Human Prostatic Acid Phosphatase Research Articles

Physico-chemistry and Cytotoxicity of Tenofovir-Loaded Acid Phosphatase-Responsive Chitosan Nanoparticles.

This study assesses the in vitro release of tenofovir (TFV)-loaded triphosphate (TPP) cross-linked chitosan nanoparticles (NPs) catalyzed by human prostatic acid phosphatase (hPAP) for 24h. The physico-chemical characterization of the NPs included particle mean diameter (PMD), zeta potential (ζ), percent drug encapsulation efficiency (% EE), Fourier transform infra-red (FTIR)spectroscopy, powder X-ray diffractometry analysis (PXRD), and drug release kinetics. The first-order rate constant (k) and activation energy (Ea) of the cross-link (TPP) are determined by the integrated rate law and Arrhenius's equations. The hPAP Michaelis-Menten constant (Km) is determined by theLineweaver-Burk's equation. The NP's safety profile is evaluated on vaginal epithelial cells (VK2/E6E7). The lyophilized drug-loaded NPs' PMD, ζ, and PDI are 149.97nm, 4.4mV, and 0.3, respectively. The % EE after lyophilization is 93.7 ± 4.4%. These NPs released drug at faster rate (63% of TFV within 6h) under the enzyme's influence. The similarity and difference factors of drug release profiles (absence vs presence of hPAP) are 56.5 and 40.3, respectively. The hPAP's Km value of 0.019mM suggests it has a good affinity for TPP at physiological pH ~ 7.4. The enhanced hydrolysis of TPP or degradation of chitosan NPs is fundamentally due to a decrease of TPP's activation energy by hPAP. In fact, the Ea value is 22.50 ± 3.06kJ/mol or 16.33 ± 0.62kJ/mol in the absence or presence of hPAP, respectively. The NPs are non-cytotoxic to the treated vaginal cell line. These hPAP-responsive NPs are promising topicalnanomicrobicides for HIV/AIDS prevention.

PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Proteomic Analysis of the Alterations in Follicular Fluid Proteins During Oocyte Maturation in Humans.

Many components in ovarian follicles (follicular fluid, cumulus cells, granular cells, etc.) dynamically change during folliculogenesis and play a positive or negative role in oocyte maturation. Infertile women who underwent intracytoplasmic sperm injection (ICSI) treatment in the reproductive medicine centre of Hangzhou Women’s Hospital between October 2018 and October 2021 were included. The ovarian follicular fluid and cumulus cells of diminished ovarian response (DOR) patients and control subjects with medical records of clinical data were collected. In total, 31 differentially expressed proteins, including 10 upregulated proteins (>1.50-fold, P<0.05) and 21 downregulated proteins (<0.67-fold, P<0.05), were identified in mature vs. immature oocytes by iTRAQ labelling coupled with 2D LC-MS/MS. GO analysis revealed that ‘cell population proliferation’ was the most diverse enrichment trend between up/downregulated proteins, while phagosome process and the PI3K-Akt signaling pathway were the two most significant pathways revealed by KEGG enrichment classification. Human prostatic acid phosphatase (PAP, ACPP) and CD5 antigen-like (CD5L) were two proteins verified by ELISA to be differentially expressed between MII and Gv oocytes (P<0.0001 and P<0.0001, respectively). Further measurement found significantly lower level of ACPP in follicular fluids and cumulus cells of DOR patients (P=0.028 and P=0.004, respectively), as an indicator of oocyte quality. Otherwise, CD5L level is upregulated in follicular fluid of DOR patients (P<0.0001). Our study provided experimental data to establish the objective indicator of oocyte maturation in the microenvironment of ovarian follicles, and also provided new insight into the measurement of oocyte quality.

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Multifunctionality of Prostatic Acid Phosphatase in Prostate Cancer Pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomic tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum, magnifying normally difficult to detect subsets of the protein of interest. For PAcP this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wild-type PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Mechanisms of Osteoblastic Bone Metastasis in Prostate Cancer: Role of Prostatic Acid Phosphatase.

