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From the * Department of Laboratory Medicine,
Division of Clinical Chemistry, Lund University, Malmö University
Hospital, Malmö, Sweden; the Department
of Clinical Laboratories, Urology, and Medicine, Memorial Sloan-Kettering
Cancer Center, New York, New York; and the
Department of Clinical Sciences, Fertility
Center, Lund University, Malmö University Hospital, Malmö,
Sweden.
| Correspondence to: Åke Lundwall, Wallenberglaboratory, 4th floor, University Hospital MAS, SE-205 02 Malmö, Sweden (e-mail: ake.lundwall{at}med.lu.se). |
| Received for publication July 6, 2007; accepted for publication January 22, 2008. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Key words: Expression, immunoassay, prostate, purification, semen
1-antichymotrypsin
(Lilja et al, 1991;
Stenman et al, 1991). There is
evidence to suggest that measurement of the proportion of fPSA to total PSA
(tPSA) enhances discrimination of men with prostate malignancy from men with
no evidence of cancer in the prostate gland
(Christensson et al, 1993;
Catalona et al, 1998). This
enhancement is incomplete, and it is still relevant to state that no serum
marker or combination of markers have the ability to both detect and
distinguish aggressive and less aggressive forms of prostate cancer with
sufficient certainty. Beta-microseminoprotein (MSP), which like PSA is a predominant protein secreted by the prostate gland, has attracted much less interest as a biomarker for prostatic disease compared with PSA (Dube et al, 1987; Abrahamsson et al, 1988, 1989). MSP is synthesized as a preprotein of 114 amino acid residues, from which a 20-residue signal peptide is cleaved off to form the mature protein; another name of MSP is prostatic secretory protein of 94 amino acids (Mbikay et al, 1987). The molecular mass is 11 kd, and the polypeptide chain is not glycosylated, despite an apparent molecular size of 16 kd on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Studies of the three-dimensional structure by nuclear magnetic resonance (NMR) have shown the molecule to consist of 2 distinct domains that form a rather extended structure (Ghasriani et al, 2006).
Early functional studies reported that MSP display inhibin activity and could suppress secretion of follicle-stimulating hormone from the pituitary gland, which resulted in the alternative name beta-inhibin. These claims were later retracted and there has been no consensus regarding the function of MSP ever since. MSP has also been suggested to be one of the immunoglobulin binding factors in seminal plasma (Liang et al, 1991; Kamada et al, 1998). More recently, 2 studies reported evidence to show that MSP forms high-affinity complexes with 2 related Cys-rich proteins: PSP94-binding protein in blood plasma and cysteine-rich secretory protein 3 (CRISP-3) in semen (Reeves et al, 2005; Udby et al, 2005). Possibly, these observations will give a clue to the function of MSP.
MSP is not solely synthesized by the prostate epithelium, in that the protein also can be detected in nonreproductive organs such as in the respiratory and gastrointestinal tracts, where the gastric mucosa especially shows high expression (Ulvsback et al, 1989; Weiber et al, 1990, 1999). Accordingly, MSP can be measured in serum of both men and women, but the levels in serum from women were found to be around two-thirds of those measured in men (Abrahamsson et al, 1989). Elevated serum concentrations of MSP are reported in hyperplasia and neoplasia of the prostate gland, and increased levels have also been seen in gastric carcinoid disease (Dube et al, 1987; Abrahamsson et al, 1989; Weiber et al, 1999). The nonprostate-specific features of MSP give rise to concerns regarding the value of MSP as a tumor marker for prostatic disease. However, of particular interest for the consideration of MSP as a prostate tumor marker are some recent observations: transcriptional down-regulation of MSP in prostate cancer (Liu et al, 1993), an inhibitory effect of MSP on growing prostate cancer cells (Lokeshwar et al, 1993; Shukeir et al, 2003), and the finding that patients with low serum concentrations of MSP have a high probability of having prostate cancer detected at biopsy (Nam et al, 2006).
