled actin
probe, which serves as a control for mRNA loading and integrity
(lower panels). With the pancreasin probe, a major band is
seen only in the lane containing pancreatic mRNA, suggesting that
the pancreas has high steady-state levels of pancreasin mRNA
compared with other tissues. Dual bands of actin hybridization seen in
the heart and skeletal muscle lanes are due to hybridization with
muscle isoforms of actin.
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Fig. 7.
Amplification of tissue-specific
cDNA. Human cDNA from the indicated range of cells and
tissues was amplified by PCR using primers (5'-GCAAAGACACCGAGTTTGGCTAC
and 5'-AGGGTATTTGAGAGGGGAGGAAG) based on pancreasin exons 5 and 6. The
amplification reaction was designed to cross intron 5 so that
cDNA-derived amplimers are distinguished from products derived from
any contaminating genomic DNA. The expected sizes of cDNA and
genomic DNA amplimers are 440 and 1154 bp, respectively. Consistent
with results of mRNA blotting in Fig. 4, the most intense bands
were obtained from cDNA from human pancreas and from the human
pancreatic carcinoma cell line, HPAC (left lane). However,
strong bands also were obtained from lung and placenta. Weak bands were
obtained from brain and liver. The right-most lane shows the
major ~1.2-kb band expected from genomic DNA. Amplimers from lung and
pancreas were isolated and sequenced, confirming their identity as
pancreasin. Thus, HPAC cells actively transcribe the pancreasin gene,
as do several tissues in addition to pancreas.
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Fig. 8.
Immunocytochemical analysis of pancreasin
expression in pancreatic ductal carcinoma cells. Cultured HPAC
cells were harvested, cytospun onto slides, and hybridized with
1:200 dilutions of pre-immune or anti-pancreasin polyclonal rabbit IgG,
followed by incubation with fluorescein-conjugated anti-rabbit IgG
secondary antibody. Images obtained by fluorescence microscopy of cells
incubated with pre-immune IgG and anti-pancreasin IgG are shown in
A and B, respectively (40× objective). Images
were captured and processed with identical parameters. Note the strong
cytoplasmic fluorescence obtained using anti-pancreasin antibody. These
findings support HPAC expression of pancreasin that is not
membrane-anchored or otherwise attached to the cell surface.
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Fig. 9.
Purification and deglycosylation of
recombinant human pancreasin expressed and secreted by CHO cells.
Medium conditioned by CHO cells transfected with a plasmid encoding
His9-tagged pancreasin was subjected to Nickel affinity
chromatography. Aliquots of the peak fraction were subjected to
SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed
with anti-pancreasin polyclonal rabbit IgG. The rhPancreasin
lane of panel A shows an immunoreactive band of 40- to
41-kDa corresponding to His9-tagged recombinant pancreasin.
The +PNGase lane shows results of incubation of an aliquot
of the same fraction with peptide N-glycosidase F, which
reduces recombinant His9-pancreasin to 31-32 kDa. Thus,
almost one quarter of CHO-expressed prostasin is comprised of
Asn-linked carbohydrate. The size of prestained marker proteins (in
kDa) is indicated. Panel B compares results of
immunoblotting (Blot lane) with the corresponding Coomassie
Blue-stained lane of an SDS-PAGE gel of the same affinity-purified
preparation. This suggests that our preparation is highly purified. The
weaker band of slightly lower size may represent partially glycosylated
pancreasin. The migration position of SDS-PAGE marker proteins (with
size in kDa) is shown to the right.
-tryptase. Unlike
-tryptase and trypsin (30),
however, pancreasin has little activity toward
tosyl-Gly-L-Pro-Lys-NA, which differs from
tosyl-Gly-L-Pro-Arg-NA only in the P1 residue. Therefore,
pancreasin appears to possess a strong preference for P1 Arg over Lys.
Pancreasin also appears to prefer peptide rather than mono-amino acid
amides, because it has less activity toward benzoyl-L-Arg-NA. This suggests that pancreasin has an
extended binding site available for productive interactions with
residues on the amino-terminal side of P1 Arg. Like tryptase, it
tolerates (and may actually prefer) substrates with P2 Pro, a somewhat
atypical preference among tryptic serine proteases (31). Further
analysis is needed to establish subsite preferences for substrate
residues P2-P4 as well as for residues on the P' side of the scissile
bond. Finally, our initial characterization of recombinant human
pancreasin suggests that the enzyme does not require calcium or heparin
for stability, in contrast to trypsin and tryptase, respectively. Indeed, pancreasin activity was undiminished by incubation for more
than 3 h at 37 °C.
