[Show abstract][Hide abstract] ABSTRACT: Renal cell carcinoma (RCC) is a highly vascular tumor associated with expression of various angiogenic growth factors. The precise process of how these growth factors are regulated in RCC is not fully understood. Recent evidence suggests that protease activated receptors (PARs), a new family of G-protein coupled receptors, play a crucial role in vascular development and tumor progression through a variety of mechanisms. However, the nature of PAR expression in human RCC tissues and its function in regulating angiogenesis in RCC are largely unknown. In this study, we investigated the expression and function of PAR-2 in RCC. RT-PCR and immunohistochemistry assays show that PAR-2 expression is significantly increased in human RCC tissue compared with the adjacent non-neoplastic kidney tissue. In RCC derived cells, PAR-2 is functional as evidenced by robust signaling through MAP kinases including ERK1/2 and JNK. Furthermore, activation of PAR-2 significantly upregulates several angiogenic cytokines, including interleukin-6 (IL-6), IL-8, monocytes chemotactic protein-1 (MCP-1) and growth-related oncogene (GRO). To our knowledge, this is the first report that characterized PAR-2 expression in RCC tissue and further demonstrated that PAR-2 has a critical role in regulating angiogenesis in RCC.
[Show abstract][Hide abstract] ABSTRACT: In prostate cancer, the mechanism by which the stromal cells surrounding the cancer epithelium become reactive and overproduce growth factors is unclear. Furthermore, the precise process of how these stromal cells stimulate the cancer epithelium is not fully understood. We recently found that protease-activated receptor-1 (PAR-1) in these reactive stromal cells is upregulated. To investigate the role of PAR-1 in the stromal-epithelial interaction, WPMY-1 stromal myofibroblasts were stimulated with PAR-1 agonists including thrombin and PAR-1 activating peptide. We show that WPMY-1 cells have functional PAR-1 by signaling through ERK1/2. Conditioned media (CM) from PAR-1 agonists-treated WPMY-1 cells stimulate the epithelial LNCaP cells leading to ERK1/2 activation and cell proliferation. Cytokine array analysis of the CM demonstrates that PAR-1 induces stromal cells to release numerous cytokines, of which interleukin 6 (IL-6) is the major factor responsible for mitogenic signaling in LNCaP cells. CM further induces expression of prostate-specific kallikrein-related peptidase-3 (KLK3/PSA) and KLK4 in LNCaP cells via the IL-6 pathway. Moreover, KLK4 functions as a potent agonist of PAR-1 by cleaving the receptor at the proper site on cell surface. KLK4 triggers transmembrane signaling and upregulates IL-6 in WPMY-1 cells through PAR-1. Immunohistochemical analysis indicates that PAR-1 is predominantly expressed in peritumoral stroma while KLK4 is produced exclusively by the epithelial cancer cells. These data provide evidence for a novel double-paracrine mechanism whereby cancer epithelium produces KLK4 to activate PAR-1 in the surrounding stroma, which in-turn releases cytokines (IL-6) that stimulate cancer cells to proliferate and increase production of KLKs.
International Journal of Cancer 02/2010; 126(3):599-610. DOI:10.1002/ijc.24904 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment.
Prostate CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder.
The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes.
CD90+ prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.
BMC Cancer 10/2009; 9(1):317. DOI:10.1186/1471-2407-9-317 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.
[Show abstract][Hide abstract] ABSTRACT: Human kallikrein-related peptidase 4 (KLK4/prostase),
a trypsin-like serine protease, is a potential
target for prostate cancer treatment because of its
proteolytic ability to activate many tumorigenic and
metastatic pathways including the protease activated
receptors (PARs). Currently there are no KLK4speciﬁc
small-molecule inhibitors available for therapeutic
development. Here were-engineer the naturally
occurring sunﬂower trypsin inhibitor to selectively
block the proteolytic activity of KLK4 and prevent
stimulation of PAR activity in a cell-based system.
The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer progression is partly facilitated by tumor-stroma interactions. We recently reported that protease-activated receptors (PAR-1 and PAR-2) are overexpressed in prostate cancer, and PAR-1 expression in peritumoral stroma is associated with biochemical recurrence. However, the nature of PAR expression in prostate tumor microenvironment is not fully understood. We therefore evaluated PAR-1 and PAR-2 expression in primary prostate cancer and bone metastasis.
PAR-1 and PAR-2 expression in normal, primary prostate cancer and the corresponding bone metastatic tissues were examined by immunohistochemistry, and double-label immunohistochemistry with the use of additional markers.
