From the
We have purified a novel human serine proteinase, designated as
prostasin, from seminal fluid (Yu et al., 1994). In the
present study, we have cloned and characterized the full-length cDNA
encoding prostasin and identified its tissue-specific expression and
cellular localization. A cDNA fragment was obtained by polymerase chain
reaction using degenerate oligonucleotide primers derived from the
NH
Human seminal fluid is a rich source of proteolytic enzymes,
many of which are involved in the postejaculatory hydrolysis of
proteins and in semen coagulation and liquefaction (Shivaji et
al., 1990). Prostate-specific antigen and acrosin are two of the
most important proteolytic enzymes found in human semen.
Prostate-specific antigen may play an important role in semen
liquefaction through hydrolyzing semenogelin, a predominant seminal
vesicle protein (Lilia, 1985). Prostate-specific antigen levels in
blood have been recognized recently as the most important marker for
prostate cancer. Acrosin is a serine proteinase present in acrosomes,
where it covers the anterior part of the sperm head (Klemm et
al., 1991). It is believed to be involved in recognition, binding,
and penetration of the zona pellucida of the ovum during interaction of
the sperm and egg (Jones et al., 1988; Topfer-Petersen and
Henschen, 1988). Recently, we have identified and purified a new serine
proteinase, designated as prostasin, from human seminal fluid (Yu
et al., 1994). At the present time, the physiological
functions of prostasin are unknown, and its physiological substrate
remains to be identified.
Prostasin has an apparent molecular mass
of 40 kDa on SDS-polyacrylamide gel electrophoresis and displays
arginine amidolytic activity. The NH
In
order to understand the structure, regulation, and function of
prostasin, it is essential to isolate and characterize its cDNA. In
this study, we have cloned the full-length cDNA encoding prostasin
through polymerase chain reaction (PCR)
In this study, we have cloned and sequenced a full-length
cDNA encoding prostasin, a human serine proteinase. This cDNA codes for
a protein of 343 amino acids with a 32-amino acid signal peptide. The
catalytic triad essential for enzymatic activity of prostasin is
His
Based on the amino acid sequence
deduced from its cDNA, the posttranslational processing sites of
prostasin have been defined. The posttranslational process of prostasin
is very similar to that of acrosin, which produces a 23-amino acid
light chain associated with the heavy chain by two disulfide bonds
(Klemm et al., 1991). Prostasin is synthesized as a
preproenzyme of 343 amino acids. During translocation into the
endoplasmic reticulum, a cleavage occurs between Gly
Like acrosin, prostasin has a unique protruding COOH terminus
(Fig. 2). Acrosin is a serine proteinase membrane-anchored
through its COOH terminus by an unknown mechanism and is then released
after cleavage (Baba et al., 1989). At the COOH terminus of
prostasin, there is a highly hydrophobic portion of 19 amino acids,
which are rich in leucine and flanked by a positively or a negatively
charged amino acid at either side (Arg and Glu). This indicates that
prostasin is likely to be a membrane-bound serine proteinase.
Similarly, angiotensin-converting enzyme has a 17-amino acid
hydrophobic portion near its COOH terminus, which has been identified
as a transmembrane domain (Wei et al., 1991). After cleavage,
angiotensin-converting enzyme is released, and its COOH-terminal
portion remains on the membrane (Beldent et al., 1993).
