| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
Institute of Biochemical Sciences,5 College of Science, National Taiwan University, Taipei 106, Taiwan Taiwan
Institute of Biological Chemistry,6 Academia Sinica, Taipei 106, Taiwan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gamete biology, male reproductive tract, male sexual function, seminal vesicles, sperm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The cDNA of P12, a Kazal-type trypsin inhibitor, was cloned from the mouse ventral prostate by Mills et al. [10]. Because P12 RNA message is detectable in the male accessory sexual glands of adult mice while its expression is constitutive in the pancreas [10], substantial progress has been made in establishing its genomic structure. As a result, the DNA-binding sites for some transcription factors such as GC2 and SP1 have been identified in this gene [11, 12]. Meanwhile, we have purified the P12 cDNA-derived protein with an inhibitory constant (Ki) of 0.15 nM to trypsin from mouse seminal vesicle secretions (SVS) [13] and recovered a recombinant P12 with full activity of the naturally occurring P12 from a chimeric polypeptide of glutathione-S-transferase and P12 (GST-P12) expressed in Escherichia coli [14]. Moreover, we demonstrated a single-type P12-binding site (1.49 x 106 sites/cell) with a Kd value of 70 nM on the anterior region of mouse sperm and showed the inhibitory effect of P12 on the Ca2+ uptake by mouse sperm [15]. We conducted this study to have a better understanding of the structural feature for the multifunction of P12.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Outbred CD-1 mice were purchased from the Charles River Laboratories (Wilmington, MA) and were maintained and bred in the animal center at the College of Medicine, National Taiwan University. Animals were treated following the institutional guidelines for the care and use of experimental animals. They were housed under controlled lighting (14L:10D) at 21–22°C and were provided with water and NIH 31 laboratory mouse chow ad libitum. Adult mice (8–12 wk old) were humanely killed by cervical dislocation. Seminal vesicles were carefully dissected to free them from the adjacent coagulating glands, and the secretion collected from 50 mice was placed directly into 50 ml of ice-cold 5% acetic acid. P12 was purified from SVS according to a previously described procedure [13, 16].
The protected oligopeptides were synthesized using stepwise solid-phase methodology on an Applied Biosystems (Foster City, CA) 433A synthesizer with Fmoc chemistry and 4-hydroxymethyl phenoxyacetic acid preloaded resin. After each amino acid coupling, an acetylation step was introduced. After cleavage, the crude sample was purified using reverse phase-HPLC (RP-HPLC) on a C18 300A column (Waters prepacked cartridge 2 x 25 x 100 mm, 15 µm; Waters Corporation, Bedford, MA) with a linear gradient of water and 20% aqueous acetonitrile, both containing 0.1% TFA (v/v), at a flow rate of 20 ml/h. The fractions corresponding to the main peaks were analyzed using RP-HPLC and electrospray ionization-mass spectrometry (ES-MS), pooled, and lyophilized.
Preparation of the Recombinant P12 Variants
For site-directed mutagenesis for P12, we generally followed the instructions of the Promega Altered Sites II in vitro Mutagenesis Systems kit (Madison, WI). Based on P12 cDNA, the mutagenic oligonucleotides used for site-directed mutagenesis included 5'-GGATCATAAATTAGGGGACATCCCGC-3' (R19L), 5'-AGGATCAACAATTCTGGGACAT-3' (Y21V), 5'-CACACAGGACCATAAATT CTGG-3' (D22G), 5'-GCGTTTCCCGTTTTCAAAGC-3' (R43G), 5'-CTCTATGCGACTCCTGTTTTC-3' (K44S), and 5'-GGCTCTATGGTTTTCCTGTTTT-3' (R45T), where the mismatched bases are underlined and the mutated proteins are indicated by a one-letter-code mutation of the amino acids in parentheses. The EcoRI/BamHI fragment of P12 cDNA was purified from an expression vector (Gfp), which was previously constructed by the insertion of P12 cDNA into pGEX-2T [14]. The DNA fragment was inserted into pALTER-1. Using single-stranded DNA of the recombinant phagemid DNA as a template, the mutant strand containing each mutagenic oligonucleotide was completed according to the manufacturer's recommendations. The phagemid from each selected transformed colony was sequenced to confirm the mutation. The procedures for construction of two truncated variants were based on PCR amplification. The cDNA of Nd10 in which the N-terminal 10 residues of P12 were deleted or Cd8 in which the C-terminal 8 residues of P12 were deleted was amplified using Gfp as a template and the primer pairs of 5'-CTCGGATCCCATGATGCAGTGGCGG-3' and 5'-CTCGAATTCTCAGCAAGG CCCAC-3' or 5'-CTCGGATCCGCTA AGGT GACTG-3' and 5'-CTCGAATTCTC AGACAGGCTCTATG-3', where