Because MSP also has extraprostatic origin, the concentrations measured in serum cannot immediately be associated only with the contribution from the prostate gland. To the best of our knowledge, no previous data are reported on whether there is an association between the secretory release of MSP into semen and the levels at which MSP occurs in serum. We have therefore determined the MSP levels in healthy young men and characterized the correlation between MSP in seminal plasma and serum and other prostate components, such as Zn2+ and PSA. To pursue this work, we developed a new MSP immunoassay and worked out procedures for the expression of recombinant MSP and its purification, which we also report in this paper.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Ejaculates and blood samples were collected from 305 young, healthy Swedish military conscripts as previously described (Richthoff et al, 2002; Sävblom et al, 2005). The sampling was not done on the same day as the examination of physical fitness. Semen samples were taken prior to the serum samples. Semen volumes were calculated from the weights of the samples, assuming a density of 1.0 g/mL. The different PSA forms in serum and seminal plasma and the Zn2+ in seminal plasma were determined in earlier studies (Elzanaty et al, 2002; Sävblom et al, 2005). The Zn2+ concentration was determined with a colorimetric method (Makino et al, 1982). A total of 100 subjects were excluded in this study because of lack of semen samples, leaving 205 cases for analysis of MSP. To investigate the risk of selection bias, the study group of 205 subjects and the excluded subjects were compared with respect to prostate parameters by the Mann-Whitney test. No significant differences were found between the 2 subgroups regarding the previously measured amount and concentration of PSA and Zn2+ in semen or the concentrations of fPSA and tPSA in serum.
Protein and Antiserum
MSP was purified from human seminal plasma essentially as previously
described (Fernlund et al,
1994). The pure protein was dialyzed against water and
lyophilized. The dry protein was weighed with an accuracy of 1% on a Cahn 28
Automatic electro balance scale (AB Ninolab, Upplands Väsby, Sweden) and
then dissolved in a buffer containing 20 mM phosphate and 50 mM NaCl, pH 7.0,
to a final concentration of 1.00 mg/mL. Aliquots of the solution were stored
at –20°C until use. The rabbit anti-human MSP serum has previously
been described (Abrahamsson et al,
1989). Biotinylated goat-anti-rabbit antibody (GAR) was purchased
from DACO Norden AB (Solna, Sweden).
Labeling with Eu3+
The labeling of MSP with Eu3+ (Eu-MSP) was made with the DELFIA
Eu labeling kit 1244-302 (Wallac Oy, Turku, Finland) according to the
manufacturer's instructions. Briefly, 0.45 mg of MSP was dissolved in 600
µL of labeling buffer (50 mM NaHCO3 and 0.15 M NaCl, pH 8.5)
containing 0.2 mg of labeling reagent and incubated for 15 hours at room
temperature. After the separation of the labeled product from excess reagent
on a Sepharose 6B column (Amersham Biosciences, Uppsala, Sweden), the yield
was estimated by measuring the delayed fluorescence on an Autodelphia 1235
automatic immunoassay system (Wallac Oy) after the addition of enhancement
solution. The MSP concentration was determined by absorbance measurements at
280 nm with an absorbance coefficient of 1.47 for an MSP concentration of 1.00
mg/mL. The mean recovery of Eu-MSP from the Sepharose 6B column was 78%.
MSP Immunoassay
All dilutions were made with a buffer containing 50 mM Tris-HCl, pH 7.75,
0.9% NaCl, 0.05% NaN3, 0.01% Tween 20, 0.05%
bovine-
-globulin, 20 µM diethylaminepentaacetic acid (DPTA), 0.5%
bovine serum albumin, and 20 µg/mL amaranth (assay buffer). Washings were
made with a buffer containing 5 mM Tris-HCl, pH 7.75, 0.9% NaCl, 0.1% germal
II, and 0.005% Tween 20. The incubations were made by shaking at room
temperature.
The amount of GAR was titrated by incubation of Streptavidin Microtitration strips (Innotrac Diagnostics Oy, Turku, Finland) with different concentrations of biotinylated GAR in 200 µL for 1 hour. After washing, 200 µL of a solution containing anti-MSP diluted 1:4000 and 50 ng of the tracer were added. After additional washes, delayed fluorescence was measured. The anti-MSP antiserum was titrated by incubating 1 ng of tracer in 200 µL of serially diluted antiserum for 4 hours. Different incubation times were then evaluated, showing the most reproducible results after 4 hours of incubation, which was then used for the assay.
