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Biochemistry and Biophysics |
Institut für Organische Chemie und Biochemie, Technische Universität München, 85748 Garching, Germany [J. P., C. U.]; PROTANA A/S, DK-5230 Odense M, Denmark [T. N. K., O. V.]; and Roche Diagnostics GmbH, 82372 Penzberg, Germany [W. H.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reason for the presence of F-PSA in human blood is not yet clear,
but this F-PSA should be a proteolytically inactive form, otherwise it
would be complexed by protease inhibitors (e.g., 
1-ACT
and 2-macroglobulin), which are present in the blood in large
amounts (9, 10, 11)
. Two different explanations have been
proposed thus far for this finding: (a) the F-PSA
could exist as an inactive form resulting from a nick in the PSA
sequence, probably at the lysine at position 145; and (b)
alternatively pro-PSA forms (zymogen forms) could be present that still
carry all or parts of the signal sequence (12
, 13)
.
Experimental evidence for the presence of a nicked form was presented
by Noldus et al. (14)
, who isolated partially
purified F-PSA from a 230-ml pool of 59 PCa sera (T-PSA, >2000 ng/ml).
Gas phase sequencing provided evidence for the presence of a form with
the regular NH2 terminus and a nicked form. There
was no indication for the presence of pro-PSA forms, but the F-PSA
yield of their preparation was only about 25%, and it still contained
impurities. Thus, the authors might have missed some minor PSA forms
that could also have been present. On the other hand, Mikolajczyk
et al. (15)
found evidence for the presence of
PSA forms larger than the F-PSA from seminal fluid when they
separated the PSA from 75 ml of pooled PCa sera (containing 50–100
ng/ml T-PSA) by a combination of immunosorption and hydrophobic
interaction liquid chromatography. The HIC high-performance
liquid chromatography showed that about 25% of the F-PSA eluted at a
similar position as the (-4) pro-PSA form from cultured prostate
cells, but there was no further characterization. The authors, using
Western blotting techniques after SDS-PAGE separation, did not find
clear evidence for the presence of nicked F-PSA.
Different pro-PSA forms have been produced and clearly identified (mostly by NH2-terminal sequencing) in several cell culture systems after the cloning of the prepro-PSA gene and isolation of the expressed PSA forms. In a Syrian hamster tumor cell line, pro-PSA forms with seven and five amino acids in the precursor part were identified by Kumar et al. (16) . Lövgren et al. (17) identified the same forms after cloning and expression of prepro-PSA in BHK cells as well as in a baculovirus system. In the latter system, a form with three amino acids corresponding to the precursor part was identified as well. Additionally a (-7)pro-PSA form was expressed and characterized in Escherichia coli cells (18) .
Here we describe the unequivocal identification of different pro-PSA forms in the serum from an individual PCa patient. The F-PSA was isolated by immunosorption, and the intact molecule and its peptides were analyzed by MALDI-TOF MS. Moreover, the relative quantities of the pro-PSA forms and the F-PSA form with the regular NH2 terminus were investigated in four other PCa serum samples.
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MATERIALS AND METHODS |
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All of the other products used were either from Roche Molecular Biochemicals or from Merck GmbH (Darmstadt, Germany) if not indicated otherwise. Among these products were PBS, 50 mM potassium phosphate, and 150 mM sodium chloride (pH 7.4).
Isolation of F-PSA by Immunosorption
The immunosorption methods used have recently been described in
detail (19)
. The following protocol was used here: a
suspension (2.5 ml) of streptavidin-coated magnetic beads (10.7
mg/ml) was placed in a 10-ml tube and washed with PBS. After the
addition of 4 ml of biotinylated anti-F-PSA-M30-IgG (monoclonal
antibody from mouse, used in the Roche Diagnostics ELECSYS test for
F-PSA; 25 µg/ml in PBS containing 1% BSA and 0.1% Tween 20), the
suspension was incubated for 30 min. The beads were collected, washed
three times with PBS containing 20 mM
N-octylglucoside and incubated with 8 ml of the PCa serum A
(containing 180 ng/ml F-PSA) for 1 h to bind the F-PSA to the
beads. The beads were collected and washed as described before,
followed by a very short washing step with 200 µl of water to remove
most of the detergent. The beads were then incubated under shaking with
500 µl of 1 M propionic acid for 1 h.