the EcoRI or BamHI site is underlined. The EcoRI/BamHI fragment of each P12 variant cDNA was purified and ligated into pGEX-2T, and each constructed vector was transformed into the Escherichia coli strain JM109. Expression of the recombinant protein followed a previously described technique [14]. The transformed cells were harvested and resuspended in 10 ml thrombin reaction buffer (50 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl2 at pH 8.0). Cells were then lysed by refreezing them three times in liquid nitrogen, followed by the addition of DNase I. After centrifugation, the supernatant was passed through an affinity column of glutathione agarose beads (Sigma, St. Louis, MO). According to a method modified from the manufacturer's instructions, the GST fusion protein in the column was mixed with an equal volume of thrombin reaction buffer containing thrombin (20 U/ml) at room temperature for 4 h. The nonbound fractions were eluted from the column, dialyzed against water, lyophilized to dryness, and redissolved in water for the RP-HPLC on a C4 300A column (Waters). The major sample peak in each chromatogram was identified using SDS/PAGE to be a single 6.0-kDa band that was immunoreactive to rabbit antiserum against P12 using the Western blot procedure (data not shown), which indicated that each P12 variant was purified to homogeneity.
Immunocytochemical Staining and Binding Assay
The rabbit antiserum against P12 was prepared [15]. Mouse spermatozoa were from the caudal epididymis and were prepared according to a method previously described [17, 18]. The spermatozoa were air dried on a glass slide and fixed with methanol. The slides were washed twice with PBS and preincubated in a blocking solution (3% nonfat skim milk in PBS) for 30 min at room temperature. Cells were further incubated with 10.0 µM P12 or its variant for another 30 min. Alternatively, cells were incubated with each ligand in PBS for 30 min before their fixation on a glass slide for the immunocytochemical analysis. For oligopeptide competition, slides were preincubated with 2.0 mM oligopeptides for 30 min before further incubation in the presence of 10.0 µM P12 for another 30 min. Slides were washed with PBS three times and then immunoreacted with the P12-induced rabbit antiserum diluted 1:200 in the blocking solution for 30 min. Slides were washed three times with PBS to remove excess antibodies before further reacting them with fluorescein-conjugated donkey anti-rabbit IgG (Pharmacia, Uppsala, Sweden) diluted 1:100 in blocking solution for 30 min. All slides were washed with PBS, covered with 50% (v/v) glycerol in PBS, and photographed under a microscope equipped with epifluorescence (AH3-RFCA; Olympus, Tokyo, Japan).
A method modified from that of Markwell [19] was followed to prepare 125I-P12 according to our method previously used [15]. Spermatozoa (2.5 x 106 cells/ml) and 100 nM 125I-P12 in PBS at pH 7.4 were incubated under specified conditions. According to our previously described procedure [20], cells were collected on a Whatman GF/C glass microfiber filter (Whatman, Maidstone, Kent, UK), and the filter was counted with a 
counter.
Analytical Methods and Spectral Measurement
The kinetic data from the digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin by bovine pancreatic type III trypsin (Sigma) in the presence of an enzyme inhibitor were analyzed using Dixon plots to determine the Ki of a tightly binding enzyme inhibitor according to a method previously described [13].
Protein concentration was determined using the BCA protein assay (Pierce, IL) according to the manufacturer's instructions. The homogeneity of the recombinant polypeptide was determined using SDS/PAGE on a gel slab (10.0 x 8.0 x 0.075 cm) according to the method of Laemmli [21]. The proteins on the gel were stained with Coomassie brilliant blue or transferred to a nitrocellulose membrane. After the transfer, protein blots were immunodetected using Western procedures with P12-induced rabbit antiserum as the primary antibody and goat anti-rabbit IgG conjugated with horseradish peroxidase (Pharmacia) as the secondary antibody.
The circular dichroism (CD) spectra were measured with a Jasco J-700 spectropolarimeter under constant flushing with N2 at room temperature. The mean residue ellipticity, 
, was estimated from the mean residue weight, which was calculated from the primary structure.
The cDNA sequence of each variant was read using the dideoxynucleotide chain termination method with a primer designed for each DNA concerned. Each base was determined at least three times using an ABI PRISM 377-96 DNA sequencer with an ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit (PE Applied Biosystem).