Carboxypeptidase digestion shows that the COOH-terminal amino acids of
the purified prostasin's heavy chain are Leu-Arg, indicating that
there is a cleavage between Arg
Compared with other serine
proteinases, the number and positions of 9 out of the 11 cysteine
residues in the catalytic chain of the translated prostasin are highly
conserved (Fig. 2). On the basis of the known disulfide bridge
arrangement in serine proteinases (Young et al., 1978), four
intrachain disulfide bonds are expected at cysteine pairs 38/54,
136/212, 169/191, and 202/230 in prostasin (Fig. 1). Except for
the single-chained
A potential N-linked glycosylation
site, Asn-Ala-Ser, has been identified at Asn
On synthetic substrates, prostasin shows
trypsin-like activities, such as arginine amidolytic activities on
D-Pro-Phe-Arg-MCA and D-Phe-Phe-Arg-MCA (Yu et
al., 1994) and lysine amidolytic activities on
succinyl-Ala-Phe-Lys-MCA and
t-butyloxycarbonyl-Val-Leu-Lys-MCA (data not shown). It has no
enzymatic activity on chymotrypsin substrates such as
succinyl-Ala-Ala-Pro-Phe-MCA (Yu et al., 1994). These results
are consistent with the fact that in the deduced prostasin, an Asp
residue is present at position 200, which is located six residues
before the active site Ser
Broad existence of
prostasin mRNA in human tissues suggests that it may have important
biological functions. Localization of prostasin mRNA in the epithelial
cells of the prostate gland indicates that prostasin is synthesized in
the cells and then secreted into the ducts. The presence of prostasin
in prostatic epithelial cells and ducts was identified by
immunohistochemistry in our previous studies (Yu et al.,
1994). Since it is likely to be a membrane-bound serine proteinase,
prostasin may be involved in some important processes on the surface of
cell membranes, such as removal of propeptides from hormones and growth
factors and the activation of proenzymes associated with membranes. In
order to understand prostasin's physiological functions, further
experimentation is needed.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. Carmelann Zintz and Gary Richards for
critical review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal and internal amino acid sequences. A
full-length cDNA sequence encoding prostasin was obtained by
amplification of the 5`- and 3`-ends of the cDNA. It contains a
1,032-base coding region, a 572-base 3`-noncoding region and a 138-base
5`-noncoding sequence. Prostasin cDNA encodes a protein of 343 amino
acids, which consists of a 32-amino acid signal peptide and a 311-amino
acid proprostasin. Proprostasin is then cleaved between Arg
and Ile
to generate a 12-amino acid light chain and
a 299-amino acid heavy chain, which are associated through a disulfide
bond. The deduced amino acid sequence of the heavy chain has
34-42% identity to human acrosin, plasma kallikrein, and hepsin.
A potential N-glycosylation site at Asn
and the
catalytic triad of His
, Asp
, and Ser
have been identified. The deduced prostasin has a unique 19-amino
acid hydrophobic portion at the COOH terminus, which makes it suitable
to anchor in the cell membrane. Carboxyl-terminal sequencing of
purified prostasin indicates that the hydrophobic portion is removed
and that there is a cleavage between Arg
and Pro
during secretion. Southern blot analysis, following a reverse
transcription polymerase chain reaction, indicates that prostasin mRNA
is expressed in prostate, liver, salivary gland, kidney, lung,
pancreas, colon, bronchus, renal proximal tubular cells, and prostate
carcinoma LNCaP cells. Cellular localization of prostasin mRNA was
identified within epithelial cells of the human prostate gland by
in situ hybridization histochemistry.
-terminal 20-amino acid
sequence of prostasin shares 50-55% identity with human
-tryptase, elastase 2A and 2B, chymotrypsin, acrosin, and the
catalytic chains of hepsin, plasma kallikrein, and coagulation factor
XI (Yu et al., 1994). It is present in many tissues and has
the highest level in the prostate gland. In the prostate gland,
prostasin has been localized in epithelial cells and ducts by
immunohistochemistry. It is believed that prostasin is synthesized in
prostatic epithelial cells, secreted into the ducts, and excreted into
the seminal fluid, where it may serve a role in fertilization. The wide
distribution of prostasin outside the prostate gland indicates that it
may also play important roles in other biological processes.
(
)
and 5`-
and 3`-rapid amplification of cDNA ends (RACE) based on its
NH
-terminal and internal amino acid sequences. We have
elucidated its primary structure and defined the posttranslational
processes that convert preproprostasin into proprostasin and prostasin.
In addition, we have identified the cleavage site where prostasin is
released from membranes. Tissue-specific expression of prostasin has
been identified by reverse transcription PCR followed by Southern blot
analysis. Cellular localization of prostasin mRNA in the human prostate
gland has been determined by in situ hybridization.
