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Articles |
1-Antichymotrypsin (PSA-ACT)
1
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany.
2
Roche Diagnostics GmbH, Nonnenwaldstrasse 2, 82372
Penzberg, Germany.
a Author for correspondence. Fax 49-8856-603341; e-mail wolfgang.hoesel{at}roche.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin (ACT) in human blood. Structural
analysis of the PSA-ACT complex is difficult, and complexation may be a
reason for biased immunological assays when compared with the analysis
of free PSA. We developed a method to cleave the PSA-ACT complex
chemically. The liberated PSA was thus available for analysis as free
PSA (F-PSA). Methods: PSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry.
Results: In vitro-prepared PSA-ACT complex was completely cleaved
by treatment with nucleophilic compounds such as ethanolamine at pH
9–10. The released PSA was stable under these conditions and could be
measured by reversed-phase HPLC as well as the ENZYMUN®
immunoassay for F-PSA. When plasma from a CaP patient [containing 190
µg/L F-PSA and 1890 µg/L total PSA (T-PSA)] was treated
under similar conditions, a concentration of 
1600 µg/L F-PSA was
measured at the end of the incubation, indicating that the PSA-ACT
complex was completely cleaved. Two benign prostatic hyperplasia and
CaP sera panels (12 and 13 sera, respectively) containing 4–45 µg/L
T-PSA were similarly treated. The concentrations of F-PSA measured
after incubation were, on average, 85% of the T-PSA values of the
untreated sera. Finally, the PSA released from the complex of the CaP
plasma was isolated by immunosorption, analyzed by MALDI-TOF mass
spectrometry, and compared to PSA obtained from semen. The intact PSA
as well as the peptides observed after digestion with endoproteinase
Lys C did not reveal any structural difference between the PSA from
these two sources.
Conclusions: PSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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15 µg/L in serum. Therefore,
PSA concentrations <15 µg/L cannot be used to distinguish between
CaP and BPH (3). Furthermore, PSA is present in human blood
as a complex mixture of several species. The main immunologically
detectable form is a covalent complex of PSA with the serine protease
inhibitor (serpin) 1-antichymotrypsin (ACT)
(4)(5). Moreover, the presence of additional
PSA-serpin complexes in serum has been reported, albeit in much lower
concentrations than the PSA-ACT complex [see, for example, Refs.
(4)(6)]. A complex of PSA with
2-macroglobulin is also present, but to
date, it has not been detectable by clinically used immunological tests
and, therefore, does not contribute to the PSA values measured by these
tests (4)(7)(8). Free (uncomplexed)
PSA (F-PSA) also is present, accounting for 5–30% of the total PSA
(T-PSA). This PSA form is enzymatically inactive and cannot form
complexes with the protease inhibitors because of internal nicking or
the existence of proPSA forms [see, for example, Ref.
(9)]. The complex distribution of PSA leads to
uncertainties in the immunological measurement of PSA (10).
It has been reported that some tests display a bias toward
overestimation of the F-PSA form because of the specificity of the
antibodies used (7)(11)(12).
Furthermore, it is difficult to standardize the tests because of the
various forms present (13). Considering that the ratio of
free to total PSA (and also the ratio of F-PSA to the PSA-ACT complex)
is being used for better differentiation between CaP and BPH than T-PSA
alone (4)(5)(14), the exact
measurement of these two markers is of importance for the value
of PSA as a tumor marker. It would therefore be desirable to be able to
measure the ratio of free to complexed PSA using only one immunological
test, thus eliminating any possible bias between two immunological
assays.
Complexes between proteases and serpins have been described as covalent
and stable (15)(16), although noncovalently
linked complexes that do not dissociate in the presence of sodium
dodecyl sulfate (SDS) have also been reported (17). There is
a considerable amount of information available on the structures and
mechanisms of formations of proteinase-serpin complexes [see, for
example, Refs. (15)(16)(18)], but
little information is available on PSA-ACT. In the first report on
complex formation of PSA with ACT, it was concluded from
electrophoretic mobility that PSA forms a covalent linkage to Leu-358
of ACT, giving an SDS-stable complex and releasing the C-terminal
peptide during SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(19). It was later shown that PSA-ACT was somewhat unstable
when stored in buffers at pH 7.5 and 35 °C over several weeks,
releasing F-PSA measurable by an immunological assay (20).