Prostate cancer (PCa) preferentially metastasizes to bone, leading to complications including severe pain, fractures, spinal cord compression, bone marrow suppression, and a mortality of ∼70%. In spite of recent advances in chemo-, hormonal, and radiation therapies, bone-metastatic, castrate-resistant PCa is incurable. PCa is somewhat unique among the solid tumors in its tendency to produce osteoblastic lesions composed of hypermineralized bone with multiple layers of poorly organized type I collagen fibrils that have reduced mechanical strength. Many of the signaling pathways that control normal bone homeostasis are at play in pathologic PCa bone metastases, including the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin system. A number of PCa-derived soluble factors have been shown to induce the dysfunctional osteoblastic phenotype. However, therapies directed at these osteoblastic-stimulating proteins have yielded disappointing clinical results to date. One of the soluble factors expressed by PCa cells, particularly in bone metastases, is prostatic acid phosphatase (PAP). Human PAP is a prostate epithelium-specific secretory protein that was the first tumor marker ever described. Biologically, PAP exhibits both phosphatase activity and ecto-5′-nucleotidase activity, generating extracellular phosphate and adenosine as the final products. Accumulating evidence indicates that PAP plays a causal role in the osteoblastic phenotype and aberrant bone mineralization seen in bone-metastatic, castrate-resistant PCa. Targeting PAP may represent a therapeutic approach to improve morbidity and mortality from PCa osteoblastic bone metastases.

Abstract B118: Development of a novel prostatic acid phosphatase-derived vaccine for the treatment of advanced prostate cancer

Abstract Our aim is to develop a new and more effective prostatic acid phosphatase (PAP) peptide-based vaccine for the treatment of advanced prostate cancer (PCa). We have previously shown that a 15mer PAP peptide-derived vaccine, when administered as a DNA vaccine, could induce PAP-specific T-cell responses and reduce the growth of implanted murine TRAMP C1-derived tumors in a syngeneic heterotopic murine model. We have subsequently developed an elongated (42mer) PAP-derived peptide in which Alanine at position 115 was replaced by Leucine.Herein, the capacity of the wild-type and mutated PAP-42mer vaccines to induce PAP-specific immune responses were compared in two pre-clinical murine models. The murine PAP-42mer sequence was evaluated in C57Bl/6 mice and the human PAP-42mer sequence in HHDII/DR1 transgenic mice. Following the identification of the most promising PAP-42mer sequence, the capacities of different delivery systems (CpG adjuvant, CAF09 adjuvant and ImmunoBody® DNA vaccine) to generate PAP-specific immunity were compared. For these studies, the responsiveness of splenocytes isolated from immunized mice following in vitro stimulation with MHC class I or class II wild-type vaccine-derived peptides (8/9 or 15 amino-acid long) was assessed using an IFNγ ELISpot assay and by immunophenotyping of splenic T-cells using flow cytometry. The ability of splenocytes to kill relevant targeT-cells after a short in vitro stimulation was assessed using a 51chromium release assay. B16 cells that had been knocked out for beta-2m gene and transfected to express chimeric HLA-A2 (HHDII), HLA-DR1 and human PAP implanted into HHDII/DR1 mice was used as a proof-of-concept model to assess the antitumor efficacy of the hPAP42mer mutated vaccine with CAF09 adjuvant in a prophylactic setting.The mutated murine and human PAP42mer-based vaccines induced a higher number of IFNγ-releasing splenocytes in response to in vitro stimulation with class I or class II vaccine-derived peptides and generated cells having a higher functional avidity. CAF09 and ImmunoBody® were superior to CpG in inducing PAP-specific immune responses, with CAF09 eliciting strong immune responses in both models, and ImmunoBody® eliciting potent immune responses in C57Bl/6 mice. Indeed, a higher proportion of CD8+ T-cells were able to release IFNγ and TNFα, to proliferate (Ki67 expression) and to degranulate (CD107a and Granzyme B expression) after short incubation with MHC class-I peptide. These vaccines strategies were also able to induce a higher proportion of memory CD8+ T-cells and the expression of PD-1 on CD8+ T-cells. Additionally, splenocytes from vaccinated mice were able to kill MHC class I peptide-pulsed targeT-cells (T2 and RMAS cells) and murine PCa cells expressing PAP (TRAMP-C1 cells) in vitro. The vaccine slowed the growth of human-PAP+-B16-derived tumors in HHDII/DR1 model and prolonged the survival (9 days delay for 30% of the mice). Splenic CD8+ T-cells and CD8+ tumor-infiltrating cells from vaccinated mice released IFNγ and TNFα, proliferated and degranulated in response to in vitro stimulation with MHC class I peptide. Finally, multimer-based flow cytometric analysis of peripheral blood mononuclear cells from patients with PCa following in vitro stimulation with PAP-derived peptides demonstrated the presence of PAP42mer-reactive CD8+ T-cells. Ongoing studies are assessing the effect of combining the vaccine with checkpoint inhibition (a PD-1 monoclonal antibody) in a therapeutic setting. In summary, we have developed a vaccine strategy which induces robust anti-PAP immunity and slows the growth of implanted PAP+ cancer cell-derived tumors in vivo. The presence of PAP-specific CD8+ T-cells in the periphery of patients with advanced PCa suggests that these patients will benefit from this new approach, especially when combined with checkpoint inhibition. Citation Format: Pauline Le Vu, Jayakumar Vadakekolathu, Dennis Christensen, Lindy Durrant, Graham Pockley, Stephanie E.B. McArdle. Development of a novel prostatic acid phosphatase-derived vaccine for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B118.