Internal Amino Acid Sequence Analysis
Prostasin
was purified from human seminal plasma as described previously (Yu
et al., 1994). Purified prostasin (40 µg) was digested
with TPCK-trypsin (Sigma) at a ratio of 1:100 (w/w) at 37 °C for 16
h after reduction by dithiothreitol and S-carboxymethylation
by iodoacetic acid according to the procedure described by Stone et
al.(1989). Generated peptide fragments were separated by a reverse
phase HPLC (model 5000 liquid chromatograph; Varian Associates, Inc.)
with a µBondapak C18 column (3.9 mm 30 cm, Water, Inc.) and
eluted by an acetonitrile gradient. The collected fractions were
concentrated by Speedvac to a desired volume and subjected to amino
acid sequencing using a gas phase protein sequenator equipped with an
on-line narrow bore phenylthiohydantoin-derivative analyzer (ABI model
470 A, Applied Biosystems Inc.).
Amplification of a Partial cDNA Fragment
A human
prostate cDNA library in gt 11 (Clontech Lab, Inc.) was amplified
with forward and backward primers corresponding to the two arms of the
phage DNA. The reaction mixture contained 1
PCR buffer (0.5
mM dNTP, 100 pmol of primers, 0.1% Triton X-100, 2
10
phage of the cDNA library, and 2.5 units of
Ampli-Taq DNA polymerase (Perkin-Elmer Corp.) in a total
volume of 50 µl. The reaction was conducted in a GeneMachine II
(USA/Scientific Plastics, Ocala, FL) using the following program: 94
°C for 1 min, 42 °C for 2 min, 72 °C for 3 min for 30
cycles followed by 5 min at 72 °C. According to the
NH
-terminal amino acid sequence of purified prostasin,
ITGGSSAVAGQWPWQVSITY (Yu et al., 1994), and the internal amino
acid sequences obtained above, two degenerate primers were designed.
The sense primer is JY-1, 5`-GTNGCNGGNCARTGGCC-3`, which corresponds to
VAGQWP in the NH
-terminal sequence; the antisense primer is
JY-T84-2, 5`-TTNGCRTCDATRTTRTA-3`, corresponding to YNIDAK in
peptide T84 (see ``Results'').
(
)
To
amplify a partial prostasin cDNA fragment, 2 µl of the amplified
human prostate cDNA library was used as template in the following PCR
reaction: 1
PCR buffer, 0.25 mM dNTP, 0.05 mM
tetramethylammonium chloride, 100 pmol of JY-1 and JY-T84-2, and
2.5 units of Ampli-Taq DNA polymerase. The cycling program was
the same as mentioned above. A second round of PCR was carried out with
10 µl of the first round product as template and the same primers
under the same conditions.
Sequence Analysis of PCR Product
After
purification, the amplified products were subjected to Southern blot
analysis using a nested degenerate oligonucleotide, JY-4, which is
located just downstream of the sense-primer JY-1. The sequence of JY-4
is 5`-CARTGGCCNTGGCARGT-3`, which corresponds to QWPWQV at the
NH terminus of the purified prostasin. A 300-base pair DNA
fragment that hybridized to JY-4 oligonucleotide was sequenced using
JY-4 as a primer with the Life Technologies, Inc. dsDNA cycle
sequencing system.
Elucidation of the Full-length cDNA Sequence of
Prostasin
A partial prostasin cDNA sequence with a size of
approximately 300 bases was obtained from the above experiment. Based
on this sequence, a sense primer, JY-F1 (5`-GTCCATGTGTGTGGTGG-3`), was
used with total RNA from human renal proximal tubular cells in the
3`-RACE reaction (Life Technologies, Inc.). A nested primer, JY-AD
(5`-CTGTCAGCTGCTCACTGC-3`) was used to sequence the product of the
3`-RACE reaction to reveal the 3`-end of prostasin cDNA. The antisense
primer JY-B1 (5`-TGGGTCTGCTGAGTTGG-3`) was exploited for reverse
transcription in the 5`-RACE reaction. After adding an oligo(dC) anchor
sequence to the 3`-end with terminal deoxynucleotidyl transferase, the
generated cDNA was amplified with a nested antisense primer, JY-UA
(5`-CTCCTGGAGGTAGCTGG-3`), and an anchor primer provided by the
manufacturer in the 5`-RACE system (Life Technologies, Inc.). The
amplified product was sequenced with primer JY-AU
(5`-GCAGTGAGCAGCTGACAG-3`) to reveal the 5`-end of prostasin cDNA.