When a 1000-fold excess of ACT was used and the pH of the buffers was
adjusted to 6.8, the cleavage could be largely prevented. Similarly, a
complex of PSA and 1-protease inhibitor (API)
formed in vitro was shown to dissociate 30–40% when kept at
37 °C for 7 days, yielding enzymatically active PSA and an inactive
API that was cleaved between Met-358 and Ser-359 (21).
Our investigations of the structural features of PSA in serum led to the development of a method for the rapid chemical release of PSA from the PSA-ACT complex, using alkaline ethanolamine treatment. After immunoaffinity purification (22), the liberated PSA was analyzed by mass spectrometry (MS) and immunological assays.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All other products used were from Roche Molecular Biochemicals or Merck, if not indicated otherwise.
The following buffers were used for the immunosorption: (a) incubation buffer [phosphate-buffered saline (PBS) containing 10 g/L bovine serum albumin and 1 g/L Tween 20, pH 7.4]; and (b) washing buffer (PBS containing 20 mmol/L octylglucoside, pH 7.4).
cleavage of psa-act reference material in buffer
PSA-ACT (4 µL of a 1 g/L solution in PBS) was added to 76 µL
of the respective cleavage buffers described below and incubated
between 0 and 240 h at 25 °C. The cleavage buffers consisted of
PBS with the nucleophiles added to the indicated concentrations
described under Results (0.1–1 mol/L). After addition of
the nucleophiles, the buffer was adjusted to pH 9 with 1 mol/L
hydrochloric acid. At the end of the incubation, the samples were
analyzed by reversed-phase HPLC and the ENZYMUN®
assays for F-PSA and T-PSA.
reversed-phase hplc
A Poros R1/H reversed-phase column (2.1 x 30 mm; Perseptive
Biosystems) was used for the analysis of the PSA-ACT cleavage assays.
The flow rate was 0.5 mL/min, and absorbance was monitored at 215 nm.
Eluent A consisted of 1 g/L trifluoroacetic acid in
distilled water, and eluent B consisted of 0.85 g/L trifluoroacetic
acid in a mixture of acetonitrile-distilled water (7:3, by volume). The
following linear step gradient was used: 0–2 min, 20% eluent B; 2–5
min, 20–55% eluent B; 5–13 min, 55–100% eluent B; 13–15 min,
100% eluent B. A 10-µL sample was injected for each analysis.
cleavage of psa-act in the CaP AND BPH SERA
A solution of 270 µL of PBS and 30 µL of 2 mol/L ethanolamine
(pH 12) was added to 300 µL of serum (final pH of 10.3) and incubated
at 25 °C for 24 h. After that time, the samples were analyzed
without further treatment by the ENZYMUN assays for F-PSA and
T-PSA.
immunological assays
The ENZYMUN assays for F-PSA and T-PSA were performed with an ES
600 automatic analyzer from Roche Diagnostics GmbH as described by the
supplier.
isolation of psa after cleavage of the psa-act complex
A suspension of streptavidin-coated magnetic beads (1.25 mL;
10.7 g/L) was placed in a 10-mL tube and washed with PBS. After
the addition of 2 mL of biotinylated anti- F-PSA IgG (monoclonal
antibody from mouse; 25 mg/L) the suspension was incubated for 30 min.
The beads were collected, washed three times, and incubated with 3.8 mL
of the CaP plasma for 1 h to bind the F-PSA to the beads. After
removal of the beads, the supernatant was incubated with 200 µL of 2
mol/L ethanolamine (pH 12) at 25 °C for 24 h. The pH of the
reaction mixture was then adjusted to 7.8 with 0.1 mol/L hydrochloric
acid, and the released F-PSA was isolated by immunosorption: A
suspension of magnetic beads (1.25 mL; c = 10.7 g/L) was again
placed in a 10-mL tube and washed as described. After the addition of
another 2 mL of biotinylated anti-F-PSA IgG (25 mg/L in incubation
buffer) the suspension was incubated for 30 min. The beads were washed
three times and incubated for 1 h with the cleavage reaction
mixture described above. The suspension was washed as described and
treated with 250 µL of 1 mol/L 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.
