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- Original Article -
Molecular mechanism of epididymal protease inhibitor
modulating the liquefaction of human semen
Zeng-Jun Wang, Wei Zhang, Ning-Han Feng, Ning-Hong Song, Hong-Fei Wu, Yuan-Geng Sui
Laboratory of Reproductive Medicine, Department of Urology, the First Affiliated Hospital of Nanjing Medical University,
Nanjing 210029, China
Abstract
Aim: To study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate
specific antigen (PSA) digesting semenogelin
(Sg). Methods: Human Sg cDNA (nucleotides 82_849) and Eppin
cDNA (nucleotides 70_423) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO.
Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was
studied by far-western immunoblot and radioautography.
In vitro the digestion of rSg by PSA in the presence or
absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding
was monitored. Results: Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino
acids 75_133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin
is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the
N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the
modulating effect of rEppin. Conclusion: Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.
(Asian J Androl 2008 Sep; 10: 770_775)
Keywords: epididymal protease inhibitor; semenogelin; prostate specific antigen
Correspondence to: Dr Zeng-Jun Wang, Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of
Nanjing Medical University, Nanjing 210029, China.
Tel: +86-25-8630-7536 Fax: +86-25-8660-4771
E-mail: zengjunwang2002@hotmail.com
Received 2007-06-03 Accepted 2007-11-25
DOI: 10.1111/j.1745-7262.2008.00393.x
1 Introduction
Epididymal protease inhibitor (Eppin) is a
testis/epididymis-specific protein. Human ejaculated spermatozoa
are coated with Eppin over both head and tail regions
before and after capacitation [1_4], which is involved in
cogulum formation in the ejaculation. Human seminal
plasma spontaneously coagulates after ejaculation. The
major component of this coagulum is semenogelin (Sg),
a 52-kDa protein expressed exclusively in the seminal
vesicles. Sg is the major protein involved in gelatinous
entrapment of ejaculated spermatozoa, which plays an
important role in the regulation of sperm motility and
fertilization. The protein is rapidly cleaved after
ejaculation by the chymotrypsin-like protease prostate-specific
antigen (PSA), resulting in liquefaction of the semen
coagulum and the progressive release of motile spermatozoa.
PSA cleaves the coagulum proteins, resulting in the
release of Sg proteolytic fragments [5]. Cleavage of Sg by
PSA during liquefaction removes Sg from the sperm
surface and results in the motility and capacitation of
spermatozoa.
During human ejaculation, Eppin binds Sg before PSA
digestion. To determine if Eppin plays an important role
in regulating the hydrolysis of recombinant Sg (rSg) by
PSA, we investigated the digestion of rSg by PSA in the
presence and absence of recombinant Eppin (rEppin) and
the effect of antibodies on Eppin-Sg binding and the
hydrolysis of rSg by PSA in vitro.
2 Materials and methods
All chemicals and reagents used in the present study
were obtained from Sigma (St. Louis, MO, USA).
Plasmid PET100 was purchased from Invitrogen (CA, USA).
Purifications of plasmid and polymerase chain reaction
(PCR) cDNAs were performed using the respective kits
from Qiagen (Valencia, CA, USA). Immobilon-P and -N
transfer membranes were purchased from Millipore (Bedford, MA, USA). Enzymatically active PSA was
obtained from EMD Bioscience (San Diego, CA, USA).
2.1 rEppin and rSg production
An Eppin cDNA (nucleotides 70_423) lacking part
of the N-terminal secretory sequence was generated by
PCR using the eppin-1/Bluescript clone [1] as template.
PCR was performed with Pfx Platinium Polymerase (Invitrogen) and cloned into pET-100D/TOPO (Invitrogen).
In a similar manner, a human Sg cDNA (nucleotides
82_849) was generated by PCR using a human seminal vesicle
cDNA library as template (a gift from Dr Frank R. French, University of North Carolina, Chapel Hill, NC,
USA) and cloned into pET-100D/TOPO.