Analysis of NH
Prostasin was purified from human
seminal fluid as described previously by Yu et al.(1994). In
order to explore whether there were two chains linked by a disulfide
bond, the purified prostasin was resolved on SDS-polyacrylamide gel
electrophoresis under nonreducing conditions and transferred to an
Immobilon-P membrane (Millipore Corp.). The protein band was cut out
for NH-terminal Amino Acid
Sequence of Purified Prostasin
-terminal amino acid sequencing.
Carboxypeptidase Digestion
Since the deduced
prostasin has a putative transmembrane domain at the COOH terminus
(Fig. 1), this hydrophobic portion is likely to be removed in the
secreted prostasin. To explore this possibility, 60 µg of purified
prostasin from human seminal fluid was dissolved in 0.2 MN-ethylmorpholine acetate (pH 8.3), and a mixture of
carboxypeptidase A and carboxypeptidase B was added to prostasin at a
molar ratio of 1:50. The reaction was carried out at 37 °C, and an
aliquot of the incubation mixture was removed for amino acid
composition analysis at 1, 5, and 19 h. After 19 h of incubation, the
pH of the test protein mixture was lowered to 6.0 with acetic acid, and
carboxypeptidase Y was added to the remaining prostasin at a 1:50
ratio. An aliquot of the incubation mixture was removed at 1 and 19 h
for amino acid composition analysis.
Figure 1:
Nucleic acid sequence of prostasin cDNA
and the deduced amino acid sequence. A variant polyadenylation signal,
ATTAAA, is underlined, and poly(A) is designated as
(A)n. A solidtriangle indicates a potential
N-glycosylation site, opentriangles indicate active sites of the catalytic triad, and stars represent a stop codon. Amino acid numbering starts with the first
amino acid of proprostasin.
Tissue-specific Expression of Prostasin
Human
tissues were obtained from autopsy and kindly provided by Dr. Sandra
Conradi at the Medical University of South Carolina. Renal proximal
tubular cells were obtained from Dr. Debra Hazen-Martin at the Medical
University of South Carolina (Detrisac et al., 1984), and
prostate carcinoma LNCaP cells were from the American Type Culture
Collection. Tissues were handled as reported by Chai et
al.(1993), and total RNA was isolated according to the procedure
described by Davis et al.(1986). Total RNAs extracted from
various tissues were subjected to reverse transcription PCR using the
sense primer JY-F1 and antisense primer JY-B1. The PCR product was
confirmed by Southern blot analysis with probe JY-AD (Maniatis et
al., 1982).
In Situ Hybridization
Prostasin cDNA was amplified
with two primers, JY-F1 and JY-B1, after reverse transcription using
total RNA from LNCaP cells. A 255-base pair fragment was gel-purified
from an agarose gel and ligated into pSP73 at the HindII site.
The recombinant plasmid was used to transform E. coli JM101.
Positive colonies were selected by colony hybridization with
oligonucleotide JY-AD, and the orientation of the cDNA fragment in the
vector was determined by DNA sequencing. The sense and the antisense
RNAs, corresponding to the partial prostasin cDNA, were synthesized by
using SP6 and T7 RNA polymerases, respectively. During the synthesis of
the RNAs, digoxigenin-labeled uridine-triphosphate (DIG-UTP) was
incorporated according to the protocol of Boehringer Mannheim. The
DIG-labeled antisense RNA (riboprobe) was used to detect prostasin mRNA
in 5-µm sections of human prostate that was formalin-fixed and
paraffin-embedded. DIG-labeled sense RNA was used as a control. An
antibody-conjugate (anti-digoxigenin alkaline phosphate conjugate) was
used to recognize the DIG-labeled RNA. A subsequent enzyme-catalyzed
color reaction was conducted by the addition of
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt and
incubated overnight at room temperature in the dark as described by the
manufacturer. After washing and dehydration, sections were mounted with
Permount.