sds-page and digestion by endo lys c
SDS-PAGE was performed under nonreducing conditions, using the
MiniPROTEAN II gel electrophoresis system and preformed 4–20%
gradient gels from Bio-Rad, essentially using the protocol described by
Laemmli (23). The gels were silver stained, and the PSA band
was digested with endo Lys C, an endoproteinase from Lysobacter
enzymogenes, as described by Shevchenko et al. (24).
maldi-tof ms
The samples were analyzed in a VoyagerTM
BiospectrometryTM Workstation VESTEC
matrix-assisted laser desorption-induced time of flight (MALDI-TOF)
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
saturated solution of ferulic acid (4-hydroxy-3-methoxycinnamic acid)
in formic acid-water-acetonitrile (1:3:2, by volume) was used for all
determinations. PSA from semen was used as a reference solution at a
concentration of 2 pmol of protein per milliliter of distilled water.
The eluates from the immunosorption procedures were dissolved in 10
µL of distilled water. An aliquot of this protein solution (0.5 µL)
was mixed with 1 µL of the matrix solution on the target plate and
allowed to dry at room temperature before insertion into the mass
spectrometer. All spectra were calibrated externally using bovine serum
albumin, [M+H]+ = 66 431 Da, and horse
skeletal apomyoglobin, [M+H]+ = 16 953 Da, as
references.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In further agreement with previous reports (19), we found
that the PSA-ACT complex is rather stable during storage in buffer.
Incubation in PBS buffer at pH 7.3 or 11.3 at 25 °C for 60 h
revealed no cleavage or loss of the complex as indicated by
reversed-phase HPLC analysis (data not shown). Similarly, F-PSA did not
show any alteration of the HPLC peak after storage under similar
conditions. However, a substantial cleavage of the PSA-ACT complex was
observed when nucleophilic compounds such as ethanolamine were added to
the storage buffer, as is illustrated in Fig. 1
.
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The PSA-ACT complex disappeared almost completely after storage at
25 °C in PBS (pH 9.0) containing 1 mol/L ethanolamine, and two peaks
representing PSA and ACT increased in size with longer storage
times. Cleavage of PSA-ACT was also achieved by other reagents
similar to ethanolamine, as shown in Table 1
.
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Interestingly, nucleophiles such as methylamine or the hydroxyl ion are not able to catalyze the cleavage effectively. Compounds with at least two nucleophilic groups seem to be required. Furthermore, strong nucleophiles such as hydroxylamine or hydrazine appear to degrade the complex altogether, because the PSA-ACT peak disappeared entirely without the concomitant appearance of PSA and ACT peaks. The cleavage rate of the PSA-ACT complex was dependent on the ethanolamine concentration. Reducing the concentration of ethanolamine from 1 mol/L to 0.1 mol/L reduced the product rate to 30% of the initial value (data not shown).
immunological analysis of the released psa
In addition to HPLC analysis, PSA was determined immunologically
in the cleavage assays to probe the released PSA for immunologically
recognizable epitopes. The measurements of total and free PSA were
performed with an ENZYMUN analyzer, and the results are displayed
in Fig. 2
.
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As shown in Fig. 2
, the PSA released from the complex was recognized in
the ENZYMUN assays for F-PSA as well as for T-PSA. In further
accordance with the HPLC analysis, no PSA could be detected after
treatment of the complex with hydroxylamine, indicating the degradation
of PSA. Ethanolamine treatment seemed to retain the immunological
properties of F-PSA, as indicated in Fig. 2
. Nearly identical values
were found in the total and free PSA ENZYMUN assays after incubation of
F-PSA for 60 h in the presence of 0.1 mol/L ethanolamine at pH 9.
cleavage of psa-act in CaP PLASMA
To determine whether the PSA-ACT complex in human blood can be
cleaved similar to the complex formed in vitro, plasma from a CaP
patient (T-PSA, 1890 µg/L; F-PSA, 190 µg/L) was treated with 0.1
mol/L ethanolamine for 60 h at different pH values, and the
release of F-PSA was monitored using the ENZYMUN assays. The results of
the incubations are shown in Fig. 3
.
