PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immuno-dominant N-terminus and an immuno-recessive C-terminus.

PSP94 ( microseminoprotein,MSP) is one of the three major proteins secreted by the normal human prostate gland. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting, PSP94 transcripts were shown in human endometrium, myometrium, ovary, breast, placenta and in the human endometrial cancer cell lines KLE and AN3 CA. Primers used in these studies were specific for human prostate PSP94, and were derived from its flanking non-coding regions. The results were confirmed by sequence analysis of two independently derived clones from normal human breast tissues and the other two from KLE cells respectively. The sequences were identical with the coding sequence of human prostate PSP94 cDNA. Using RNA from the endometrial tissues, two different transcripts of 487 bp, equivalent to prostate PSP94 and 381 bp, corresponding to prostate PSP57, its alternately spliced form, were amplified by RT-PCR. Human ovary, breast, placenta and endometrial cancer cell lines (KLE, AN3 CA), however, showed only the full length, 487 bp, PSP94 transcript. We further demonstrated by in situ hybridization that PSP94 mRNA is expressed specifically in the glandular epithelial cells, and not in the stroma of both the human endometrial and breast tissues. Further, using image analysis of in situ hybridization data, the levels of PSP94 mRNA in the cycling endometrial tissues and in breast confirmed the differential levels of expression in the cycling endometrium (P<0·005). This study distinctly demonstrated significant expression of PSP94 mRNA in human uterine, breast and other female reproductive tissues as well in the endometrial cancer cell lines, suggesting that it may have a role in these tissues as a local autocrine paracrine factor.

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PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immuno-dominant N-terminus and an immuno-recessive C-terminus.