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Clinical Chemistry 48: 2187-2194, 2002;
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2002;48:2187-2194.)
© 2002 American Association for Clinical Chemistry, Inc.

Structural Diversity of Cancer-related and Non-Cancer-related Prostate-specific Antigen

Takahiro Isono1a, Tsutomu Tanaka2, Susumu Kageyama2 and Tatsuhiro Yoshiki2

1 Central Research Laboratory and
2 Department of Urology, Shiga University of Medical Science, Seta, Otsu, Japan 520-2192.

aAuthor for correspondence. Fax 81-77-548-2049; e-mail isono{at}belle.shiga-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Heterogeneity among the various molecular forms of prostate-specific antigen (PSA) has not been well characterized, despite the critical importance of PSA in the detection of prostate cancer. The purpose of this study was to examine PSA heterogeneity in cancerous and noncancerous materials by extensive and systematic protein analysis.

Methods: A catalog of molecular forms of PSA was established with the PSA purified from seminal fluid. This catalog was used to analyze PSA heterogeneity in cancerous and noncancerous materials by immunoblotting with polyclonal antibodies.

Results: PSA from noncancerous materials showed a wider range of molecular mass, from 6000 to 28 000 Da. PSA from cancerous materials did not contain lower molecular mass forms.

Conclusions: The PSA protein catalog may be useful for the analysis of differences among PSA forms in men with and without prostate cancer and for analysis of antibodies used to detect PSA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Measurements of prostate-specific antigen (PSA)1 in serum are used to detect prostate cancer (PC), but PSA is produced not only by malignant cells, but also by noncancerous prostate secretory cells. Serum concentrations of PSA in men with benign prostatic hyperplasia (BPH) and those with PC overlap (1)(2)(3). The serum ratio of free (noncomplexed) to total PSA (which includes PSA complexes with {alpha}1-antichymotrypsin) has been introduced to better differentiate BPH and PC (4)(5)(6). The different ratios seen in BPH and PC may reflect different concentrations of {alpha}1-antichymotrypsin in benign and malignant prostatic tissues (7) or different routes of PSA egress into the circulation (8). The heterogeneity among the molecular forms of PSA on which these different ratios are based remains unexplained at present.

We (9) and others (10)(11)(12) have identified alternatively spliced PSA gene transcripts. Detailed analyses of the different molecular forms and modifications of the PSA protein are lacking.

Numerous studies of the molecular forms of the PSA protein have been reported since the initial purification of PSA from the prostate (13). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of PSA under reducing conditions shows a major 35-kDa band and several smaller bands that correspond to proteolytic cleavage products (14). Two-dimensional polyacrylamide gel electrophoresis (2DE) allows the separation of spots corresponding to different PSA proteins, and more spots are identified with this method than can be detected by immunoblot analysis (15). Ion spray mass spectrometry (MS) experiments have indicated a molecular mass of 28.43 kDa for the primary molecular form of PSA (16). N-Terminal sequencing of PSA has confirmed that there are three internal cleavage sites, at residues 85–86, 145–146, and 182–183 (14). Analysis of these data has not been fully integrated, and it is still unclear whether a specific difference exists between cancer-related and non-cancer-related forms of PSA.

