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1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
2
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5G 1L5 Canada.
3
Department of Pharmacy, University of Patras, 26500 Patras, Greece.
aAddress correspondence to this author at: Mount Sinai Hospital, Department of Pathology and Laboratory Medicine, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada. Fax 416-586-8628; e-mail ediamandis{at}mtsinai.on.ca.
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Methods: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections.
Results: The hK13 immunoassay had a detection limit of 0.05 µg/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0–20 µg/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70–98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (
30 kDa) form.
Conclusions: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.
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KLK13, previously known as KLK-L4, is one of the newly identified kallikrein genes. KLK13 was cloned with use of the positional candidate approach. The gene resides on chromosome 19q13.4, spans 10 Kb of genomic DNA sequence, and has five coding exons and four introns (7). The protein has 47% and 45% sequence identity with PSA and hK2, respectively (7). At the mRNA level, KLK13 expression is highest in the mammary gland, prostate, testis, and salivary glands (7). The function of the protein is still unknown. KLK13, like all other members of the human kallikrein family, is predicted to encode a secreted serine protease that is likely present in biological fluids. However, no suitable method for measuring hK13 protein with high sensitivity and specificity has been described. Given the prominent role of PSA as a cancer biomarker and the recent demonstration that other members of this gene family are also potential cancer biomarkers (8)(9)(10)(11)(12), we hypothesized that hK13 may also have utility as a disease biomarker. To further investigate the diagnostic and other clinical applications of this protein, it will be necessary to develop highly sensitive and specific procedures for its quantification in biological fluids and tissues. Because no known natural source of hK13 exists, we expressed this protein in yeast, purified the recombinant protein to homogeneity, generated monoclonal and polyclonal antibodies, and developed an immunofluorometric method for hK13 quantification. Using this assay, we studied the tissue expression of hK13 and preliminarily report that hK13 is overexpressed in ovarian cancer tissues.
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Materials and Methods |
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A by standard procedures (13). The sequence of the construct was confirmed with an automated DNA sequencer.
production of HK13 in yeast
The pPICZA vector containing the KLK13 cDNA sequence was introduced into the yeast strains X-33, KM71, and GS115. A stable clone was selected from the X-33 strain according to the manufacturer’s recommendations (Invitrogen). hK13 was produced by growing the stable yeast clone in a medium containing 10 g/L yeast extract, 20 g/L peptone, 100 mmol/L potassium phosphate (pH 6.0), 13.4 g/L yeast nitrogen base, and 40 mg/L biotin in a 30 °C shaking incubator (250 rpm). hK13 production was induced with 5 mL/L methanol over 6 days. The cells were subsequently pelleted by centrifugation, and the supernatant was collected. A hK13 rabbit polyclonal peptide antibody (produced in house) was used to monitor hK13 production by Western blot analysis.
characterization of HK13 by mass spectrometry
Polyacrylamide gels were stained with Coomassie G-250 for visualization, and selected bands were subsequently excised and destained with 300 mL/L acetonitrile in 100 mmol/L ammonium bicarbonate. Each band was then reduced (10 mmol/L dithiothreitol in 50 mmol/L ammonium bicarbonate, pH 8.3) and alkylated (50 mmol/L iodoacetamide in 50 mmol/L ammonium bicarbonate, pH 8.3) before overnight trypsin digestion. Peptide fragments were then extracted with 50 mL/L acetic acid, evaporated dry on a Savant concentrator, and reconstituted in 10 µL of a solution of methanol–water–acetic acid (500:495:5 by volume).
All nanoelectrospray mass spectrometry (MS) experiments were conducted on a Q-star (PE/Sciex) hybrid quadrupole/time-of-flight instrument, for high resolution and online tandem MS (MS/MS) experiments (14). Conventional mass spectra were obtained by operating the quadrupole in a radiofrequency-only mode while a pusher electrode was pulsed at a frequency of 7 kHz to transfer all ions to the time-of-flight analyzer. MS/MS experiments on trypsin-digested peptides identified in survey scans were conducted with use of a nanoelectrospray source. Precursor ions were selected by the first quadrupole while a pusher electrode was pulsed (frequency 7 kHz) to transfer fragment ions formed in the radiofrequency-only quadrupole cell to the time-of-flight analyzer. Mass spectral resolution was typically 9000–10 000. Scan durations of 1 and 2 s were set for conventional and MS/MS mass spectral acquisition, respectively. Collisional activation was performed using nitrogen collision gas with typically a 30-V offset between the DC voltage of the entrance quadrupole and the radiofrequency-only quadrupole cell. Data were acquired and processed by LC Tune and Biomultiview programs from PE/Sciex.