The final assay was done with an Autodelphia 1235 automatic immunoassay system (Wallac). As above, all incubations were made at room temperature by shaking. Each well of the streptavidin plates was coated with 300 ng of the biotinylated GAR diluted in 200 µL of assay buffer for 1 hour. After 2 wash steps, 50 µL of sample, 1 ng of Eu-labeled MSP in 50 µL assay buffer, and 100 µL of antiserum diluted 1:16 000, were added to the wells, and the plates were incubated for another 4 hours. After 4 wash steps, 200 µL of enhancement solution was added, and the plates were incubated for 5 minutes prior to measurement of the delayed fluorescence. The standard curve comprised 7 points, with concentrations from 0 to 130 µg/L assay buffer, and calculations were made by the Multicalc program (Wallac). Serum samples were analyzed without prior dilution, but the seminal plasma samples were routinely diluted 1:50 000, in some cases 1:10 000, in assay buffer. The following control samples were included in each assay run: 3 levels of MSP in assay buffer, 2 levels of MSP added to serum, and pooled seminal plasma from 7 donors. All study and control samples were run in duplicate. The standards and controls were prepared from stock solutions, diluted in assay buffer, and frozen in aliquots that were thawed just before use and then handled in the same way as the serum and seminal plasma samples.
Assay Validation
The sensitivity, or lower limit of detection of the assay, was defined as
the concentration obtained by the mean count minus 2 standard deviations of 32
zero-standard readings in the same assay. The intra-assay coefficient of
variation (CV) was determined at 3 different levels. Each sample was run 20
times in 1 assay, and the mean value with CV was calculated. The intra-assay
CV was also estimated for every sample because of duplicates. The interassay
variation was estimated by analysis of variance from the values for the
duplicates in 10 consecutive assays.
The linearity and recovery were evaluated by adding MSP to a female sample, with an endogenous MSP value of 11 µg/L, to a final concentration of 200 µg/L. The sample was then serially diluted with either serum or assay buffer, and the recovery was reported as the ratio between the expected concentration and the measured concentration.
The stability of Eu-MSP was tested by comparing the values of assay standards in runs with aliquots that were newly thawed or stored in the refrigerator overnight.
Statistical Analysis
The statistical analyses were done with the SPSS 13.0 software (SPSS Inc,
Chicago, Illinois). The correlations were calculated using Spearman 
method.
Recombinant Protein Expression
MSP was produced by recombinant expression in insect cells according to the
Bac-to-Bac system (Invitrogen AB, Stockholm, Sweden). The generation of a cDNA
encoding human MSP has been described elsewhere
(Ghasriani et al, 2006). The
cDNA was cloned into the EcoRI site of the transfer vector pFastBac,
and the insertion was verified by DNA sequencing. The recombinant plasmid was
introduced in Escherichia coli H10Bac, carrying a bacmid with the
baculo virus genome. Colonies containing recombinant baculo virus were
identified by blue/white selection and the insertion was confirmed by
polymerase chain reaction using the 17-mer and 16-mer M13/pUC universal and
reverse sequencing primers (New England Biolabs, In Vitro Sweden AB,
Stockholm, Sweden). Mini preparations of recombinant bacmid DNA were
transfected into Sf 9 insect cells using Cellfectin (Invitrogen AB, Stockholm,
Sweden). Recombinant baculo virus was harvested from the supernatant and
titrated by viral plaque assay. The infection of Trichoplusia ni H5
cells was done essentially according to the manual provided by the supplier,
with several pilot expressions to optimize the time and multiplicity of
infection (MOI) for expression. The expression of recombinant MSP was
monitored with the immunoassay and by Western blot as previously described
(Valtonen-André et al,
2005).