PAR-1 was expressed in peritumoral stroma in the majority of primary cancer tissues (83%). Serial sections and double-label immunohistochemistry determined that these PAR-1 expressing stromal cells were predominantly myofibroblasts, the primary cell type in reactive stroma. Analysis of cancer glands revealed that PAR-1 expression was significantly increased in the reactive stroma around higher Gleason grade cancers. PAR-2 was predominantly expressed in the primary cancer cells as well as smooth muscle cells but not in reactive stroma. In bone metastasis, PAR-1 expression in cancer cells was elevated compared to the primary site from the same patient. In the bone reactive stroma, PAR-1 was present in vascular endothelial cells and fibroblasts, while both PAR-1 and PAR-2 were expressed in osteoblasts and osteoclasts.
In primary prostate cancer and bone metastasis, PAR-1 is upregulated in reactive stroma and PAR-2 is uniformly overexpressed in carcinoma cells, suggesting these receptors may play potentially different roles in prostate cancer development and metastasis.
The Prostate 05/2009; 69(7):727-36. DOI:10.1002/pros.20920 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Benign prostatic hyperplasia (BPH) is characterized by abnormal epithelial and stromal proliferation causing urinary obstruction. Prostate growth is regulated by a variety of growth factors secreted from the stroma, including fibroblast growth factor 7 (FGF-7), a potent epithelial-specific growth factor which is increased in hyperplastic prostate. However, the mediator(s) of FGF-7 over-expression is unclear. Protease-activated receptor-1 (PAR-1) is a G-protein coupled receptor known to induce multiple biological processes, but its effect on BPH pathogenesis is mostly unknown. The aim of this study was to investigate the role of PAR-1 as a mediator of BPH development.
PAR-1 expression was investigated in BPH and normal prostate tissues by immunohistochemistry. Prostate stromal cells were isolated from BPH specimens, cultured and immunohistochemically characterized. Cultured stromal cells were stimulated with PAR-1 agonists, and extracellular-signal regulated kinase (ERK1/2) activation and cell proliferation were examined. PAR-1 mediated FGF-7 production by cultured stromal cells was assessed by RT-PCR and immunoassays, and verified by small interfering RNA (siRNA).
PAR-1 expression was increased in BPH stroma. In stromal cells isolated from BPH tissues, PAR-1 agonists activated ERK1/2 in a time- and concentration-dependent manner and with resultant enhanced cell proliferation. Pertussis toxin-sensitive G protein/(betagamma-subunits)-phosphatidylinositol 3-kinase and protein kinase C pathways were involved in ERK1/2 phosphorylation. PAR-1 activation strikingly induced FGF-7 production from cultured stromal cells mediated predominantly via ERK1/2 signaling pathway, and PAR-1 siRNA decreased the elicited FGF-7 upregulation.
The expression and function of PAR-1 in BPH stroma indicate PAR-1 may play important roles in BPH pathogenesis.
The Prostate 07/2008; 68(10):1064-75. DOI:10.1002/pros.20767 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer.
Molecular Cancer Research 07/2008; 6(6):1043-51. DOI:10.1158/1541-7786.MCR-08-0096 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.
Protein Expression and Purification 03/2008; 57(2):280-9. DOI:10.1016/j.pep.2007.09.018 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-tuberous sclerosis complex (TSC), non-lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.
Human Pathlogy 10/2007; 38(9):1361-71. DOI:10.1016/j.humpath.2007.01.028 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although protease-activated receptors (PARs) have been described to play a role in different malignancies, their expression and biological activity in prostate cancer are mostly unknown.
PAR expression in radical prostatectomy specimens was investigated by immunohistochemistry (IHC, 40 patients) and RT-PCR. Their role in LNCaP prostate cancer cell migration and Rac1/Cdc42 signaling was assessed with Boyden chamber analysis and Western blot, respectively.
PAR mRNA expression was higher in cancer, and protein expression was increased in PAR-1 (45%), PAR-2 (42%), and PAR-4 (68%), compared to normal glands. Increased PAR-1 (periglandular stroma) was associated with higher rates of biochemical recurrence (median follow-up, 5 years; P = 0.006). LNCaP migration was enhanced twofold and Rac1/Cdc42 signaling was activated by stimulation of PAR-1 and PAR-2.
PARs are overexpressed in prostate cancer and may serve as potential predictors of recurrence. The data suggest potential role of PARs in autocrine and paracrine mechanisms of prostate cancer.
The Prostate 05/2007; 67(7):743-56. DOI:10.1002/pros.20503 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study 41 men treated for prostate cancer with unilateral nerve-sparing radical prostatectomy and contralateral sural nerve grafting from January 2000 to September 2003.