All constructs were verified by sequencing and
expressed in DH5-α. Bacterial lysates were purified on
Ni-NTA agarose (pET-100D/TOPO) or anti-FLAG-M2 affinity gels (pFLAG-MAC; Siama).
2.2 Antiserum production
Affinity-purified rabbit antisera to N-terminal amino
acids 20_39 of mouse Eppin were made by Bethyl
Laboratories (Montgomery, TX, USA). Cysteine residue 33
was changed to an alanine. These antisera (anti-Q20E)
reacted with both mouse and human Eppin.
2.3 Western blot analysis
Proteins were separated on reducing 10%_20 %
gradient gels (Bio-Rad, Hercules, CA, USA) or on reducing
NuPAGE 4%_12% Bis-Tris gels (Invitrogen) and transblotted to Immobilon-P (Millipore) and either stained
for protein with amido black or blocked with Tris
buffered saline (TBS) (50 mmol/L Tris, pH 7.4,
150 mmol/L NaCl) containing 3% BSA for 60 min at room
temperature and probed with primary antibodies as described
[2]. Two micrograms of recombinant protein were loaded
per lane. Primary antibodies were used at a 1:2 000
dilution and secondary antibodies (goat anti-rabbit IgG or
goat anti-mouse IgG, 1:2 000) were either alkaline
phosphatase labeled and developed with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate
or peroxidase labeled and developed with
chemiluminescence using Supersignal West Pico Chemiluminescent
Substrate (Pierce, Rockford, IL, USA) according to the
manufacturer's instructions.
For far-western blots, proteins were immobilized on
Immobilon-P, blocked as above, incubated for 1_2 h or
overnight in protein probes, washed and detected with
primary and secondary antibodies as described above.
The protein concentrations were determined using
Micro BCA Protein Detection Reagents (Pierce).
2.4 Labeling and quantitative binding assay
Labeling of 20 μg of rEppin or rSg with
125I was carried out using the Iodo-gen direct method (Pierce),
according to the manufacturer's instructions, and the
unbinded 125I was removed with a micro Bio-spin 6
chromatography column (Bio-Rad). Proteins were
immobilized on Immobilon-P, blocked as above, incubated for
1_4 h in either 125I-rEppin or
125I-rSg, and exposed for autoradiography overnight.
In vitro 125I-rSg binding assay and
4 μg of rEppin were immobilized on a
nitrocellulose membrane (0.45 μm) using a Bio-Dot microfiltration
apparatus (Bio-Rad) and the membrane was washed with
TBS-Tween (TBST) (50 mmol/L Tris, pH 7.4,
150 mmol/L NaCl with 0.05% Tween 20) and blocked with 5% BSA
in TBST. Triplicate bio-dots on a membrane with or
without Eppin (control) were incubated in increasing
amounts of 125I-rSg overnight at 4°C, then washed in
TBST, cut into 1 cm squares, each containing a single
dot, and counted in a r-counter. To demonstrate the
competition for binding, increasing amounts of unlabeled rSg
were added to the 125I-rSg and
125I-Eppin bio-dot incubation mixtures.
2.5 Sg hydrolysis
All hydrolysis reactions of rSg with commercial
native PSA in the presence or absence of rEppin, were
performed in 1 mol/L NaCl, 0.1 mol/L Tris-HCl, pH 8.3 at a
1:50 enzyme/substrate ratio overnight at 37°C. rEppin
was incubated with rSg for at least 2 h before PSA was
added. The hydrolysis product was analyzed using
10_20% precast SDS-PAGE (Criterion gels, Bio-Rad) and
the gel stained overnight with 0.01% Bio-Rad R-250
Coomassie (Bio-Rad) in 10% acetic acid. Protein
concentrations were determined using micro BCA protein
detection reagents (Pierce), using BSA as a standard. To
test the effects of specific anti-Eppin antibodies on the
PSA hydrolysis of rSg, either anti-Q20E or
anti-C-terminal Eppin was incubated with rEppin for 2 h before rSg
was added. After a further 2-h incubation, PSA was
added for varying times at 37°C.