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Fig. 10.
Peptide substrate preferences and inhibitor
susceptibility profile of recombinant human pancreasin.
A, comparison of rhpancreasin substrate preferences with
those of pancreatic trypsin. Nine peptidyl-NA substrates were examined.
Four of these are tryptic substrates (GPR,
tosyl-Gly-L-Pro-Arg-NA; GPK,
tosyl-Gly-L-Pro-Lys-NA; VGR,
benzoyl-L-Val-Gly-Arg-NA; KGR,
benzoyl-L-Lys-Gly-Arg-NA; PFR,
H-D-Pro-L-Phe-Arg-NA; and R,
benzoyl-L-Arg-NA), two are chymotryptic (FPF,
succinyl-L-Phe-Pro-Phe-NA; AAPF,
succinyl-L-Ala-Ala-Pro-Phe-NA), and one is elastolytic
(AAPV, succinyl-L-Ala-Ala-Pro-Val-NA). Results
are graphed as percentage of cleavage rate compared with the most
avidly cleaved substrate, which is GPR for both pancreasin and trypsin
among this group of substrates. Note the relative difference in rates
between the two enzymes, neither of which has detectable chymotryptic
or elastolytic activity. Pancreasin prefers Arg to Lys at the site of
hydrolysis in an otherwise matched pair of tripeptidyl substrates (GPR
versus GPK), but is less active toward a monoamino acid
substrate (R). This suggests that pancreasin possesses an
extended substrate-binding cleft with substrate preferences distinct
from those of trypsin. B, comparison of the inhibitor
susceptibility profile of rhpancreasin to that of trypsin. Data are
expressed as the percentage of activity remaining after preincubation
with inhibitor in comparison with rate of hydrolysis of GPR in the
absence of inhibitor ( ). Inhibitors examined include soybean trypsin
inhibitor (SBTI), benzamidine (Benz),
tosyl-Lys-chloromethylketone (TLCK), aprotinin
(Aprot), leupeptin (Leu), pepstatin
(Pep), E-64,
D-Phe-L-Pro-Arg-chloromethylketone
(PPACK), and p-amidinophenylmethanesulfonyl
fluoride (APMSF). The results reveal that pancreasin is
comparatively inhibitor-resistant, especially toward large
inhibitors.
-tryptases, which are cousins of pancreasin, as
shown in Fig. 3. Human
-tryptases resist large inhibitors by forming
non-covalently associated, heparin-stabilized oligomers, which
compartmentalize active sites within a central pore accessible only to
smaller inhibitors (17). At least one relative of tryptase, canine
mastin, forges disulfide links between catalytic subunits to stabilize
the inhibitor-resistant conformation (32). Based on results of
non-reducing SDS-PAGE (not shown), there is no evidence of the
formation of intersubunit disulfide links in our preparations of
pancreasin. However, the potential role of non-covalent oligomerization in pancreasin's resistance to aprotinin and inhibitors circulating in
the bloodstream merits further investigation. Similarly, it will be
helpful to identify low molecular weight inhibitors more potent than
benzamidine for future pharmacological explorations of pancreasin
function. Human pancreasin's resistance to aprotinin and its secretion
as a soluble enzyme lessens the likelihood that it plays a
channel-activating protease or prostasin-like role in regulating
epithelial sodium channel function. This is because functional data
from cultured epithelia suggest that the endogenous regulator of
Na+ flux via the amiloride-sensitive sodium channel is
sensitive to aprotinin and other large inhibitors and that a membrane
anchor may be required for channel-activating function (24, 33,
34).
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FOOTNOTES |
---|
* This work was supported by Grant HL-24136 from the National Institutes of Health, by Grant 9RT-0152 of the University of California Tobacco-Related Disease Research Program, and by the Research and Development Program of the Cystic Fibrosis Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY030095 and AF542056.
To whom correspondence should be addressed: Cardiovascular
Research Institute, University of California at San Francisco, CA
94143-0911. Tel.: 415-476-9794; Fax: 415-476-9749; E-mail: ghc@itsa.ucsf.edu.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209353200
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; NA, nitroanilide; DISP, distal intestinal serine protease; CHO, Chinese hamster ovary; HPAC, human pancreatic adenocarcinoma; RACE, rapid amplification of cDNA ends; UTR, untranslated region; PBS, phosphate-buffered saline; rhpancreasin, recombinant human pancreasin.
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