Isolation of Recombinant MSP
Frozen culture medium (200 mL) from cells infected with the recombinant
baculo virus was used for purification of MSP. Immediately after thawing, 94.4
g of (NH4)2SO4 was added to yield 70%
saturation (ie, a concentration of 2.8 M). The solution was stirred gently
with a magnetic stirrer at room temperature for 1 hour and then centrifuged at
5000 x g for 20 minutes. The precipitate was discarded, and 1
volume of 99.5% ethanol was added to 4 volumes of the supernatant. The mixture
was stirred as above for 30 minutes and again centrifuged as above. The upper
ethanol phase was saved, and 1 volume of 99.5% ethanol was added to 16 volumes
of the remaining water/salt phase, followed by incubation and centrifugation
as above. The ethanol phases from the first and second extraction were
combined, and 1 volume of n-butanol was added to 4 volumes of the
pooled ethanol phase. After 30 minutes of stirring, the phases were separated
by centrifugation at 5000 x g for 10 minutes. The lower water
phase was saved, whereas the ethanol phase was subjected to a new round of
extraction with the same amount of n-butanol. Again, a water phase
was formed, which was isolated as above and then combined with the water phase
from the first extraction. The water phase was dialyzed against 50 mM Tris pH
7.4 and 0.1 M NaCl concentrated by ultrafiltration with an Omega 3K filter
(PALL Life Sciences, Ann Arbor, Michigan) to a final volume of approximately
1–2 mL and subjected to gel filtration at 4°C using a 1.6 x
100 cm Sephadex G-75 column (Amersham Biosciences). The column was eluted at a
rate of 4.9 mL/h with a buffer containing 25 mM Tris-HCl pH 8.0, 0.15 M NaCl,
and 0.02% NaN3, and 2.4-mL fractions were collected. The recovery
was estimated by the immunoassay. The purification was also monitored by
silver-stained SDS-PAGE with 12% polyacrylamide gels using the Mini PROTEAN II
system (Bio-Rad Laboratories AB, Sundbyberg, Sweden)
(Laemmli, 1970).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Seven standard points were chosen in the range of 0–130 µg/L, and
a standard curve was generated with the logarithmic concentration on the
x-axis plotted against the ratio between the detected signal and the
maximal signal on the y-axis
(Figure 1). The sensitivity, or
lower limit of detection of the assay, was 4.9 µg/L. The intraassay CV for
samples on 3 levels were 13% (
= 5.5
µg/L), 3% ( = 47 µg/L), and 4%
( = 97 µg/L). The interassay CV
for each of the 3 levels was 6% ( =
5.0 µg/L), 3% ( = 55 µg/L), and
3% ( = 88 µg/L). The analytical
recovery of added purified MSP ranged from 102% to 109% and was independent of
whether serum or buffer was used as dilution matrix, and dilution series of
the seminal plasmas down to 1:50 000, had good reproducibility. Also, the
stability of Eu-MSP was high, yielding comparable values in the assay
independent of whether the tracer was freshly thawed or stored in the
refrigerator overnight.
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MSP in Serum and Seminal Plasma
The MSP concentration was measured in serum and seminal plasma from 205
young males, and the median values were calculated. Five serum values were
below the analytical detection limit of the assay and were reported as 0
µg/L. The median MSP concentration in serum was 12 µg/L (2.5–97.5
percentile, 4.9–26 µg/L) and in seminal plasma 0.53 g/L
(2.5–97.5 percentile, 0.13–2.0 g/L). The total amount of MSP in
the seminal plasmas showed a median of 1.8 mg (2.5–97.5 percentile,
0.32–6.6 mg). The MSP concentration in serum correlated significantly to
both the concentration (r = .47, P < .001) and the amount
(r = .50, P < .001) of MSP in seminal plasma
(Figure 2). Hence, the levels
of MSP in serum normally corresponds to less than 10–4 of the
levels in semen.