We analyzed PSA heterogeneity by 2DE, MS, and amino acid sequencing and created a catalog of variants of purified PSA, including novel peptide forms of PSA with unique cleavage sites. The various PSA forms were compared with this catalog by immunoblot analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
preparation of samples for 2de
Seminal fluid was obtained from healthy, cancer-free volunteers. PSA was purified from clarified seminal fluid according to a previously reported chromatographic method (17) with phenyl-Sepharose HP and Sephacryl S-200 (Amersham Pharmacia Biotechnology). Although some forms of PSA may be removed and some modifications of PSA may occur during the purification process, e.g., by further proteolysis, this established chromatographic method was applied for purification of PSA as in many other studies. The cell extracts containing PSA were prepared from the LNCaP cancer cell line (18) and BPH tissues. BPH tissues were obtained from patients who had undergone transurethral resection of the prostate or retropubic prostatectomy. Serum 1 (3.5 mg/L PSA) and serum 2 (1.9 mg/L PSA) were obtained from patients with PC. Although it has not been determined whether there is any difference in composition between the gray zone PSA (4~10 µg/L) and high-density PSA (>1000 µg/L), serum samples with high-density PSA were used to make the analysis easier. These 2DE samples were suspended in lysis/rehydration buffer consisting of 8 mol/L urea, 20 g/L CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}, 10 g/L dithiothreitol, 20 mL/L Pharmalyte 3–10 (Amersham Pharmacia Biotechnology), and 100 mL/L glycerol. Protein was loaded onto gels at 15–50 µg of protein for each gel.

2de
2DE was performed with the Immobiline-polyacrylamide system as described previously (19). Isoelectric focusing was on linear, wide-range immobilized polyacrylamide gel strips (17 cm; pH 3–10; Bio-Rad Laboratories) in the first dimension and on 15% polyacrylamide gels (20 cm x 22 cm x 1 mm) in the second dimension. Gels were then stained with Coomassie Brilliant Blue (CBB; Nacalai tesque) or with silver nitrate (20). Glycoproteins in gels were stained by PAS carbohydrate staining using a GelCode Glycoprotein Staining Kit (Pierce). Isoelectric points (pIs) were inferred by use of pI marker proteins as calibrators (Bio-Rad Laboratories).

peptide mass fingerprinting analysis
Spots visualized by CBB or silver staining were digested in the gel by modified trypsin (Promega) (20). The extracted samples were analyzed by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) MS using an {alpha}-cyano-4-hydroxycinnamic acid matrix. Spectra were acquired with a Voyager RP mass spectrometer (Applied Biosystems) operated in reflectron mode with delayed extraction. External calibration was performed with des-Arg-bradykinin and adrenocorticotropic hormone (18–39 clip) as calibrators.

analysis of molecular mass
The spots visualized by CBB staining were excised, and the peptides within the spots were eluted electrophoretically by a Centrilutor (Millipore Corporation). The eluates were analyzed by MALDI-TOF MS using a 3, 5-dimethoxy-4-hydroxycinnamic acid matrix. Spectra were acquired as described above in linear mode. Internal calibration was performed with bovine serum albumin as the calibrator.

amino acid sequencing
Protein spots in the gels were electroblotted semidry onto Sequi-Blot PVDF membranes (Bio-Rad Laboratories), and those stained by CBB were excised and analyzed on an ABI-473A (Applied Biosystems) protein sequencer.

immunoblot analysis
After 2DE, proteins in the gels were electroblotted semidry onto Immuno-Blot PVDF membranes (Bio-Rad Laboratories) as described previously (21). Anti-PSA polyclonal antibodies (provided by Wako Pure Chemical Industries, Osaka, Japan) were produced after immunization of rabbits with PSA prepared by an established chromatographic method (17). For this experiment, polyclonal antibodies were preferable to a monoclonal antibody because polyclonal antibodies can recognize a wide range of PSA forms. The working dilution for anti-PSA polyclonal antibodies was 1:20 000 (50 µg/L), and the immunoreactive spots were detected by use of goat anti-rabbit immunoglobulins (working dilution, 1:10 000) conjugated with horseradish peroxidase (Medical and Biological Laboratory) and an enhanced chemiluminescence detection system (Amersham Pharmacia Biotechnology).

