Androgen Regulation of an Elastase-Like Protease Activity in the ...

BIOLOGY OF REPRODUCTION 52, 1059-1065 (1995)

Androgen Regulation of an Elastase-Like Protease Activity in the Seminal Vesicle' SCOT[ HARVEY, 3 ANNE VRABEL,3 STEVE SMITH, 4 and ERIC WIEBEN 2'3

Department of Biochemistry and Molecular Biology3 and Department of Internal Medicine Division of Endocrinology,4 Mayo Foundation,Rochester, Minnesota 55905 ABSTRACT The processing of secretory proteins in the guinea pig (GP) seminal vesicle epithelium (SVE) is altered by castration and restored by treatment of animals with androgens. To test the hypothesis that the changes in protein processing are due to changes in the activity of specific proteases, we examined the GPSVE for protease activities capable of cleaving a synthetic elastase substrate, succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc(Ala) 3pNA). We found that the GPSVE does contain a Suc(Ala) pNA3 cleaving activity that is sensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP) and to the elastase inhibitor elastatinal. Furthermore, the amount of protease activity per milligram of SVE protein is reduced to about 50% of control levels by castration. The activity is completely restored within four days by treatment of castrated animals with androgens, but is not restored by treatment with estradiol, progesterone, or dexamethasone. Although the SVE enzyme did not yield a pattern of specific cleavage products when incubated with a secretory protein substrate in vitro, this enzyme activity was competitively inhibited by a peptide whose primary sequence included the cleavage site used by the processing machinery in vivo.

INTRODUCTION Despite great progress in understanding the molecular mechanisms by which androgens influence the transcription of target genes, relatively few androgen-regulated regulatory genes have been identified. There are several reports of protein-processing events that are regulated by androgens. Processing of progonadotropin-releasing hormone has been shown to be regulated by testosterone in the rat [1], and there is evidence that the proteolytic processing of renin is similarly influenced by androgens [2]. In the course of our studies of the regulation of gene expression by androgens in the guinea pig, we have noted that secretory protein processing is responsive to androgens. The guinea pig (GP) seminal vesicle epithelium (SVE) is an androgen-dependent tissue that synthesizes and secretes four abundant secretory proteins (SVP 1-4) [3, 4]. The largest secretory protein, SVP-1, is a clotting protein that becomes highly cross-linked to itself in the presence of a transglutaminase produced by the prostate [5, 6]. The function of the other three secretory proteins is still not clear. Previous studies of the biosynthesis of these proteins in the GPSVE indicated that the four mature secretory proteins are cleaved from only two primary translation products in a complex series of protein-processing reactions [7, 8]. At least three proteolytic cleavages are required to produce the largest secretory protein, SVP-1, from the 45-kDa precursor. Processing of the same 45-kDa precursor also yields SVP-3 and SVP-4 (Fig. 1). The first proteolytic event in SVP-1 proAccepted December 13, 1994. Received June 20, 1994. 'Supported by NIH grant HD 9140 to E.D.W. 2 Correspondence: Eric Wieben, Department of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street South West, Rochester, MN 55905. FAX: (507) 284-9759.

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duction results in the removal of the signal peptide (cleavage 1 in Fig. 1), which is highly conserved between the two secretory protein precursors. The second cleavage occurs after a dibasic Lys-Arg sequence that is also conserved between the secretory protein precursors (cleavage 2 in Fig. 1). This cleavage leads to the production of an SVP-1 processing intermediate of 24.5 kDa with an amino terminus nine amino acids longer than that of mature SVP-1 [9]. The other product of this cleavage is mature SVP-3 (-4; SVP-3 and SVP-4 differ only with respect to an unidentified posttranslational modification). The third proteolytic cleavage is unusual in that it occurs after a pair of alanine residues in the 24.5-kDa intermediate (cleavage 3 in Fig. 1). These three proteolytic cleavages have been confirmed by direct sequencing of the protein products. However, the details of the processing reactions remain obscure, and it is possible that additional proteolytic cleavages are required for full processing of the secretory protein precursors (see question marks in Fig. 1). Studies of secretory protein gene expression have revealed that androgens do not directly regulate the expression of secretory protein genes in adult animals [10]. However, data from previous studies suggests that the processing steps required to produce SVP-1 from the 24.5-kDa intermediate are less efficient in tissue from castrated animals, and that normal processing can be restored by treatment of castrated animals with testosterone [9]. Our long term goal is to understand the molecular mechanisms involved in the regulation of protein processing by androgens. As a first step towards this goal, we tested the hypothesis that the GPSVE contains an androgen-regulated protein-processing activity. As a focus for these studies, we have been studying the activity of SVE proteases that are capable of cleaving an alanine-containing synthetic substrate. Our results indicate that the GPSVE does contain a serine protease