Internal Amino Acid Sequence Analysis
Purified
prostasin was subjected to TPCK-trypsin cleavage, and as many as 15
peptide fragments were isolated by reverse phase HPLC with a C18
column. Five of the peptide fragments were subjected to
NH-terminal amino acid sequencing. The peptides are T45
(LGAHQLDSYSE), T59 (ASSYASWIQSK), T75 (NRPGVYTLASSYAS), T77
(YIRPIXLPAAXASFP), and T84
(ETXNXLYNIDAKPEEPHFVQ), where X represents
an undetermined amino acid. A degenerate oligonucleotide,
JY-T84-2, with the least degree of degeneracy at 96 was
synthesized as an antisense primer corresponding to the amino acid
sequence YNIDAK in peptide T84. Two degenerate sense primers, JY-1 and
JY-4, with degrees of degeneracy at 128 and 16, respectively, were
designed according to the previously obtained NH
-terminal
amino acid sequence ITGGSSAVAGQWPWQVSITY (Yu et al., 1994).
Cloning and Analysis of Full-length cDNA Encoding Human
Prostasin with Degenerate Oligonucleotide Primers
Fig. 1
shows the nucleic acid sequence of prostasin cDNA and its
deduced amino acid sequence. Prostasin cDNA consists of a coding region
of 1,032 bases, a 3`-noncoding region of 572 bases, and a 5`-noncoding
region. The coding region starts with an ATG codon, which is present in
a sequence of GTCCTGGCCATGG, similar to the consensus sequence
GCCGCCRCCATGG for the eukaryotic translational initiation site (Kozak,
1987). The cDNA encodes a 343-amino acid polypeptide, including a
32-amino acid signal peptide, a 12-amino acid light chain, and a
299-amino acid heavy chain beginning with Ile-Thr-Gly-Gly, the
NH-terminal amino acid sequence of purified prostasin
reported by Yu et al.(1994). A potential N-linked
glycosylation site has been identified as Asn
in a
sequence of Asn-Ala-Ser, and a variant polyadenylation signal, ATTAAA,
was found 12 bases upstream of the poly(A). Fig. 2compares the
deduced prostasin amino acid sequence with other serine proteinases.
Prostasin shares 34-42% identity to human plasma kallikrein,
coagulation factor XI,
-tryptase, hepsin, plasminogen, and
acrosin. The catalytic triad of the deduced prostasin has been
identified as His
, Asp
, and Ser
according to the multiple sequence alignment. In alignment with
other serine proteinases (Fig. 2), prostasin contains an Asp
residue at position 200, indicating that it has trypsin-like activity.
A hydropathy plot of the deduced prostasin identifies two hydrophobic
regions, one located at the NH
terminus and the other at
the COOH terminus (Fig. 3). The one located at the NH
terminus is likely to be a signal peptide, which could direct
newly synthesized prostasin to enter the endoplasmic reticulum. The
other region located at the COOH terminus is indicated as a putative
transmembrane domain and is double-underlined in Fig. 1.
Figure 2:
Comparison of prostasin sequence with
other serine proteinases. The amino acid sequences of these serine
proteinases correspond to the mature forms of -tryptase
(Vanderslice et al., 1990) or the catalytic chains of acrosin
(Adham et al., 1990), plasma kallikrein (Chung et
al., 1986), coagulation factor XI (Fujikawa et al.,
1986), serine protease hepsin (Leytus et al., 1988), and
plasminogen (Forsgren et al., 1987). Amino acid residues that
are highly conserved are shaded, and the catalytic triad of histidine,
aspartic acid, and serine of the catalytic triad are indicated by
triangles. Dots represent gaps to bring the sequences
to better alignment.