3 Results
3.1 rEppin
rEppin and its C-terminal and N-terminal were
transferred onto Immobilon-P Polyvinylidene Difuoride
(PVDF) membrane by Western blot (Figure 1A). The
membrane was incubated into rSg. Far-western immunoblot analysis demonstrates that the C-terminal of
rEppin binds to rSg (Figure 1B).
3.2 rSg
rSg and its N-terminal and C-terminal were
transferred onto PVDF membrane by Western-blot. The
membrane was incubated into 125I-rEppin. Autoradiograph
analysis demonstrates that rSg164_283 fragment binds
125I-rEppin (Figures 2 and 3).
3.3 Digestion of rSg by PSA
Digestion of rSg by PSA initially produces several
lower molecular weight fragments (< 10 kDa) (Figure 4,
lane 5). In the presence of rEppin (Figure 4, lane 1),
rEppin was bound to rSg, then digested by PSA, producing incomplete digestion and a 15-kDa fragment
(Figure 4, lane 2, asterisk). Analysis of the protected
fragment by MS/MS revealed that it contained
cys239, the necessary residue for rEppin binding. Anti-Q20E
(N-terminal) had no effect on Eppin-Sg binding, as
monitored by PSA digestion of rSg (data not shown).
Antibodies to the C-terminal of rEppin make rEppin lose the
modulating function and the protected 15-kDa fragment
of rSg disappears. MS analysis of the protected
fragment of rSg by rEppin.
The complete Sg sequence was showed as follows:
mkpniifvls lllilekqaa vmgqkggskg r/lpsefsqfp hgqkgqhysg qkgkqqtesk;
gsfsiqytyh vdandhdqsr ksqqydlnal hkttksqrhl ggsqqllhnk qegrdhdksk;
ghfhrvvihh kggkahrgtq npsqdqgnsp sgkgissqys nteerlwvhg lskeqtsvsg;
aqkgrkqggs qssyvlqtee lvankqqret knshqnkghy qnvvevreeh sskvqtslcp;
ahqdklqhgs kdifstqdel lvynknqhqt knlnqdqqhg r/kankisyqs ssteerrlhy;
gengvqkdvs qssiysqtee kaqgksqkqi tipsqeqehs qkankisyqs ssteerrlhy;
gengvqkdvs qrsiysqtek lvagksqiqa pnpkqepwhg enakgesgqs tnreqdllsh;
eqngrhqhgs hggldiviie qeddsdrhla qhlnndrnpl ft.
When rSg was bound to rEppin, an Sg fragment with
an approximate molecular weight of 15 kDa was protected from PSA digestion. Reduced and
carboxymethylated rSg did not bind rEppin. When reduced and
carboxymethylated rSg was digested with PSA, an Sg
fragment with an approximate molecular weight of 15
kDa was also protected from PSA digestion. The sequence of the fragment protected from digestion was as
follows:
nteerlwvhg lskeqtsvsgaqkgrkqggs qssyvlqtee lvankqqret knshqnkghy qnvvevreeh sskvqtslcpahqdklqhgs
kdifstqdel lvynknqhqt knlnqdqqhg r
In the presence of anti-Eppin antibody Q20E
(anti-Eppin peptide, amino acids 20_39) bound to Eppin, the
fragments were still protected from PSA digestion. In
the presence of antibody 9714 (anti-Eppin peptide epitope,
amino acids 90_98), the fragments were not protected
from PSA digestion (Figure 4, Lane 1).
4 Discussion
Recombinant protein purification is facilitated using
high expression systems. The solubility and yield of pure
protein are highly dependent on various combinations of
chemical additives, ionic and non-ionic detergents and
salts, with solubilizing agents followed by refolding of
denatured protein into its active form. As the extraction
of the purified protein from high expression systems
requires denaturation and a subsequent refolding step,
careful balancing steps were needed to develop under
different controlled conditions. Here the purified fragments
of refolded proteins were screened to select the
conditions that yield the activity having native conformation.