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FIG. 1. Proteolytic cleavages required for processing of SVP-1 precursors in GPSVE. SVP-1 (hatched bar) is cleaved from carboxyl terminus of 45-kDa primary translation product. On the basis of sequences of processing intermediates, at least three different proteases are thought to be involved in production of mature SVP-1. Sites of action of these proteases are depicted by numbers enclosed in circles. Possible additional processing activities involved in production of SVE secretory proteins are depicted by question marks (?). Amino terminal sequences of 24.5-kDa processing intermediate, SVP-1 IB, and mature SVP-1 are also given.

whose activity is rapidly and specifically regulated by androgens. MATERIALS AND METHODS Animals Adult male guinea pigs weighing 500-850 g were obtained from SASCO (Omaha, NB). Where indicated, animals were castrated via the scrotal route, under ether anesthesia. For hormone repletion studies, animals received i.m. injections of 2 mg testosterone propionate in sterile sesame oil every other day. In some cases, animals were given identical courses of treatment with dihydrotestosterone (DHT), estradiol, dexamethasone, or progesterone instead of testosterone. Each hormone was administered by i.m. injection of 2 mg of hormone in sesame oil/10% ethanol every other day. Unless otherwise noted, all data were obtained from pooled tissue samples from two animals. All experiments were conducted according to the guidelines of the Mayo Foundation Institutional Animal Care and Use Committee. ProteaseAssays Assays for elastase-like protease activity utilized the N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (Suc(Ala) 3 pNA) substrate developed by Bieth et al. [11] (Sigma Chemical Co., St. Louis, MO). For the standard assay, SVE was prepared from isolated seminal vesicles as described previously [8], and an SVE extract was prepared by homogenization of the SVE in PBS. After clarification of the extract by centrifugation at 13 000 rpm for 15 min, the protein content of the supernatant was determined by the BCA assay

(Pierce, Rockford, IL), with BSA used as a standard [12]. Standard assays used 100 g of SVE protein in a final volume of 150 Rl of PBS containing 1.67 mM Suc(Ala) 3pNA. The optical density at 410 nm was recorded for 5 min at 25°C. Activity calculations used E = 8800 M-l cm-' forpnitroaniline at 410 nm. Trypsin assays were performed similarly, with p-toluenesulfonyl-L-arginine methylester used as a substrate. One unit of trypsin-like activity yields 1 pRmol of product per minute at 25 0C. Electrophoretic analysis of plasminogen activators was performed according to Roche et al. [13]. Unless noted otherwise, results are expressed as mean + SEM. Statistical significance was evaluated by Student's ttest, withp < 0.05 taken as significant. In Vitro Labeling of Seminal Vesicle Proteins Pulse-chase analysis of secretory protein synthesis in isolated SVE was performed as described in Norvitch et al. [8], except that [3 5S]methionine was used instead of [3H]leucine. Radioactively-labeled proteins were analyzed on 12% SDSpolyacrylamide gels, and fluorographed by means of ENHANCE (New England Nuclear, Boston, MA). Construction of the 24.5-kDa Substrate The DNA template used to transcribe an mRNA coding for a 24.5-kDa SVP-1 processing intermediate was constructed by use of polymerase chain reaction (PCR) mutagenesis. The full-length cDNA clone for the 45-kDa primary translation product was used as the template [7]. The 5' oligonucleotide had the sequence 5'-gcgcgaattc atg GAT CCC ATA GCA GCA CTA-3', and the 3' oligonucleotide had the sequence 5'-cgatcaccc GGGGGTCTCTTTATTG-3', where sequences shown in all capitals correspond to residues found in the natural SVP-1 cDNA sequence (GP1 [7]). Amplification was carried out for 35 cycles (94°C, 1 min; 50°C, 2 min; 72°C, 3 min). The products were cloned into pGEM4 Blue after digestion of both the PCR product and the plasmid with EcoRI and Sma I. The resulting plasmid was sequenced to verify orientation and the absence of PCR-induced mutations. When linearized with Sma I, this plasmid serves as a template for the production of a synthetic mRNA using SP6 RNA polymerase. When translated in rabbit reticulocyte lysate, the mRNA codes for a protein that has a primary sequence identical to the natural 24.5-kDa intermediate except for the substitution of a methionine for a glutamine at the amino terminus. Translation products from reticulocyte lysates supplemented with [35S]methionine and programmed with this mRNA were used without further purification as substrates for the SVE protease or porcine pancreatic elastase. InhibitorStudies The 24.5-kDa peptide used for inhibition studies was synthesized by the 9-fluorenylmethoxycarbonyl (FMOC)