After magnetic separation of the beads, the supernatant was removed,
lyophilized in a vacuum concentrator, and stored at -20°C if further
analysis was not performed immediately after lyophilization. In case of
the other four PCa sera (F-PSA > 600 ng/ml) a volume of
100 µl of the sera was used for immunosorption, and the amounts of
reagents were adjusted accordingly. For these sera, biotinylated IgG
from the monoclonal antibody M36, which recognizes free as well as
complexed PSA and is used in the Roche Diagnostics ELECSYS test for
T-PSA was used for the immunosorption, because we wanted to isolate the
PSA/ACT complex as well as the F-PSA by separation on SDS-PAGE
(see Fig. 4
).
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MS
Determination of the Molecular Mass of F-PSA.
The samples were analyzed in a Voyager Biospectrometry Workstation
MALDI mass spectrometer equipped with delayed extraction, operating in
the positive mode of detection. The spectrometer contains a nitrogen
laser operating at 337 nm. TOF spectra were produced at 25 kV
acceleration voltage by averaging 80 single spectra. A matrix
consisting of a solution of ferulic acid (4-hydroxy-3-methoxy cinnamic
acid; 10 mg/ml) in formic acid:water:acetonitrile (1:3:2, v/v/v) was
used for all determinations. PSA from semen was used as a reference
solution at a concentration of 2 pmol of protein/µl of distilled
water. The eluates from the immunosorption procedures were dissolved in
10 µl of distilled water. An aliquot (0.5 µl) of this protein
solution was mixed with 1 µl of the matrix solution on the target
plate and allowed to dry at room temperature, prior to insertion into
the mass spectrometer. All of the spectra were calibrated externally
using the singly charged ion of BSA ([M + H], 66,431
Da) and the doubly charged ion of horse skeletal apomyoglobin
([M + 2H]2+, 16,953 Da) as references.
Analysis of F-PSA Peptides.
The endo Lys-C in-gel digests of the purified F-PSA were analyzed by
MALDI-MS on either a Bruker Reflex III or a PerSeptive Biosystems
STR mass spectrometer both equipped with delayed extraction and
reflectors. All of the spectra were acquired in positive ion mode using
a reflector mode for higher resolution. Samples were prepared on top of
a fast evaporating thin-layer preparation of
4-hydroxy--cyano-cinnamic acid (25 g/liter) and nitrocellulose (2
g/liter) in acetone. Sample (0.75 µl) was mixed with 0.75 µl of 2%
trifluoroacetic acid, allowed to dry, and washed with 10 µl of
ultra-high-quality water. Two hundred single-shot spectra were averaged
and externally calibrated using Angiotensin I.
Sequencing of pro-PSA peptides was carried out using nanoelectrospray ionization Q-TOF MS, carried out on a SCIEX Qstar. The sample were desalted on a Poros R2,20 column packed in a GEloader tip (Eppendorf) and eluted into a nanoelectrospray capillary (Protana) by 50% methanol, 5% formic acid. Spectra were acquired in positive ion mode.
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RESULTS |
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. This molecular mass is about 600 Da above the value for
F-PSA from seminal fluid (28,580 Da). This reference spectrum is shown
in Fig. 1A
.
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Analysis of F-PSA Peptides.
F-PSA (180 ng) isolated from PCa serum A separated by SDS-PAGE. The
silver-stained band of F-PSA was cut out, alkylated under reducing
conditions, and digested by endo Lys-C as described under
Methods. Fig. 2
shows the peptide patterns obtained by MALDI-TOF MS from this band
(A) and a PSA band from seminal fluid (B) which
had been treated similarly. Table 1
contains a list of molecular masses for all of the PSA peptides
observed in these two preparations and the expected theoretical values.