Optimized electrochemical biosensor for human prostatic acid phosphatase

An innovative and sensitive label-free electrochemical biosensor platform for detecting human prostatic acid phosphatase (hPAP) is proposed herein. The methodology uses impedance-derived immittance functions (ImFs) for monitoring the interfacial changes in a hPAP bio-recognition layer constructed using a self-assembled monolayer of PEGlate thiol (low-fouling materials) and 11-ferrocenyl undecanethiol (redox probe confined). We evaluated the response toward the target primarily in phosphatase-buffered saline, at varying concentrations and across a wide range of frequencies. The optimum frequency for each ImF parameter was selected based on its sensitivity and linearity within a target concentration ranging from 20 pM to 5000 pM and a limit of detection (LoD) below the cut-off value for this cancer biomarker (10 pM). The best ImF selected (inverse of imaginary capacitance, 1/C″) was then successfully used to monitor hPAP in human blood serum and showed a linear range from 50pM to 104pM, with an LoD of 11.2±2.6pM. Additionally, both the assay and analysis of the results can be performed in a few minutes without the need to add an interfering redox probe to the biological samples, thus providing a rapid, sensitive, and label-free test for prostate cancer.

Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture.

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

Homotropic allostery of nucleotidase activity of human prostatic acid phosphatase

The steady-state kinetics of hydrolysis of four purine ribonucleotides: 3′-AMP, 5′-AMP, 5′-GMP and 5′-IMP catalysed by human prostatic acid phosphatase (PAP; EC 3.1.3.2) in vitro was examined in this study. It has been shown for the first time that nucleotidase activity of PAP exhibits positive cooperativity, or homotropic allostery, in binding the purine ribonucleotides. Therefore, these substrates are homotropic positive effectors, or homotropic allosteric activators, of PAP-catalysed reaction. Enzyme-substrate saturation curves described by Hill equation are sigmoidal and the values of Hill cooperativity coefficient h are higher than 1. The affinity of PAP to substrates, the degree of cooperativity and the efficiency all depend on the chemical nature of the ribonucleotide and they increase in the following order: 5′-AMP<5′-IMP<5′-GMP<3′-AMP, but the enzyme reactivity remains almost equal. Therefore, 3′-AMP is the most efficient PAP substrate while 5′-AMP is the least efficient one of the four ribonucleotides studied. The noncovalent, homoallosteric regulation of the nucleotidase activity of PAP in vitro discovered in this study may be physiologically important in vivo.

Screening and characterization of a novel RNA aptamer that specifically binds to human prostatic acid phosphatase and human prostate cancer cells.

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2'-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2'-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.

Label-free Capacitive Diagnostics: Exploiting Local Redox Probe State Occupancy

An electrode surface confined redox group contributes to a substantial potential-dependent interfacial charging that can be sensitively probed and frequency-resolved by impedance-derived capacitance spectroscopy. In utilizing the sensitivity of this charging fingerprint to redox group environment, one can seek to generate derived sensory configurations. Exemplified here through the generation of mixed molecular films comprising ferrocene and antibody receptors to two clinically important targets, the label-free methodology is able to report on human prostatic acid phosphatase (PAP), a tumor marker, with a limit of detection of 11 pM and C-reactive protein with a limit of detection of 28 pM. Both assays exhibit linear ranges encompassing those of clinical value.

Consequences of the lack of CD73 and prostatic acid phosphatase in the lymphoid organs.