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strategy by means of an Applied Biosystems 431A automated synthesizer (Foster City, CA). Protocols and reagents used were those suggested by the manufacturer. Peptides were purified by reverse-phase HPLC on a Vydac 22-mm x 25-cm preparative column (The Separation Group, Hesperia, CA), with a trifluoroacetate/acetonitrile buffer system employed before use. Peptide integrity was monitored by either amino acid analysis or mass spectroscopy. The sequence of this peptide is given in Figure 7. The control peptide (EA) corresponds to amino acids 59-72 of a human snRNP protein [14] prepared similarly, and has the sequence: DDAEEIHSKTKSRK. This control peptide did not significantly inhibit the SVE protease, even when added at a 2-fold molar excess of the concentration that led to significant inhibition by the 24.5-kDa peptide. All other inhibitors were obtained from Sigma. The enzyme preparation for inhibitor studies was partially purified from a GP seminal vesicle extract before use by acetone precipitation and phenyl Sepharose chromatography. Briefly, 88 g flash-frozen GP seminal vesicles (Keystone Biologicals, Cleveland, OK) were homogenized in 250 ml of 20 mM triethanolamine HC1, pH 7.4. The resulting homogenate was centrifuged for 10 min at 5000 rpm, and the supernatant was fractionated by the addition of 0.7 volumes ice-cold acetone. After centrifugation as before, additional cold acetone was added to the supernatant to make the final volume of acetone equal to that of the homogenate. The precipitate was collected by centrifugation, and the pellet was lyophilized overnight. The 1-volume acetone pellet was resolubilized in 10 ml of 20 mM triethanolamine HCl, pH 7.4. An equal volume of 2.2 M (NH4)2S0 4 + 0.2 M NaP04, pH 7.0, was then added. The solution was clarified by centrifugation at 8000 rpm in a Sorvall SS-34 rotor, and the supernatant was filtered through a 0.45-jm syringe filter. Further purification was achieved by phenyl Sepharose chromatography. A single peak of activity was eluted from the phenyl Sepharose column with use of a decreasing step gradient of (NH 4 )2S0 4 in 0.1 M NaPO4 , pH 7. Peak fractions were pooled and desalted by ultrafiltration before use. This procedure typically yields 20-50% of the starting activity with a purification of approximately 100-fold over that of the crude homogenate.

FIG. 2. Pulse-chase analysis of secreted proteins synthesized by minced whole seminal vesicle from intact and castrated guinea pigs. Secretory protein precursors were labeled by incubation of minced seminal vesicles in [35Slmethionine for 6 min. At end of incubation period, medium containing [35Slmethionine was removed and replaced with fresh medium containing unlabeled methionine. Incubation was then continued for additional period as indicated. Samples of medium were removed for analysis at end of initial labeling period (lanes 4 and 5), and after 4 (lanes 3 and 6), 8 (lanes 2 and 7), and 12 min (lanes 1 and 8) of cold methionine chase. Samples in lanes 9 and 10 are from separate experiment where chase was extended to 1.5 h. Lanes 5-8 and 10 utilized tissue from intact animals. Lanes 1-4 utilized tissue from animals that had been castrated for 12 days. Sample in lane 9 is from 9-day castrated animal. Similar results were obtained in two independent replicates using seminal vesicles from 5- and 6-day castrates.

secretory protein processing (SVP-3/-4 and the 24.5-kDa intermediate). By 8 min after the initiation of the chase (lanes 2 and 7), these products are found to be labeled to approximately the same degree in both intact and castrate samples. However, there was a noticeable shift in the pattern of SVP-1-related products that were secreted between 4 and

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RESULTS The kinetics of seminal vesicle secretory protein processing in intact and castrated animals was investigated by means of a pulse-chase strategy. After a 6-min incubation of minced seminal vesicles in [3 5S]methionine, the radioactive medium was replaced with fresh medium containing an excess of unlabeled methionine. Samples of secreted proteins were taken at the end of the 6-min labeling period, and at 4-min intervals for the next 12 min. As shown in Figure 2, castration does not result in a major change in the rate of synthesis and secretion of the initial products of