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). All of
the other peptides originating from PSA in the two samples were
identical. Moreover, the peptide patterns that were obtained from PSA
released from the PSA/ACT complex (22)
and PSA from
seminal fluid were identical to each other and showed only the
NH2-terminal peptide amino acids 1–9. In these
cases, pro-PSA forms were absent. The sequence coverage obtained by endo Lys-C digestion was calculated to be 83%. One minor peptide (amino acids 168–170; Mr 346.2) and the peptide carrying the predicted N-glycosylation site were missing (amino acids 10–46). We assume that the N-glycopeptide was not observed because of signal suppression. Because all of the mass spectrometric experiments were carried out in reflector mode and the glycans were not derivatized, significant metastable losses of the glycans may have occurred, and these altered fragments are normally not detected in reflector mode MALDI-MS.
Sequencing of the Pro-PSA Peptide (Amino Acids -7 to +9) by
Electrospray Ionization Q-TOF MS.
To unequivocally identify the pro-PSA forms the longest pro-PSA peptide
(amino acids -7 to +9; Mr 1827.97)
was sequenced by Q-TOF MS. The resulting spectrum is displayed in Fig. 3
, and the observed masses were assigned to the resulting
sequences as shown in Table 2
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Analysis of Additional PCa Sera for the Presence of Pro-PSA Forms.
Four other sera (S1, S5, S6, and S9) of PCa patients with a high
content of T-PSA (>6000 ng/ml) were further examined for the presence
of pro-PSA peptides. The analysis followed the same protocol as
described above for the analysis of F-PSA peptides, and Fig. 4
shows a representative silver-stained SDS-PAGE gel of the PSA fractions
isolated from these four sera. The biotinylated monoclonal antibody
M36, which recognizes T-PSA, and the immunosorption method with 1
M propionic acid elution, as described above, were used in
these studies. The bands representing the PSA/ACT complex and F-PSA
could clearly be identified on the gel, although some other
contaminating protein bands were still present. The F-PSA bands were
cut out and treated as described above, digested with endo Lys-C and
the resulting peptides analyzed by MALDI-TOF MS. The result of the
analysis is shown in Table 3
, where the relative signal heights of the
NH2-terminal PSA peptides obtained from the F-PSA
of these four sera are compared with the peptide signals obtained from
F-PSA of PCa serum A (in each case calculated as percentage of the
total amount of F-PSA present in that sample).
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should be regarded with caution, considering that
they were obtained in different experiments and that quantification of
MALDI-TOF MS is generally problematic. However, it can be assumed that
the F-PSA form with a regular NH2 terminus
represents a major part of the F-PSA of the sera S1 and S 5. The (-3)
and (-6) pro-PSA forms could not be detected in any of the
samples investigated thus far. |
DISCUSSION |
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1-ACT and
2-macroglobulin). Whether this inactivity is related to internal
nicking of the PSA or the presence of pro-PSA forms has largely been a
matter of speculation (12
, 13)
. Some evidence for the
presence of a nicked form came from data by Noldus et al.
(14)
, but this was not corroborated by Mikolajczik
et al. (15)
, who did not find substantial
amounts of a nicked form. Instead their HIC high-performance
liquid chromatography data provided evidence that about 25% of the
F-PSA seemed to be present as pro-PSA forms when compared with the
elution of a pro-PSA reference obtained from cell cultures. A
structural analysis of that peak was not reported. The contradictory
results may be related to the use of pooled PCa sera. Moreover only low
amounts of partially purified F-PSA were available, which prevented a
thorough structural analysis (14)
. In a recent paper by
Hilz et al. (23)
, it was reported that pro-PSA
forms could not be detected in the F-PSA preparation that was isolated
by immunosorption from blood aspirated during transvesical
prostatectomy of BPH patients. This was concluded from the sequencing
of the F-PSA by Edman degradation on polyvinylidene difluoride
membranes. Furthermore, it was stated in that paper that no pro-PSA
forms could be detected in PCa and BPH sera when probing with an
antibody that had been raised against the propeptide moiety of the
zymogen. The development of efficient immunopurification procedures using streptavidin-coated magnetic beads (19) enabled us to isolate sufficient amounts of F-PSA from individual PCa sera for an analysis using state-of-the-art MS. Thus, we could unequivocally demonstrate the presence of pro-PSA forms in the serum of a PCa patient and identify their structures. Moreover, by looking at other PCa sera, we found that F-PSA with a regular NH2 terminus can also be present. This could explain the results obtained by Noldus et al. (14) as well as those of Mikolajczik et al. (15) , because F-PSA, with a regular NH2 terminus, as well as pro-PSA forms can be present in PCa sera and were, therefore, very likely present in the pooled PCa sera used by those authors. In contrast to Mikolajczik et al. (15) , the presence of pro-PSA forms with seven and five amino acids in the pro-PSA part could be detected in all of the PCa sera investigated by us. However, the form with the regular NH2 terminus was only present in some of the sera. Thus far, we have not investigated the lack of a complex produced by protease inhibitors of this F-PSA form carrying the regular NH2 terminus. It is tempting to speculate about the occurrence of a nicked form, but this needs to be examined in additional experiments, using an approach different from the one described above (e.g., using SDS-PAGE separation under reducing conditions in combination with suitable specific detection methods for the analysis of F-PSA forms, which has not been done by us thus far).