Two monoclonal antibodies, Tandem-MP PSA (purchased from Beckman Coulter) and PSA213-1 (provided by Wako Pure Chemical Industries), were then tested for reactivity with various PSA forms. Both antibodies were used to measure total PSA. An enhanced chemiluminescence detection system using goat anti-mouse immunoglobulins (working dilution, 1:5000) conjugated with horseradish peroxidase (Medical and Biological Laboratory) was used. The working dilutions for Tandem-MP PSA and PSA213-1 were 1:300 (~10 µg/L) and 1:20 000 (50 µg/L), respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
2de of purified psa and peptide mass fingerprinting analysis of each spot
A typical silver-stained gel produced by 2DE of chromatographically purified PSA (17) is presented in Fig. 1 . The silver-stained gels contained ~30 spots. The peptides in these spots were subjected to peptide mass fingerprinting analysis after in-gel digestion with trypsin and were then analyzed by MALDI-TOF MS. The peptide masses, which were derived from many spots in the MALDI-TOF MS analysis, agreed with the masses of peptides calculated from the sequences of mature PSA fragments produced by digestion with trypsin (Table 1 ).



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  		Figure 1. Silver-stained two-dimensional gel of chromatographically purified PSA.

The spots containing peptide fragments derived from PSA are numbered.


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  		Table 1. Peptide map of mature PSA.

A typical MALDI-TOF MS spectrum is presented in Fig. 2 . Twenty spots corresponded to peptide fragments derived from PSA (Table 2 ), and these spots were num-bered 1–20 (see Fig. 1Up ). Spots 1–5 contained fragments derived from the N-terminal as well as the C-terminal regions of PSA, but spots 6, 7, 11–14, and 16–18 contained no fragments derived from the C-terminal region of PSA, and spots 8–10, 15, 19, and 20 contained no fragments from its N-terminal region. The other silver-stained spots did not correspond to any fragments derived from PSA (data not shown). These spots may have derived from proteins that contaminated the purified PSA material. The polypeptide fragments derived from a previously reported, alternatively spliced PSA form (7)(8)(9)(10) were not found in the present analysis. The polypeptide fragments derived from human kallikrein 2 were also not found.



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  		Figure 2. MALDI-TOF MS spectrum of the tryptic digest of spot 1 (Fig. 1Up ).

The single letters indicate the peptide fragments derived from the PSA described in Table 1Up .


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  		Table 2. Characterization of PSA peptides within each spot in Fig. 1Up .

characterization of psa peptides in each spot
The PSA peptides within each spot were characterized by analyses of molecular mass, amino acid sequencing, glycosylation, and pI. The properties of these PSA peptides are summarized in Table 2Up . The amino acid range and composition corresponding to each peptide were estimated by combining all of the available data, and the results are listed in Table 2Up . The gp28 group contains the entire mature PSA protein (amino acid residues 1–237) plus a carbohydrate residue. The p26 group contains the full-length PSA protein without any carbohydrate residues. The gp22 group contains PSA residues 1–182 plus a carbohydrate residue. The gp18 group contains PSA residues 1–145 plus a carbohydrate residue. The gp12 group contains PSA residues 1–85 plus a carbohydrate residue. The p16, p10, and p6 peptides have N-terminal amino acids corresponding to positions 86, 146, and 183 in the PSA sequence, respectively. The p20 group contains peptides beginning with residues between positions 56 and 61 in the PSA sequence. This group represents novel forms of PSA peptides that have not been reported previously. Spots 13 and 14 were included in the gp18 group, although the existence of carbohydrate residues in these spots was not confirmed by PAS carbohydrate staining. This extensive and systematic analysis of PSA proteins and fragments demonstrated the existence of many peptide forms, including variations in glycosylation, and identified new PSA digestion products corresponding to novel cleavage sites. These new data were linked to 2DE data and included in an updated PSA protein catalog.