Molecular Modeling
Following the first approach model reported in the homology-modeling method by Schwede et al. [22], we submitted the amino acid sequence of P12 to the SWISS-MODEL (http://swissmodel.expasy.org/). Template selection, alignment, modeling process, and evaluation were completely automated by the server. The automated modeling procedure started when at least one modeling template was available that had a sequence identity of more than 25% of the P12 sequence. These steps could be iteratively repeated until a satisfying model structure was achieved.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The group of Kazal-type serine protease inhibitors is characterized by a well-preserved amino acid sequence containing three disulfide bridges [23]. Despite that the strict coincidence of the polypeptide chain folding does not occur throughout the inhibitor molecules, their extensive sequence homology suggests a similarity of their overall three-dimensional structures. In the absence of an x-ray structure of P12, we extrapolated a comparative model of its tertiary structure from the homologous modeling using the SWISS-MODEL, which automatically selected the known three-dimensional structures of related family members, including the porcine trypsin inhibitor [24], human pancreatic trypsin inhibitor [25], and pig intestine protease inhibitor [26] as the templates (see Materials and Methods). The high homology of target-template sequences increased the model reliability. According to the molecular model (Fig. 1), C10, C17, and C25 were, respectively, cross-linked with C39, C36, and C57 in the formation of three disulfide bonds. R19, Y21, D22, R43, K44, R45, R52, and K53 were distributed over the entire protein surface. Around 40% of the total amino acid residues formed secondary structures that included one 
helix, which stretched from E35 to R43, and a small antiparallel ß sheet, which comprised the three twisted strands of 23PVCG26, 27TDGI30, and 53KGGP56. In addition, there appeared to be a type I reverse turn centered on G26 and T27, an irregular reverse turn at the C-terminus of the helix, where 43RKR45 allowed a sharp inversion of the chain path, and a reverse turn at the C-terminal region, which were stabilized by a disulfide bond between C25 and C57. The remainder of the molecule may have contained no definite secondary structure. In particular, residues 1–22 may have adopted an extended conformation that stretches across the entire molecule. Homologous alignments of P12 to the known structures of other protease inhibitors suggested that the reactive site for protease inhibition was at the peptide bond between R19 and I20, and 43RKR45 was the regulatory site for temporary inhibition [27]. It was noted that residues 1–10 of the N-terminal region, 43RKR45, and residues 50–57 of the C-terminal region were three-dimensionally distant from R19, Y21, and D22 in the loop of residues 17–24.
|
Protein Conformation of P12 Variants
Based on the structural features of the molecular model, we prepared eight P12 variants, which were purified to homogeneity (see Materials and Methods). Six were made from single-site mutations, including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutations of amino acids. The other two, Nd10 and Cd8, were made by, respectively, deleting 10 residues of the N-terminus and 8 residues of the C-terminus.
The CD profile of each P12 variant was compared with that of P12 in PBS (Fig. 2). The spectra of P12 in the wavelengths of 200–250 nm had two negative bands with magnitudes of -8.1 x 103 and -1.31 x 104 deg·cm2·dmol-1 around 220 nm (band I) and 205 nm (band II), respectively, which suggested a considerable amount of ordered structures including a helix and a mixture of ß-forms and ß-turns in the P12 molecule, based on the CD spectra of the protein conformation [28–30]. The secondary structure shown in the molecular model of Figure 2 accounts for the characteristic CD. R19L, Y21V, D22G, R43G, K44S, R45T, and Nd10 shared very similar spectra with P12. On the other hand, the protein conformation of Cd8 changed remarkably as evidenced by the disappearance of bands I and II of the native protein and the appearance of a strong negative band below 200 nm, which indicated that Cd8 became unfolded. Apparently, the ß strand of 53KGGP56 is important to maintaining the P12 conformation.
|
The Reactive Site for Trypsin Inhibition and the Sperm-Binding Site on P12
The inhibitory effect of each P12 variant on the hydrolysis of N-benzoyl-Phe-Val-Arg 7-amino-4 methyl coumarin during the course of trypsin digestion was compared with its parent protein (Fig. 3). The Ki of each enzyme inhibitor was determined from the kinetic data (Fig. 3, top). P12 produced a Ki of 0.15 nM. Except that R19L entirely lost the ability to inhibit the protease, the other variants remained active in protease inhibition. Apparently, R19 is indispensable but the N-terminal 10 residues, Y21, D22, R43, K44, R45, and the C-terminal 8 residues are not essential for the protease inhibition.