The results presented above demonstrate that F-PSA in sera from PCa patients occurs as a combination of different pro-PSA forms, and that a form with the regular NH2 terminus can be present as well in some sera. Among the individual sera, the presence of, and the relative amounts of, the different PSA forms seem to vary. The origins of these differences are not known. The five sera used in this study contained very high amounts of PSA. In PCa and BPH sera, in which there are low amounts of PSA (e.g., in the diagnostically interesting range of 2–15 ng/ml), the relative amounts of the various F-PSA forms could differ significantly from samples with a high PSA content or might not be present at all. For this reason comparisons to the results obtained by Mikolajczik et al. (15) and Hilz et al. (23) , who were using sera with a much lower PSA concentration, have to be made with much caution because they may or may not be relevant. However, to be able to analyze sera with low PSA content for the presence of pro-PSA forms, we have prepared a monoclonal antibody that binds to the three pro-PSA forms (-7,-6,-5), and used it to develop an immunoassay that specifically recognizes these precursor forms. A preliminary analysis of panels of PCa and BPH sera of high and low PSA content showed that the pro-PSA forms could be detected almost in all of the sera analyzed.6 Whether this test can be used to better differentiate between BPH and PCa in the diagnostically gray area of 2–15 ng/ml T-PSA compared with the state-of-the-art methods (e.g., by using the ratio of F-PSA:T-PSA) will be evaluated.
In this context, a very recent report from Mikolajczik et al. (24) , dealing with the identification of pro-PSA forms in PCa and benign transition zone prostate tissue, is also shedding light on the issue of F-PSA isoforms. As reported earlier for sera (15) , they identified the (-4) and (-2) forms of pro-PSA in tissue extracts after immunopurification by NH2-terminal sequencing. In contrast to our report, they again did not find pro-PSA forms with the longer precursor sequences (e.g., -7 or -5), and the reason for this is not clear to us at this time. Interestingly enough, the pro-PSA forms were present more in PCa tissues than in BPH. Whether this reflects a similar situation in the sera has to be elucidated in the future using a panel of specific antibodies for the different pro-PSA forms.
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FOOTNOTES |
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1 Present address: National Institute of
Environmental Health Sciences (NIH/NIEHS), Building 101, Room F011,
Research Triangle Park, NC 27709. 
2 Present address: Lehrstuhl für
Bioorganische Chemie, Universität Bayreuth, Gebäude NW 1,
95440 Bayreuth, Germany.
3 To whom requests for reprints should be
addressed, at Roche Diagnostics GmbH, Nonnenwaldstrasse 2, 82372
Penzberg, Germany. Phone: 49-8856-603274; Fax: 49-8856-603341; E-mail: wolfgang.hoesel{at}roche.com
4 The abbreviations used are: PSA,
prostate-specific antigen; F-PSA, free PSA; T-PSA, total PSA; PCa,
prostate cancer; BPH, benign prostatic hyperplasia; MALDI,
matrix-assisted laser desorption ionization; TOF, time-of-flight; MS,
mass spectrometry; Q-TOF MS, quadrupole TOF MS; endo Lys-C,
endoproteinase from Lysobacter enzymogenes; ACT, antichymotrypsin.
5 The direct immunosorption method was used in
this case because it gives higher yield of a reasonably pure F-PSA
compared with the indirect method using digoxigenin-Lys.HCL for
elution, which provides higher purity but lower yield (see Ref.