The refolded recombinant protein was analyzed by
RP-HPLC, showing a purity of 99%. The size exclusion
chromatography profile shows that there are minimal
aggregates in the active protein and the percentage of
renaturation is approximately 99%.
During liquefaction of semen, PSA cleaves Sg bound
to the sperm surface, releasing the sperm motility
inhibitory factor (amino acids 69_160) [5_8]. We now know
that Sg on the sperm surface is bound to Eppin and,
therefore, the cleavage of Sg by PSA must occur while
Sg is bound to Eppin. Consequently, we compared
in vitro the digestion of rSg by PSA in the presence or
absence of rEppin. As shown in Figure 4, when rSg (Sg,
lane 4) is digested with PSA, many low molecular weight
fragments are produced (lane 3). However, when rEppin
is bound to rSg, digestion by PSA is modulated,
producing incomplete digestion and a 15-kDa fragment (asterisk,
lane 2). This experiment suggests that Eppin has an
important function in ejaculated semen liquafication, sperm
capacitility and motility.
Our understanding of Eppin's essential role in sperm
survival during transfer from male to female
reproductive tracts prior to fertilization stems from an analysis of
anti-Eppin antibody binding sites (epitopes) on Eppin. As
described previously [9_12], sera from the infertile male
monkeys immunized with Eppin recognizes two
predominant epitopes: N-terminal (QGPGLTDWLFPRRCPKIRE;
amino acids 20_38) and C-terminal
(TCSMFVYGGCQGNNNNFQSKANCLN; amino acids 101_125). Production
of antibodies to N-terminal amino acids 20_39
(anti-Q20E) [1, 2], and to C-terminal rEppin have been
described [1, 2]. To test the effect of specific anti-Eppin
antibodies on the PSA hydrolysis of Sg as it might occur
in vivo, either anti-Q20E or anti-C-terminal Eppin was
incubated with Eppin. Incubation continued with the
addition of Sg, and finally PSA was added for a final
incubation period. Addition of anti-Q20E had no effect
on Eppin-Sg binding, as monitored by PSA digestion of
Sg. Therefore, antibody binding to the N-terminal of
Eppin did not affect Sg digestion. However, addition of
antibodies to the C-terminal of Eppin resulted in blocking
PSA activity modulation. Consequently, digestion with
PSA produced many low molecular weight fragments and, notably, the protected 15-kDa fragment (Figure 4,
lane 2, asterisk) was absent (Figure 4, lane 1). Analysis
of the protected fragment by MS/MS revealed that it
contained cys239, the residue necessary for Eppin binding.
Moreover, the Sg N-terminal sequence containing the
sperm motility inhibiting peptide [13] had been cleaved
from the cys239 containing fragment by PSA into very
small fragments, which would presumably no longer be
anchored to Eppin. Although sperm motility inhibiting
peptide is bound to sperm, it remains immotile and its
removal is necessary for resumption of motility and
subsequent capacitation [14_16].
We can hypothesize from our analysis of anti-Eppin
epitopes on Eppin that when anti-Eppin antibodies in the
infertile male monkeys entered the epididymal fluid and
bound to Eppin on the sperm surface, they blocked the
binding site for Sg [10, 11]. Blocking the binding of Sg
had two consequences. First, as a result of not being
bound to Eppin, Sg in the ejaculate was quickly
hydrolyzed into small fragments; no modulation of PSA
activity and no semen coagulum was observed. Second,
having anti-Eppin bound to Eppin on the sperm surface
mimicked the physiological effect of having sperm
motility inhibiting peptide bound to the surface, namely, a
loss of forward motility, which was observed in semen
from infertile men. The second consequence predicts
that the removal of anti-Eppin antibodies from the sperm
surface would allow spermatozoa to recover their motility.
Further studies are underway to verify it.
Acknowledgment
This study was supported by grant from National
Key Project of Scientific and Technical Supporting
Programs (No. 2006BAI03B12).
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