Figure 3:
Hydropathy plot of the deduced prostasin.
The hydropathy of prostasin's amino acid sequence translated from
its cDNA was predicted with MacProMass program and plotted with the
Kyte & Doolittle Hydropathic index using a window size of 10
residues. Amino acid numbering begins with the start codon
Met.
Analysis of Prostasin NH
Under nonreducing conditions, two distinct
signals were observed on chromatograms in each Edman degradation cycle
of the purified prostasin. However, under reducing conditions only one
signal was obtained (Yu et al., 1994). The two amino acid
sequences obtained are ITGGSSAVAGQW, which is the same as the
previously published NH-terminal Amino
Acid Sequence and Defining the Cleavage Sites of the
Preproprostasin
-terminal amino acid sequence under
reducing conditions (Yu et al., 1994), and
AEAPXGVAPQ. Examination of the deduced prostasin amino acid
sequence from its cDNA indicates that AEAPXGVAPQ is located
just upstream from ITGGSSAVAGQW and that the X is a cysteine
residue. This result shows that the nascent prostasin is a
preproenzyme, which is converted to proenzyme by removing a 32-amino
acid signal peptide. The generated proprostasin is then activated by
the cleavage of a peptide bond between Arg
and Ile
to give rise to active prostasin, which contains a light chain of
12 amino acids and a heavy chain of 299 amino acids. The two chains are
held together by a disulfide bond.
Identification of COOH-terminal Residues of Purified
Prostasin
The result of carboxypeptidase digestion indicates
that the first released amino acid residue is Arg, followed by either
Leu or Ala. No release of His, the last amino acid
residue of prostasin deduced from its cDNA, can be observed. The total
amount of released Arg is substantially higher than that of Leu or Ala,
indicating that there are two carboxyl-terminal Arg residues.
Considering that a light chain and a heavy chain exist in the purified
prostasin, the signals obtained from carboxypeptidase digestion
represent two COOH-terminal sequences. Since the last two amino acids
of the light chain are Ala-Arg, the COOH terminus of the heavy chain
must be Leu-Arg. Compared with the deduced prostasin sequence, there is
a Leu-Arg sequence just upstream from the putative transmembrane domain
at the COOH terminus. Therefore, it is believed that a cleavage occurs
between Arg
and Pro
.
Tissue-specific Expression of Human
Prostasin
Prostasin mRNA expression was detected in several
human tissues with reverse transcription PCR followed by Southern blot
analysis using a specific oligonucleotide probe. The result shows that
prostasin is expressed in the human prostate gland, liver, salivary
gland, kidney, lung, pancreas, colon, bronchus, renal proximal tubular
cells, and LNCaP cells, but not in the testis, ovary, spleen, uterus,
cortex, muscle, atrium, ventricle, aorta, vein, artery, umbilical vein
endothelial cells, lymphocytes, and polymorphonuclear cells
(Fig. 4).
Figure 4:
Tissue-specific expression of prostasin
mRNA. A specific reverse transcription PCR was conducted using total
RNA from 24 human tissues or cells. Toppanel,
prostate, liver, testis, salivary gland, kidney, lung, pancreas, ovary,
spleen, uterus, colon, and cortex. Bottompanel,
muscle, atrium, ventricle, bronchus, aorta, vein, renal proximal
tubular cells, human umbilical vein endothelial cells, LNCaP cells,
lymphocytes, and polymorphonuclear cells. The reverse transcription PCR
products were detected by a nested oligonucleotide probe specific for
prostasin.
In Situ Hybridization of Prostasin mRNA in the Prostate
Gland
Cellular localization of prostasin mRNA has been
identified within the epithelial cells of the human prostate gland
using the antisense riboprobe by in situ hybridization
histochemistry (Fig. 5a). No staining was observed in
the control section using the sense riboprobe (Fig. 5b).