application of the psa protein catalog to the analysis of psa heterogeneity in various materials
The heterogeneity of PSA in various materials was assessed by immunoblot analysis using anti-PSA polyclonal antibodies (Fig. 3 ). The molecular forms corresponding to entries in the PSA protein catalog are summarized in Table 3 . In the control sample of purified PSA, all of the cataloged forms were detected, and several other unexpected spots were found as well. Most of these extra spots did not correspond to PSA fragments, i.e., they did not contain the peptides derived from PSA by peptide mass fingerprinting analysis. These spots may be derived from contaminating proteins and may result from nonspecific reactions with the polyclonal antibodies. However, some of these new spots may indicate previously unrecognized forms of PSA. A detailed characterization of these spots awaits further study. PSA that was detected immunologically in the seminal fluid showed a pattern similar to that seen in the PSA protein catalog, whereas the gp18 group of peptides from this source showed higher variability than that seen in the PSA catalog. This higher variability may be derived from some PSA variants that are removed during the PSA purification procedure used here. PSA from BPH tissue showed a pattern similar to that of the PSA protein catalog. Again, the gp28 group of peptides from this tissue showed higher variability than that seen in the catalog. PSA from extracts of the PC cell line LNCaP contained primarily the higher molecular mass forms, and the gp28 group of peptides again showed higher variability than indicated by the catalog. PSA in two serum samples from patients with PC contained primarily higher molecular weight forms, similar to the LNCaP cells, but the gp28 group of peptides in these sera did not show higher than normal variability. These results suggest that lower molecular mass forms of PSA are undetectable in the materials derived from PC, although this detection system may have had limited sensitivity against PC serum samples. In particular, the p16 and gp12 group forms, which were produced by cleavage between amino acid residues 84 and 85, were undetectable. Spot 8 was detected only in purified PSA. This result suggests that spot 8 may correspond to a modification of PSA that occurred during this purification process.



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  		Figure 3. 2DE immunoblotting with polyclonal antibodies for the mature PSA protein from various sources.

PSA, purified PSA; SF, seminal fluid.


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  		Table 3. Detection of various PSA molecular forms by immunoblotting using polyclonal antibodies against the full-length, mature PSA protein.1

application of the psa protein catalog to the discrimination of psa molecules detected by different psa assays
Two monoclonal antibodies that are used in different PSA assays were analyzed for PSA immunoreactivity by immunoblot analysis (Fig. 4 ). The Tandem-MP PSA monoclonal antibody reacted with the gp28 group peptides and with the p26 peptide (Table 4 ). This result showed that the Tandem-MP PSA monoclonal antibody required an intact, mature form of PSA for immunoreactivity. On the other hand, the PSA213-1 monoclonal antibody reacted with gp28 group peptides, the p26 peptide, the gp22 group peptides, the p20 group peptides, and the p16 peptide (Table 4 ). This indicated that the PSA213-1 monoclonal antibody required amino acid residues 86–182 for full immunoreactivity.



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  		Figure 4. 2DE immunoblotting of purified PSA with Tandem-MP PSA and PSA213--1 monoclonal antibodies.


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  		Table 4. PSA peptides detected by two different PSA monoclonal antibodies.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, a protein catalog of purified PSA was established through extensive and systematic protein analysis, including 2DE, MS, and amino acid sequencing, yielding new information on various forms and proteolytic products of this important molecule.

The first set of experiments led to the identification of novel peptide forms of PSA (the p20 group) that have N-terminal amino acids between residues 56 and 61. These peptides must be produced in vivo because they were detected not only in purified PSA but also in clinical materials. PSA fragments that had N-terminal amino acids corresponding to positions 55 and 58 were related to PSA derived from BPH (22)(23). It will be interesting to pursue the question as to whether these novel peptide forms of PSA influence the ratio of free to total PSA in serum.

The second set of experiments showed that there are significant variations in the glycosylation patterns of PSA forms. Two PSA forms (28 430 and 28 284 Da) that each contain a carbohydrate residue have been reported (16). These forms may be identical to members of the PSA gp28 group because they are similar in molecular mass. The PSA gp22, gp18, and gp12 groups are likely to be derived from the PSA gp28 group by proteolytic cleavage. Significant differences were found in the glycosylation patterns between PSA derived from cancerous and noncancerous materials, as detected by immunoblot analysis. Modifications of the carbohydrate residues and the ratios of different carbohydrate residues have been reported in association with other cancers (24). Further studies analyzing the carbohydrate residues in PSA are strongly urged to improve the diagnosis of PC.