19
).
6 J. Peter, C. Hübner-Parajsz, P. Stieber,
and W. Hoesel. Development and clinical evaluation of an immunoassay
for the measurement of pro-PSA forms in BPH and PCa sera. Manuscript in
preparation.
Received 8/ 9/00. Accepted 11/20/00.
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REFERENCES |
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1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostate cancer: assay of the complex improves clinical sensitivity for cancer.. Cancer Res., 51: 222-226, 1991.1-antichymotrypsin.. Clin. Chem., 37: 1618-1627, 1991.2-macroglobulin.. Clin. Chem., 44: 2471-2479, 1998.1-antichymotrypsin in blood samples.. Urology, 48(Suppl.6A): 81-87, 1996.[Medline]
1-antichymotrypsin complex. Clin. Chem., 41: 1480-1488, 1995.1-antichymotrypsin (PSA-ACT).. Clin. Chem., 46: 474-482, 2000.This article has been cited by other articles:
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  C. STEPHAN, H. RITTENHOUSE, H. CAMMANN, M. LEIN, M. SCHRADER, S. DEGER, K. MILLER, and K. JUNG New Markers and Multivariate Models for Prostate Cancer Detection Anticancer Res, July 1, 2009; 29(7): 2589 - 2600. [Abstract] [Full Text] [PDF]
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 G. Tabares, C. M. Radcliffe, S. Barrabes, M. Ramirez, R. N. Aleixandre, W. Hoesel, R. A. Dwek, P. M. Rudd, R. Peracaula, and R. de Llorens Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA Glycobiology, February 1, 2006; 16(2): 132 - 145. [Abstract] [Full Text] [PDF]
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 K. Jung, J. Reiche, A. Boehme, C. Stephan, S. A. Loening, D. Schnorr, W. Hoesel, and P. Sinha Analysis of Subforms of Free Prostate-Specific Antigen in Serum by Two-Dimensional Gel Electrophoresis: Potential to Improve Diagnosis of Prostate Cancer Clin. Chem., December 1, 2004; 50(12): 2292 - 2301. [Abstract] [Full Text] [PDF]
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S. D. Mikolajczyk, W. J. Catalona, C. L. Evans, H. J. Linton, L. S. Millar, K. M. Marker, D. Katir, A. Amirkhan, and H. G. Rittenhouse Proenzyme Forms of Prostate-Specific Antigen in Serum Improve the Detection of Prostate Cancer Clin. Chem., June 1, 2004; 50(6): 1017 - 1025. [Abstract] [Full Text] [PDF]
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R. Peracaula, G. Tabares, L. Royle, D. J. Harvey, R. A. Dwek, P. M. Rudd, and R. de Llorens Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins Glycobiology, June 1, 2003; 13(6): 457 - 470. [Abstract] [Full Text] [PDF]
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H. J. Linton, L. S. Marks, L. S. Millar, C. L. Knott, H. G. Rittenhouse, and S. D. Mikolajczyk Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease Clin. Chem., February 1, 2003; 49(2): 253 - 259. [Abstract] [Full Text] [PDF]
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T. Isono, T. Tanaka, S. Kageyama, and T. Yoshiki Structural Diversity of Cancer-related and Non-Cancer-related Prostate-specific Antigen Clin. Chem., December 1, 2002; 48(12): 2187 - 2194. [Abstract] [Full Text] [PDF]
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P. Niemela, J. Lovgren, M. Karp, H. Lilja, and K. Pettersson Sensitive and Specific Enzymatic Assay for the Determination of Precursor Forms of Prostate-specific Antigen after an Activation Step Clin. Chem., August 1, 2002; 48(8): 1257 - 1264. [Abstract] [Full Text] [PDF]
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 S. D. Mikolajczyk, K. M. Marker, L. S. Millar, A. Kumar, M. S. Saedi, J. K. Payne, C. L. Evans, C. L. Gasior, H. J. Linton, P. Carpenter, et al. A Truncated Precursor Form of Prostate-specific Antigen Is a More Specific Serum Marker of Prostate Cancer Cancer Res., September 1, 2001; 61(18): 6958 - 6963. [Abstract] [Full Text] [PDF]
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