Figure 5:
Localization of prostasin mRNA by in
situ hybridization in the human prostate gland. a, a
digoxigenin-labeled antisense RNA of prostasin was used as a probe.
b, a digoxigenin-labeled sense RNA of prostasin was used for
hybridization as a control. The labeled RNA probes were detected with
an enzyme-linked immunoassay (magnification,
80).
, Asp
, and Ser
. The
sequences around these active sites are highly conserved in the serine
proteinases as shown in Fig. 2, and the heavy chain of prostasin
shares 34-42% identity with them. A potential glycosylation site,
Asn
, has been identified. A putative transmembrane domain
is present at the COOH terminus, suggesting that prostasin is likely to
be a membrane-anchored serine proteinase. Prostasin is expressed in a
variety of human tissues, and its mRNA is localized within epithelial
cells of the prostate gland.
and Ala
to remove the 32-amino acid signal peptide
generating proprostasin. This cleavage site agrees with the motif that
requires small neutral amino acid residues at positions -3 and
-1 (von Heijne, 1990). The generated proenzyme with 311 amino
acids is then activated by a specific cleavage between Arg
and Ile
to produce an active, two-chain form. The
two chains are held together by an interchain disulfide bond between
Cys
in the light chain and a Cys residue in the heavy
chain.
and Pro
in
the deduced prostasin. There are two Arg residues in the COOH terminus
of the deduced prostasin, Arg
and Arg
.
Obviously, the peptide bond between Arg
and Pro
is preferred over that between Arg
and Val
since Pro
cannot be detected after carboxypeptidase
digestion. This result was verified by immunoblot analysis using a
specific antiserum against an 11-amino acid peptide,
Pro
-Gln
-Thr
-Gln
-Glu
-Ser
-Gln
-Pro
-Asp
-Ser
-Asn
,
which recognized purified prostasin (data not shown). Analysis of the
amino acid composition of purified prostasin's heavy chain also
suggests that the Leu-rich hydrophobic portion is missing since the Leu
content is much lower than that in the deduced one (data not shown).
From these results, we conclude that prostasin loses its COOH-terminal
hydrophobic portion during secretion.
-tryptase, Cys
is conserved in
all of the serine proteinases listed in Fig. 2. This cysteine
residue has been found to be involved in the formation of an interchain
disulfide bond with the noncatalytic chain in plasma kallikrein,
coagulation factor XI, and acrosin (McMullen et al., 1991a;
McMullen et al., 1991b; Topfer-Petersen et al.,
1990). Considering that purified prostasin consists of two chains held
by a disulfide bond, we conclude that an interchain disulfide bond
exists between Cys
and Cys
. In addition,
prostasin has two unique cysteine residues at positions 171 and 274
(Fig. 1). Whether these cysteine residues form a disulfide bond
remains to be explored.
. The
presence of this site explains why purified prostasin under reducing
conditions displayed a larger size (40 kDa) on SDS-polyacrylamide gel
electrophoresis than the deduced heavy chain of 32 kDa.
Asn
, which was included in tryptic peptide T77 and
described as X, could not be identified in amino acid
sequencing although the signals before and after the Asn residue were
very strong (data not shown). Furthermore, Asn residues in tryptic
peptide T84 were easily identified. This phenomenon indicates that
Asn
has been modified by carbohydrates which make it
nondetectable. In addition to N-linked glycosylation, it is
likely that O-linked glycosylation is present in prostasin,
since there are many serine and threonine residues in the deduced amino
acid sequence of prostasin. This modification might also contribute to
the discrepancy between the molecular masses of the deduced and the
purified protein.
. This Asp residue, which is
located at the bottom of the substrate-binding pocket in trypsin, is
involved in an interaction with the Arg or Lys residue of a substrate
(Ruhlmann et al., 1973). In addition, Gly
and
Gly
are conserved in prostasin. The counterparts of these
two Gly residues in trypsin are present at the entrance of the
substrate-binding pocket and permit entry of large amino acid side
chains. Thus, these features in prostasin's primary structure
determine its trypsin-like cleavage preference.
/EMBL Data Bank with accession number(s) L41351.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.