In the third set of experiments, immunoblot analysis combined with the PSA protein catalog showed that the lower molecular mass forms of PSA were undetectable in materials related to PC. In particular, the p16 and gp12 forms of PSA, which are produced by cleavage between amino acid residues 85 and 86, were undetectable. It is possible that these results may be used to improve the diagnosis of PC. Recently, many proteases have been isolated from the prostate gland (25). Although some of these enzymes are involved in proteolysis of PSA, the exact reason for the observed fragmentation pattern is as yet unknown. There may be different activities or amounts of proteolytic enzymes between PC and BPH cells. For example, human kallikrein 2 was shown to be up-regulated in PC (26). However, this result must be confirmed by the analysis of larger numbers of samples. The immunoblot analysis system used in this study revealed new spots that could not be detected in the PSA protein catalog. New spots in clinical materials may be derived from some PSA variants that are removed by the PSA purification procedure. Further analysis of these novel spots will make the PSA protein catalog even more useful. Although spots containing peptide fragments derived from the alternatively spliced PSA (9)(10)(11)(12) were not detected in this study, with further analysis it may become possible to identify these forms as well. The detailed characterization of minor and unidentified spots in cancerous materials may lead to the identification of new forms of PSA that can serve as cancer markers.

Analyses of PSA by the combination of 2DE and immunoblotting without MS analysis were reported previously (15)(27). Our PSA protein catalog with MS data will add useful information for comparing immunoblot analysis data. These reports will require further analysis of new spots that could not be detected in the PSA protein catalog. In addition, the higher mass complexes containing PSA covalently bound to protease inhibitors that were detected in these previous reports were not detected in this study. Additional fine-tuning of some experimental conditions will be required to solve this apparent discrepancy.

The application of a protein catalog to analyze the immunoreactivity of different monoclonal antibodies against PSA was informative. The protein regions containing epitopes of denatured PSA that react with specific monoclonal antibodies were clearly identified. However, the immunoreactivities of monoclonal antibodies against PSA are changed after treatments to reduce and denature PSA (28). Usually the native PSA forms are analyzed by clinical immunoassays. Therefore, a more exact assessment of monoclonal antibody immunoreactivities will require the combination of an immunoassay system for the native PSA forms with use of the PSA protein catalog.

The PSA precursor form (pPSA) has been reported to be a significant subpopulation of PSA in both cancerous and noncancerous samples (26)(29)(30)(31)(32). However, we never found pPSA in our meticulous analysis of purified PSA prepared chromatographically from normal seminal fluid. However, gp28 group peptides from BPH tissue and LNCaP cells showed high variability by immunoblot analysis. Some of these unidentified spots may correspond to pPSA. Further examination will be required to solve this problem and to make the PSA protein catalog more complete.

In conclusion, we believe that "PSA" consists of many molecular forms, that the protein catalog of PSA is useful for the analysis of PSA heterogeneity in molecular PSA forms, and that the updating of this catalog by the characterization of new spots will aid in efforts to improve the accuracy of PC diagnoses.


   Acknowledgments
 
We thank Dr. Hirokazu Inoue (Department of Microbiology, Shiga University of Medical Science) for fruitful discussions. We also thank Noboru Urusiyamam, Ryouhei Okamoto, and Gyouzan Yamazaki (Central Research Laboratory, Shiga University of Medical Science) for technical assistance. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the Public Trust Haraguchi Memorial Cancer Research Fund, and a grant from the Foundation for Promotion of Cancer Research in Japan.


   Footnotes
 
1 Nonstandard abbreviations: PSA, prostate-specific antigen; PC, prostate cancer; BPH, benign prostatic hyperplasia; 2DE, two-dimensional polyacrylamide gel electrophoresis; MS, mass spectrometry; CBB, Coomassie Brilliant Blue; and MALDI-TOF, matrix-associated laser desorption/ionization time-of-flight.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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