©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning, Tissue-specific Expression, and Cellular Localization of Human Prostasin mRNA (*)

Jack X. Yu, Lee Chao, and Julie Chao (§)

From the (1) Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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-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.

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)() 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.


EXPERIMENTAL PROCEDURES

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-terminal Amino Acid Sequence of Purified Prostasin

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 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.


RESULTS

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-terminal Amino Acid Sequence and Defining the Cleavage Sites of the Preproprostasin

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 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).




DISCUSSION

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, 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.

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 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.

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 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.

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 -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.

A potential N-linked glycosylation site, Asn-Ala-Ser, has been identified at Asn. 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.

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. 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 29397 and DE 09731. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) L41351.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-4321; Fax: 803-792-4322.

The abbreviations used are: PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; RACE, rapid amplification of cDNA ends; TPCK-trypsin, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin; MCA, 4-methylcoumarin-7-amide; DIG, digoxigenin.

N represents A, C, G, or T; R represents A or G; and D represents A, G, or T.


ACKNOWLEDGEMENTS

We thank Dr. Carmelann Zintz and Gary Richards for critical review of the manuscript.


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