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Expression and Purification of Recombinant MSP
Recombinant MSP was produced in insect cells using the baculo virus
expression system. Infection at a MOI of 100 yielded an MSP concentration of
35 mg/L in the culture medium after 96 hours of growth. The recombinant
protein displayed the same size as native MSP on SDS-PAGE and gave a strong
and specific reaction with the polyclonal antiserum in Western blotting. A
2-step method based on ethanol extraction and gel filtration was developed for
the purification of recombinant MSP from the culture medium. The recovery of
MSP from the ethanol extraction step was 80%–90%. The extraction also
reduced the volume considerably, to around 5%–10% of the initial cell
culture volume. The purity of the preparation, assessed with silver staining
after SDSPAGE, showed enrichment of MSP and some other low- and high-molecular
mass proteins after the ethanol extraction. To further purify MSP, the
material was subjected to gel filtration on a Sephadex G-75. This final step
yielded a homogeneous MSP preparation with very few low abundant contaminants,
as shown on the silver-stained SDS-PAGE gel
(Figure 5).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In an earlier study, it was shown that MSPs in serum of women have a median concentration of around 65% of the values in men (Abrahamsson et al, 1989). The source of this nonprostatic MSP could be the trachea or the stomach because the expression in these organs is high. Such extraprostatic MSP most likely affects the serum values measured in this study, but the contribution of prostate-secreted MSP is presumably high, given the correlation between the serum and seminal plasma values. The median value of 12 µg/L reported for the serum MSP concentration in our study of military conscripts is double that of the median value reported in a previous study (Abrahamsson et al, 1989). This could be at least in part because of differences in the composition of each study cohort, but most likely it is also due to differences in assay design, and comparison between values obtained with dissimilar methods which are not uniformly standardized should be interpreted with great caution.
As expected, the prostate parameters measured in seminal plasma all closely correlate with MSP in seminal plasma. The strongest correlation was that between MSP and PSA, but their covariance in seminal plasma is not currently known to reflect any functional relationship. It is well known that the Zn2+ found in seminal plasma is secreted mainly by the prostate, and the concentration in the ejaculate is around 100-fold higher than that in blood plasma (Mann and Lutwak-Mann, 1981; Shivaji et al, 1990). In semen, Zn2+ also strongly correlates with MSP in this study. Whether this cation has any biological role associated with MSP is not known, and because MSP has no Zn2+ binding sites, the correlation could merely reflect their common glandular secretory origin.
MSP is a stable protein in various laboratory settings, and an investigation of the proteolytic degradation of human seminal plasma proteins under acidic conditions has shown that MSP was one of the proteins most resistant to proteolysis by the aspartic protease progastricsin (Szecsi and Lilja, 1993). Stability in the face of proteolytic degradation, combined with covariation with other prostate-secreted components and to the serum levels of MSP, suggests that MSP could be a suitable semen marker for the secretory function of the prostate. A major advantage with the newly developed MSP assay is the microtiter plate format, which has enabled automation of the assay. Its usefulness was clearly demonstrated with the samples from military conscripts because the sensitivity of the assay made it possible to measure MSP in 98% of the serum samples and, after appropriate dilution, in all of the seminal plasma samples.
An MSP assay for clinical use, requires reliable access to protein for preparation of reagents, and the generation and purification of recombinant MSP described in this paper enables us to produce considerable amounts of the protein without having to rely on sperm donors. The baculo virus system was selected because of the high yield and the use of eukaryotic cells, with their higher probability of correct folding of the peptide chain that contains 5 disulfides. The expression in insect cells could affect posttranslational modifications, but because MSP is nonglycosylated and also is lacking other modifications, this was not a problem. It has previously been shown that ethanol added to seminal plasma containing 2.8 M (NH4)2SO4 forms an organic phase that is enriched with MSP (Fernlund et al, 1994). We used the same strategy to enrich MSP from the culture medium. By adding n-butanol to the organic phase, the polarity is decreased in such a way that a water phase is recreated and simultaneously MSP is quantitatively transferred to the water phase. A similar procedure has been described for the purification of Green Fluorescent Protein from the jellyfish Aequorea victoria (Yakhnin et al, 1998). The modified method used in this study presents an excellent yield of MSP after ethanol extraction, and the subsequent gel filtration chromatography gives an MSP product of high purity.
In this study, we developed an immunoassay that we used to measure MSP in serum and seminal plasma of 205 young males. The levels of MSP in serum were found to reflect the prostate release of MSP, and strong correlations were also found in seminal plasma between MSP and the prostate parameters PSA and Zn2+. Furthermore, the methods described for the generation and purification of recombinant MSP will facilitate future MSP studies and assay designs.
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Acknowledgments |
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Footnotes |
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