|
The sperm-binding ability of each P12 variant was measured. Mouse spermatozoa from the caudal epididymis were incubated with each ligand, and its appearance on the cell surface was examined using an indirect immunofluorescence technique. The cells fixed on a glass slide were incubated with P12 or the cells were incubated with P12 before their fixation on a slide, and no differences in the immunocytochemical patterns were examined. Figure 4 displays the fluorescein fluorescence attributed to each ligand on the cell surface. A crescent fluorescence zone on the anterior region of the head of P12-treated sperm was seen, indicating P12-binding sites on the acrosomal region (Fig. 4, row 1). Fluorescence intensity as strong as that of P12-treated cells was visible on the cells preincubated with Nd10 and R19L (Fig. 4, rows 2 and 3). Likewise, immunochemical stainability was maintained on cells pretreated with R43G, K44S, or R45T (data not shown). On the other hand, very weak fluorescence appeared on Y21V-treated cells (Fig. 4, row 4), and neither D22G nor Cd8 immunochemically stained the sperm acrosome after the cell was preincubated with either variant (Fig. 4, rows 5 and 6). P12 barely stained cells after their preincubation with a 200-fold molar excess of G-18PRIYDPV24 for 30 min (Fig. 5, row 2). Substitution of this oligopeptide with either G-11HDAVAG16 or 47EPVLIRKGGP56-G in the cell preincubation did not hinder the binding of P12 to the cell surface (Fig. 5, rows 1 and 3). This rules out the sperm-binding site being within these two peptide regions. Meanwhile, 125I-P12 was used for quantitative characterization of the ligand-sperm binding. The sperm-binding ability of each ligand was assayed by its inhibitory effect on the binding of 125I-P12 to the cells. Figure 6 displays data of one representative determination from cell incubation in the presence of 100 nM 125I-P12 and each unlabeled ligand. The IC50 determined for each ligand is given at the top of Figure 6. The radiolabeled P12 bound to the cell surface was completely inhibited by unlabeled P12 with an IC50 of 98 nM, but was not inhibited by D22G or Cd8 even when a large excess of each ligand to 125I-P12 was present in the cell incubation. Y21V seemed to have very weak sperm-binding ability, because the concentration of up to 10 µM in the cell incubation inhibited less than 40% of 125I-P12-sperm binding. R43G was comparable with P12 in the inhibition of 125I-P12-sperm binding. Nd10, R19L, K44S, and R45T were as strong as P12 in binding sperm. Furthermore, the addition of G18-PRIYDPV24 to the cell incubation in a final concentration of 100 µM completely inhibited 125I-P12-sperm binding (data not shown).
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Winnica et al. [9] reported that excess rat caltrin I or P12 suppressed the proteolytic activity of individual rat or mouse epididymal spermatozoa. This may be a consequence of acrosome inhibition, blockage of proacrosin/acrosin activation, or acrosin release from acrosome. As shown previously [15], P12 is exclusively secreted from male accessory sexual glands and it binds to the plasma membrane overlaying the acrosome. Because acrosin/proacrosin should not be exposed on intact acrosomes, the principal acrosomal protease is unlikely to be the P12-binding site on the spermatozoal head. More studies are needed to clarify this aspect. Based on the molecular model shown in Figure 1, the binding of D22/Y21 on the P12 molecule to its receptor on the sperm head from one direction in the ejaculated semen would turn R19 to the other direction. Such a structural feature allows for the binding of a trypsin-like protease from another direction. Therefore, P12 may dissociate from the sperm head when ejaculated spermatozoa encounter a trypsin-like protease in the female reproduction tract under natural circumstance, considering that P12 gave a Ki value of 0.15 nM for the trypsin inhibition and a Kd value of 70 nM for the sperm binding. Determining the way in which P12 and the protease regulates the activity of sperm during their transit in the female reproductive tract is worthy of future study.
|
FOOTNOTES |
|---|
2 Correspondence: Yee-Hsiung Chen, P.O. Box 23-106, Taipei, Taiwan. FAX: 886 2 23635038; bc304{at}gate.sinica.edu.tw
3 Current address: Stanford University School of Medicine, Department of Gynecology and Obstetrics, 300 Pasteur Dr., Room S385, Stanford, CA 94305-5317
4 Current address: Functional Nucleic Acids Group, Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305 8566, Japan
Received: 23 June 2003.
First decision: 23 July 2003.
Accepted: 24 November 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Nd10; 
, Y21V; 
, D22G; 
, Cd8; 
, R19L. Except for R19L being unable to inhibit the enzyme, the kinetic data for trypsin activity in the presence of substrate at 5.0, 10.0, and 25.0 µM were analyzed by Dixon plot to determine the inhibitory constant (Ki, at the top of the figure)