Patients were considered for sural nerve grafting if they were considered at high risk of extracapsular extension before or during surgery, were younger than 70 years of age with good preoperative erectile function, were sexually active, and had no significant risk factors for erectile dysfunction. Potency was assessed by patient-reported questionnaires, including the International Index of Erectile Function erectile domain and Rigiscan testing.
The mean follow-up was 27.4 +/- 14.5 months. At 24 months, 24 (63.2%) of 38 men had erections sufficient for intercourse, with or without phosphodiesterase type 5 inhibitor use. Four men had partial erections that were occasionally satisfactory (10.5%), and 10 men reported no sexual activity, no spontaneous erections, or partial erections unsatisfactory for intercourse (26.3%). In contrast, in a group of 49 men who underwent unilateral nerve-sparing prostatectomy without nerve grafting during the same period at our institution, 13 (26.5%) had rigid erections adequate for intercourse with or without phosphodiesterase type 5 inhibitor use at 24 months of follow-up.
At 24 months of follow-up, men who had undergone unilateral nerve-sparing prostatectomy with contralateral sural nerve interposition graft repair of a cut cavernosal nerve had a greater rate of return of erectile function than men undergoing unilateral nerve-sparing prostatectomy alone.
[Show abstract][Hide abstract] ABSTRACT: Prostase/KLK4 is a member of the human kallikrein (KLK) gene family that is expressed in prostate epithelial cells under the regulation of androgenic hormones. In this study, we sought to characterize the substrate specificity of KLK4 in order to gain insight into potential physiological roles of the enzyme.
A chimeric form of KLK4 was constructed in which the pro-region of KLK4 was replaced with the signal and propeptide sequence of trypsinogen (proT-KLK4) to create an activation site susceptible to enterokinase cleavage. proT-KLK4 was expressed in Drosophila S2 cells, purified, and activated with enterokinase to generate mature KLK4. The extended substrate specificity of KLK4 was defined by screening tetrapeptide positional scanning synthetic combinatorial libraries (PS-SCL).
The preferred P1-P4 positions as determined by PS-SCL were: P1-Arg; P2-Gln/Leu/Val; P3-Gln/Ser/Val; P4-Ile/Val. The trypsin-like specificity of KLK4 was further confirmed using synthetic chromogenic peptides. Based upon the optimal cleavage site residues, a database search for potential KLK4 substrates identified several proteins with potential roles mediating normal prostate physiology or neoplastic growth including KLK3/PSA, parathyroid hormone-related peptide (PTHrP), and members of the bone morphogenetic protein (BMP) family. Recombinant KLK4 was able to activate pro-PSA/KLK3 and degrade members of the insulin-like growth factor (IGF) binding protein (IGFBP) family.
These results identify potential KLK4 substrates that may serve to define the role of this protease in normal prostate physiology, and facilitate studies of the consequences of KLK4 expression in pathological conditions.
The Prostate 01/2005; 62(1):1-13. DOI:10.1002/pros.20101 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malignant mesothelioma of the tunica vaginalis is a rare tumor managed principally by radical surgical resection. Chemotherapy and radiotherapy have limited efficacy. We report on a 67-year-old man with severe debilitation from multiple scrotal and inguinal recurrences of a malignant mesothelioma originating in the right tunica vaginalis. Local pain from extensive tumor spread prevented ambulation. Aggressive surgical debridement (total penectomy and scrotectomy) and perineal urethrostomy afforded the patient significant improvement in his quality of life before he finally died of the disease 3 years after diagnosis.
[Show abstract][Hide abstract] ABSTRACT: Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.
[Show abstract][Hide abstract] ABSTRACT: hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.
[Show abstract][Hide abstract] ABSTRACT: MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >107 M-1 s-1, similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>106 M-1 s-1), mast cell chymase (>105 M-1 s-1), chymotrypsin (>106 M-1 s-1), and prostate-specific antigen (PSA), which had the slowest rate constant at ∼104 M-1 s-1. Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys344, which corresponds to Met358, the P1 site of α1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe343, was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P1 and P2 positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.
[Show abstract][Hide abstract] ABSTRACT: Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving
protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family
of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern
blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide,
a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain,
a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains.
VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn2+ and Ca2+ ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent
binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the
protein at different positions after the protease domain. Alternative splicing may have functional significance, producing
proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.
[Show abstract][Hide abstract] ABSTRACT: A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned. The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme. The transcript has a signal peptide of 15 amino acid residues. The mature enzyme has 41% sequence identity with prostate specific antigen (PSA). Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies. The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222). Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond. These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.