Wan-Ming Zhang

Complex Formation Between PSA Isoenzymes and Protease Inhibitors

PURPOSE We have studied complex formation between the isoenzymes of prostate specific antigen (PSA) and protease inhibitors in vitro. MATERIALS AND METHODS Ion exchange chromatography and hydrophobic interaction chromatography (HIC) were used for rapid separation of PSA isoenzymes from inhibitors and for characterization of the complex formation. Immunofluorometric assays (IFMA) specific for free PSA, the PSA-alpha 1-antichymotrypsin (PSA-ACT) complex and for both of these (total PSA) were used to measure various forms of PSA. Loss of free PSA immunoreactivity was used to estimate complex formation with alpha 2-macroglobulin (A2M) and ACT, which also was measured by PSA-ACT IFMA. RESULTS Complex formation between PSA and A2M was more rapid than with ACT. After extended incubation, about 75% of PSA reacted with ACT and 85% with A2M. When added to a mixture of ACT and A2M at concentrations corresponding to those in plasma, only 17% of PSA formed a complex with ACT while 17% remained free and 66% was undetectable, indicating complex formation with A2M. After extended incubation of PSA-ACT at 37C, a significant proportion of PSA was released as free active PSA. When A2M was included in the reaction mixture, the loss of PSA-ACT was not accompanied by appearance of free PSA, an indication that it complexed with A2M. Five to 18% of nicked PSA complexed with ACT whereas 54 to 67% reacted with A2M. CONCLUSIONS alpha 2-macroglobulin is the major inhibitor of PSA when it reached the circulation. Contrary to earlier assumptions, nicked PSA can bind to A2M rendering it inaccessible to antibodies.

Expression and characterization of trypsinogen produced in the human male genital tract.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

Prostate-specific antigen forms a complex with and cleaves α1-protease inhibitor in vitro

Complexes between prostate‐specific antigen (PSA) and α1‐protease inhibitor (API) occur in serum and they are of potential interest in the diagnosis of prostate cancer. Pure PSA‐API complexes are needed for development of specific assays, but complex formation has not earlier been achieved in vitro.

Dual-Label Immunoassay for Simultaneous Measurement of Prostate-specific Antigen (PSA)-α1-Antichymotrypsin Complex Together with Free or Total PSA

BACKGROUND A major portion of prostate-specific antigen exists in circulation as a complex with alpha(1)-antichymotrypsin (PSA-ACT), whereas a minor part is free (fPSA). The proportion of PSA-ACT is increased in prostate cancer (PCa), but immunologic determination of PSA-ACT is hampered by a background produced by nonspecific adsorption of ACT to the solid phase. To reduce the nonspecific interference, we produced an antibody specific for complexed ACT and developed immunofluorometric assays (IFMAs) for simultaneous measurement of fPSA + PSA-ACT (fPSA/PSA-ACT) and PSA-ACT + total PSA (tPSA, PSA-ACT/tPSA). METHODS Monoclonal antibodies (MAbs) were produced by immunization with PSA-ACT. The dual-label time-resolved IFMAs for fPSA/PSA-ACT and PSA-ACT/tPSA used a capture MAb to tPSA, an Eu(3+)-labeled MAb to fPSA or complexed ACT, and an Sm(3+)-labeled MAb to complexed ACT or to tPSA as tracer antibodies. The clinical utility was evaluated using serum samples from individuals with or without PCa with PSA concentrations of 2.0-20.0 micro g/L. RESULTS One MAb (1D10) showed low cross-reactivity with free ACT and cathepsin G-ACT. A sandwich assay for PSA-ACT with 1D10 as tracer had a detection limit of 0.05 micro g/L, and with this assay, PSA-ACT was undetectable in female sera. The detection limit for fPSA was 0.004 micro g/L. Determinations of the ratio of fPSA to PSA-ACT and the proportions of fPSA/tPSA and PSA-ACT/tPSA provided the same clinical specificity for PCa and provided significantly better clinical specificity than did tPSA. CONCLUSIONS Background problems observed in earlier PSA-ACT assays are eliminated by the use of a MAb specific for complexed ACT as a tracer. The same clinical validity can be obtained by determination of fPSA or PSA-ACT together or in combination with tPSA.

Prostate-specific antigen and other prostate cancer markers

P antigen (PSA) is the most important serum marker for prostate cancer. It is increasingly used for screening and early detection, although 65% to 75% of moderately elevated PSA results are caused by benign prostatic hyperplasia (BPH).1 Of the immunoreactive PSA in serum, 65% to 95% occurs in complex with alpha1antichymotrypsin (ACT), and measurement of the proportion of free or complexed PSA reduces the frequency of false-positive results by 20% to 30%.1–3 However, additional improvement is desirable and appears to be possible using some new markers. A few percent of PSA in serum is complexed to alpha1-protease inhibitor (API)4 and 2% to 30% with alpha2-macroglobulin (A2M).5 PSAAPI, PSA-A2M, and human kallikrein-2 (hK2)6,7 have provided promising results. In addition to these prostate-specific markers, insulin-like growth factor-1 (IGF-1)8 and vascular endothelial growth factors (VEGF)9 may provide information on prostate cancer risk. Efficient use of these new markers requires the use of statistical methods for calculation of the combined impact of multiple variables.