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It can be seen that only a minor release of F-PSA is observed after incubation with 0.1 mol/L ethanolamine at pH 8.4. Raising the pH to 9.3 increases the value of F-PSA considerably within 24 h of incubation. After incubation for 48 h at pH 10.3 in the presence of ethanolamine, the F-PSA value is close to the T-PSA value of 1890 µg/L. The reaction proceeded at an appreciable velocity, as indicated by the 0 h values at pH 9.3 and 10.3, which already were well above the starting value of 190 µg/L. Release occurred between the addition of the ethanolamine and the withdrawal and freezing of the sample aliquot for the immunological analysis.
cleavage of psa-act in CaP AND BPH SERA WITH LOWER PSA
CONTENT
We applied the ethanolamine cleavage of PSA-ACT to the measurement
of T-PSA, using only the assay for F-PSA. Thus, a panel of BPH and CaP
sera was incubated for 24 h in the presence of 0.1 mol/L
ethanolamine as described in Materials and Methods, and the
F-PSA content was analyzed by the ENZYMUN assay. In addition, the free
and total PSA values of the untreated sera were determined in parallel.
The results of these experiments are shown in Table 2
. The T-PSA values measured as F-PSA after treatment correlated
very well with the values determined in the ENZYMUN assay for T-PSA
(r = 0.97 for both panels).
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maldi-tof ms analysis of psa released from psa-act
complex of CaP PLASMA
The PSA released from the PSA-ACT complex of the CaP plasma used
above was isolated by immunosorption as described previously
(22). F-PSA was first removed from the plasma by
immunosorption, confirming that no F-PSA remained. Subsequently, the
plasma was treated with ethanolamine as described above, and the
released PSA was isolated by immunosorption using a biotinylated
antibody with high affinity to F-PSA only. The PSA was isolated by
SDS-PAGE, analyzed by MALDI-TOF MS, and compared to the PSA reference
material from semen. The results are displayed in Fig. 4
.
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The two samples showed almost identical molecular masses and similar
peak patterns. An additional shoulder at 28.2 kDa was present in the
spectrum of the PSA obtained from the complex. This was later shown to
be caused by contamination with apolipoprotein A, which bound
nonspecifically to the magnetic beads and moved similarly to PSA in
SDS-PAGE (compare also Fig. 5
).
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After SDS-PAGE separation, the F-PSA from the two sources was compared
in detail by protease digests with endo Lys C. Fig. 5
shows that the peptide patterns of the two PSA samples were
very similar and matched the theoretical values predicted from the PSA
sequence (78% coverage of the sequence).
The additional peptides observed in the PSA sample matched the peptides
predicted for the endo Lys C digest of human apolipoprotein A, thus
revealing that this protein of 28.2 kDa was a contaminant of the PSA
sample originating from the CaP plasma (see Fig. 5
). Altogether, the
data confirm that the PSA released from the PSA-ACT complex is very
similar, if not identical, to the reference material obtained from
semen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and also for the glycosylated peptide (amino
acids 11–45), although this could not be observed in the
peptide pattern because of the low response in the MALDI-TOF MS at low
concentrations. However, the glycopeptide obtained from reference PSA
(semen) was detected by MALDI-TOF MS, and it showed the sialylated
complex biantennary N-glycan structure described previously
(26). Because the intact PSA molecules of the two sources
showed an almost identical molecular mass and a similar peak pattern in
HPLC and MALDI-TOF MS, it can be assumed that only minor differences
are present between these two molecules. Regarding the structural
integrity, it will be interesting to learn whether the released PSA
still displays protease activity, and if it does, to what extent.
Furthermore, it will be worthwhile to probe the released PSA with more
antibodies, specifically those recognizing conformational epitopes to
probe their integrity.