CD73, ecto-5′-nucleotidase, is the key enzyme catalyzing the conversion of extracellular AMP to adenosine that controls vascular permeability and immunosuppression. Also prostatic acid phosphatase (PAP) possesses ecto-5′-nucleotidase/AMPase activity and is present in leukocytes. However, its role related to immune system is unknown. Therefore, we analyzed enzymatic activities and leukocyte subtypes of CD73 and PAP knockouts and generated CD73/PAP double knockout mice to elucidate the contribution of CD73 and PAP to immunological parameters. Enzymatic assays confirmed the ability of recombinant human PAP to hydrolyze [3H]AMP, although at much lower rate than human CD73. Nevertheless, 5′-nucleotidase/AMPase activity in splenocytes and lymphocytes from PAP−/− mice tended to be lower than in wild-type controls, suggesting potential contribution of PAP, along with CD73, into lymphoid AMP metabolism ex vivo. Single knockouts had decreased number of CD4+/CD25+/FoxP3 + regulatory T cells in thymus and CD73/PAP double knockouts exhibited reduced percentages of CD4+ cells in spleen, regulatory T cells in lymph nodes and thymus, and CD4+ and CD8+ cells in blood. These findings suggest that PAP has a synergistic role together with CD73 in the immune system by contributing to the balance of leukocyte subpopulations and especially to the number of regulatory T cells in lymph nodes and thymus.

Human prostatic acid phosphatase: structure, function and regulation.

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.

High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well–based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

Emerging Roles of Human Prostatic Acid Phosphatase

Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed.

Immunization with a prostate cancer xenoantigen elicits a xenoantigen epitope-specific T-cell response

Vaccines encoding xenoantigens, “non-self” proteins that are highly homologous to their autologous counterparts, have been investigated as a means to increase immunogenicity and overcome tolerance to “self” antigens. We have previously shown that DNA vaccines encoding native prostatic acid phosphatase (PAP) were able to elicit PAP-specific T cells in both rats and humans, but required multiple immunization courses. In this study, we investigated in a preclinical model whether immunizations with a DNA vaccine encoding a xenoantigen could elicit a cross-reactive immune response to the native protein, potentially requiring fewer immunizations. Lewis rats were immunized with a DNA vaccine encoding human PAP and splenocytes from immunized rats were screened with a human peptide library containing overlapping, 15-mer PAP-derived peptides using T-cell proliferation and interferon γ (IFNγ) release as measures of the immune response. One dominant PAP-specific, RT1.Al-restricted, epitope was identified. Direct immunization with the immunodominant peptide (HP201–215) containing this epitope demonstrated that it included a naturally presented MHC Class I epitope recognized by CD8+ T cells in Lewis rats. However, no cross-reactive immune response was elicited to the corresponding rat peptide despite a difference of only three amino acids. Immunization with DNA vaccines encoding rat PAP (rPAP) in which this foreign dominant epitope was included as well as with DNA vaccines coding for a variant of the xenoantigen from which this epitope was deleted, did not elicit responses to the native antigen. Overall, these results indicate that the immunization with a xenoantigen-coding DNA vaccine can lead to an immune response which potentially favors foreign epitopes and hence limits any cross-reactive response to the native antigen.

Secretion and N-Linked Glycosylation Are Required for Prostatic Acid Phosphatase Catalytic and Antinociceptive Activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6–7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This “cellular” construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia—a yeast strain that is used to generate biologics for therapeutic purposes.

Intracerebroventricular injection of human prostatic acid phosphatase has potent neuroprotective effects against transient focal cerebral ischemia in rats

Though the potential use of adenosine as a neuroprotective agent has long been realized, there are currently no adenosine-based therapies for the prevention or treatment of cerebral ischemia and reperfusion injury. Prostatic acid phosphatase (PAP), an enzyme that has long served as a diagnostic marker for prostate cancer, has been recently demonstrated to exhibit ecto-5′-nucleotidase activity, and dephosphorylate endogenous extracellular AMP to adenosine. We therefore tested the hypothesis that PAP has sustained and potent neuroprotective effects against cerebral ischemia in the rat model of middle cerebral artery occlusion. We found that hPAP produced significant neuroprotection against focal cerebral ischemia, as evident from significant reduction in cerebral infarction and neurological deficits. The therapeutic time window for hPAP in rat focal cerebral ischemia model was limited from 6 h before ischemia to 1.5 h after reperfusion. The present study suggested that PAP is a potential candidate for the prevention and treatment of cerebral ischemic injury, especially during perioperative period.