Standardization of PSA determinations.

Standardization of determinations for prostate specific antigen (PSA) has become an important issue due to the widespread use of these determinations for prostate cancer screening. Standardization of this assay is complicated due to the occurrence of two major forms of PSA in serum, the free antigen and a complex between PSA and alpha 1-antichymotrypsin (PSA-ACT). These two forms of PSA are recognized differently by different antibodies, but by careful selection of antibodies, it is possible to design methods that measure each form equally. It is suggested, that standards for PSA and PSA-ACT should be prepared and established as international standards. Furthermore, reference methods should be established on the basis that these standards and carefully selected reference antibodies.

Reactivity of 77 antibodies to prostate-specific antigen with isoenzymes and complexes of prostate-specific antigen.

Seventy-seven antibodies submitted to the ISOBM TD-3 PSA Workshop (TD-3.1 and TD-3.2) were characterized by measuring their reactivity with isoenzymes of free prostate-specific antigen (PSA), PSA complexed to α1-antichymotrypsin (PSA-ACT) and α1-proteinase inhibitor (PSA-API). Antibodies were classified into 15 distinct groups according to their reaction profiles with the various isoenzymes. Some antibodies recognizing both free and complexed PSA were inaccurate in measuring total PSA. Eight of the 9 free PSA-specific antibodies cross-reacted more with PSA-API than with PSA-ACT, while 1 antibody reacted less with PSA-API than PSA-ACT. From the panel of antibodies 39 reacted with both free and complexed PSA and were classified as total PSA antibodies.

Nexin-1 inhibits the activity of human brain trypsin.

Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural development, plasticity and neurodegeneration. Their activity is modulated by serine protease inhibitors, serpins. However, for human brain trypsin, trypsin-4, no brain-derived inhibitors have been described. Here, we report that nexin-1 inhibits trypsin-4, and forms stable complexes only with this trypsin-isoenzyme. This result suggests that nexin-1 could modulate trypsin activity in brain where both nexin-1 and trypsin-4 are expressed.

Characterization and determination of the complex between prostate-specific antigen and α1-protease inhibitor in benign and malignant prostatic diseases

Prostate-specific antigen (PSA) is a tissue-specific serine protease which forms complexes with protease inhibitors such as f 1 -antichymotrypsin and f 2 - macroglobulin. We have studied the interaction between PSA and f 1 -protease inhibitor (API) in vitro and found that 15% of the added PSA binds to API while the majority of API is cleaved between Met358 and Ser359 when PSA is incubated with a 5-fold excess of API at 37 C for 7 days. The complex between PSA and API (PSA- API) formed in vitro displays the same chromatographic behavior, molecular size and immunoreactivity as endogenous PSA- API occurring in serum, indicating that they are identical. PSA- API can be detected in serum by a time-resolved immunofluorometric assay (IFMA), in which a monoclonal antibody to PSA is used as a catcher and a polyclonal antibody to API labeled with a Eu-chelate is used as a tracer. Purified PSA- API formed in vitro is used as a calibrator. PSA- API in serum represents 1.0- 7.9% (median 2.4%) of total PSA (tPSA) in prostate cancer (PCa, n= 82) and 1.3- 12.2% (median 3.6%, p<0.01) in patients with benign prostatic hyperplasia (BPH, n= 66). The IFMA for PSA- API in serum is hampered by a variable background, which is caused by non-specific adsorption of the huge excess of API in serum to the solid phase. The background can be determined by an assay using the same tracer as in the IFMA for PSA- API but PSA-unrelated antibody on the solid phase. The background signal is subtracted from the PSA- API signal. The clinical utility of PSA- API in serum has been evaluated in PSA-positive subjects from the Finnish PCa screening trial. After subtraction of the background, the proportion of PSA- API in relation to tPSA is lower in PCa than in controls, 0.9% vs. 1.6%, respectively (p<0.001). Logistic regression analysis showed that the concentration of PSA- API was independent of the proportion of free PSA as a diagnostic variable among subjects with a tPSA of 4- 10 w g/l (p= 0.009). The probability of PCa calculated by logistic regression using the concentration of PSA- API and the proportion of free PSA in serum significantly improved cancer specificity at high sensitivity levels (85 - 95%) as compared to the proportion of free PSA alone.

The clinical importance of free prostate-specific antigen (PSA).

The proportion of free prostate-specific antigen (PSA) in serum relative to total PSA (F/T) is lower in patients with prostate cancer than in those with elevated levels of PSA due to benign prostatic disease. When applied to early diagnosis and screening for prostate cancer, the proportion of free PSA can be used to reduce the number of false-positive results by 20-40%. The utility of F/T is better in men with a small prostate volume, i.e. in relatively young men, who are most likely to benefit from early diagnosis and treatment of prostate cancer. The concentrations of PSA and especially free PSA are affected by considerable intra-individual variation and sample stability. Assay standardization is variable and it is therefore important to establish reference values for the methods used. Better control of these factors is likely to improve the diagnostic accuracy. The utility of determining free PSA can be improved by evaluating the combined impact of free and total PSA by logistic regression analysis or neural networks.