The data obtained for the cleavage of the PSA-ACT complex in human
plasma or sera (see Fig. 3
and Table 2
) suggest that the complex was
cleaved to near completion. On average, the values measured after
ethanolamine treatment were 85% of the total PSA values detected in
the untreated sera. There could be several effects contributing to this
slightly lower value: (a) A small amount of PSA-ACT complex
could still be present because of incomplete cleavage. (b)
Other PSA-serpin complexes, which are also present in human sera
(4)(6) but represent <5% of T-PSA
(30), might not be cleaved by the treatment. (c)
The released PSA might form complexes with
2-macroglobulin or serpins. However, new
complex formation does not seem to take place to a substantial extent
because otherwise the amount of F-PSA present at the end of the
incubations would be much smaller. This inability to form new complexes
could be attributable to the loss of proteolytic activity of the
released PSA or the "cleavage conditions" that are always present
in the assays. This is in contrast to the reported cleavage of the
PSA-API complex, where the released PSA seemed to form complexes again
when the cleavage was performed in serum (21). Which of
these possible reasons hold true as explanations for the lower value
detected need to be analyzed in further investigations.
It can also be concluded from the data that the complex of PSA and
2-macroglobulin present in serum does not seem
to release PSA under the cleavage conditions because otherwise the PSA
values after ethanolamine treatment should be higher than the T-PSA
values of the untreated sera.
The data also reveal that the presence of serum was favorable for the
cleavage of PSA-ACT, because it was almost complete after 24 h,
whereas only 40% of PSA-ACT was cleaved when PBS buffer with the
same pH and ethanolamine concentration was used. A similar favorable
effect of serum was also reported for the cleavage of PSA-API
(21). Incidentally, the addition of bovine serum albumin (10
g/L) to the assays also seemed to accelerate the cleavage of PSA-ACT in
buffer (data not shown).
The novel procedure described allows the measurement of free and "total" PSA with one type of assay, thus eliminating any bias that might exist when two types of assays are used (7)(11)(12)(31). Whether the ratio of F-PSA to T-PSA determined by this method might somewhat improve the differentiation between BPH and CaP compared with the determination of the ratio using two different types of assays is uncertain and needs to be analyzed in larger panels of BPH and CaP sera. However, the main value of this new method must be regarded more in the fact that it makes PSA complexed to ACT in serum amenable for studies in the form of F-PSA, which allows further structural analysis (e.g., of glycosylation), which to date has been very difficult or not possible.
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Footnotes |
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2 Present address: Lehrstuhl für Bioorganische Chemie,
Universität Bayreuth, Gebäude NW 1, 95440 Bayreuth,
Germany.
3 Nonstandard abbreviations: PSA, prostate-specific antigen; CaP, prostate cancer; BPH, benign prostatic hyperplasia; ACT, 1-antichymotrypsin; F-PSA and T-PSA, free and total PSA; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; API, 1-protease inhibitor; PBS, phosphate-buffered saline; and MALDI-TOF MS, matrix-assisted laser desorption-induced time of flight mass spectrometry.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin is the major form of prostatic-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.1-antichymotrypsin. Clin Chem 1991;37:1618-1627.2-macroglobulin. Clin Chem 1998;44:2471-2479.1-antichymotrypsin. Potential reference material for international standardization of PSA immunoassays. Clin Chem 1995;41:1273-1282.1-antichymotrypsin complex. Clin Chem 1995;41:1480-1488.1-protease inhibitor in vitro. Prostate 1997;33:87-96.[ISI][Medline]
[Order article via Infotrieve]
1-antichymotrypsin. J Biol Chem 1983;258:12749-12752.1-protease inhibitor in serum. Clin Chem 1999;45:814-821.The following articles in journals at HighWire Press have cited this article:
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  M. Tajiri, C. Ohyama, and Y. Wada Oligosaccharide Profiles of the Prostate Specific Antigen in Free and Complexed Forms from the Prostate Cancer Patient Serum and in Seminal Plasma: a Glycopeptide Approach Glycobiology, January 1, 2008; 18(1): 2 - 8. [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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 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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J. Peter, C. Unverzagt, T. N. Krogh, O. Vorm, and W. Hoesel Identification of Precursor Forms of Free Prostate-specific Antigen in Serum of Prostate Cancer Patients by Immunosorption and Mass Spectrometry Cancer Res., February 1, 2001; 61(3): 957 - 962. [Abstract] [Full Text]
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 W. Hoesel, J. Peter, H. Lenz, and C. Unverzagt Purification of Prostate-specific Antigen from Serum by Indirect Immunosorption and Elution with a Hapten Clin. Chem., September 1, 2000; 46(9): 1490 - 1491. [Full Text] [PDF]
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