purification of HK13 with cation-exchange and reversed-phase chromatography
The recombinant hK13 was purified from yeast culture supernatants by cation-exchange chromatography using CM-Sepharose fast flow (Pharmacia Biotech) and reversed-phase liquid chromatography using a C4 column (0.45 x 5 cm; Vydac). The presence of hK13 in various fractions was identified by Western blotting with an anti-hK13 peptide antibody. In summary, CM-Sepharose beads previously activated with 1 mol/L KCl were equilibrated in 10 mmol/L MES buffer (pH 6.5). The yeast culture supernatant was first absorbed on CM-Sepharose beads by incubation at 4 °C overnight under agitation. The beads were then washed with 10 mmol/L MES buffer (pH 6.5), and hK13 was eluted with 300 mmol/L KCl in 10 mmol/L MES buffer (pH 6.5). Trifluoroacetic acid as ion-pairing agent was added into this eluate (final concentration, 10 mL/L), which was then loaded on a C4 column equilibrated with 1 mL/L trifluoroacetic acid in water. A linear gradient (1% per min) of acetonitrile from 15% to 50% in 1 mL/L trifluoroacetic acid was then performed. The fraction containing hK13 was evaporated on a SpeedVac (Savant). The purified material was then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue to assess its purity and its molecular mass. The protein concentration of the purified hK13 was determined by the bicinchoninic acid method, which uses bovine serum albumin (BSA) as calibrator (Pierce Chemical Co.).
assessment of HK13 protein glycosylation
hK13 protein glycosylation was assessed by treatment with the deglycosylation enzyme PNGaseF and the Gel Code glycoprotein staining reagent set according to the manufacturer’s recommendations (Pierce).
assessment of HK13 enzymatic activity
The enzymatic activity of recombinant hK13 protein was assessed with use of the fluorogenic substrates FSR-7-amido-4-methylcoumarin (AMC) and VPR-AMC. We used 10 mmol/L Tris (pH 7.5), 0.1 mol/L NaCl, 5 g/L BSA as a buffer and mixed 5 µL of 10 mmol/L substrate solution, 1 µL of enzyme (1–600 ng), and 94 µL of buffer. We then incubated the samples at 37 °C for 30 min and added 700 µL of 125 mL/L acetic acid to stop the reaction. We measured the fluorescence at an excitation wavelength of 300 nm and emission of 460 nm. We used trypsin as the positive control. The fluorogenic substrates were purchased from Bachem.
n-terminal sequencing of HK13 recombinant protein
N-Terminal sequencing of purified recombinant hK13, blotted on polyvinyldifluoride membranes and stained with Coomassie blue, was performed with the Edman degradation method on an automated amino acid sequencer.
production of polyclonal antibodies against HK13
The purified recombinant hK13 protein was used as an immunogen to immunize rabbits and mice. hK13 (100 µg) was injected subcutaneously into BALB/c female mice and New Zealand White female rabbits. The protein was diluted 1:1 in complete Freund’s adjuvant for the first injection and in incomplete Freund’s adjuvant for the subsequent injections. Injections were repeated six times at 3-week intervals. Blood was drawn from the animals and tested for antibody generation. To test for production of anti-hK13 polyclonal antibodies in mice and rabbits (see below), we used the following immunoassay. Sheep anti-mouse or goat anti-rabbit IgG (Jackson ImmunoResearch) was immobilized on 96-well white ELISA plates. The mouse/rabbit serum was then applied to the plates at different dilutions ranging from 1:500 to 1:50 000 [100 µL/well; diluted in 60 g/L BSA, 50 mmol/L Tris (pH 7.80), 0.5 g/L sodium azide]. After incubation (1 h) and washing, biotinylated recombinant hK13 was then added to each well (5–10 ng/well). Finally, after incubation (1 h) and washing, alkaline phosphatase-conjugated streptavidin was added, the wells were incubated (15 min) and washed, and the alkaline phosphatase activity was detected with time-resolved fluorescence (for details, see below).
production and characterization of monoclonal antibodies
Female BALB/c mice were immunized with recombinant hK13 protein. The immune splenocytes were fused with murine myeloma cells by standard hybridoma technology. Briefly, 100 µg (200 µL) of hK13 was diluted 1:1 in complete Freund’s adjuvant for the first injection and in incomplete Freund’s adjuvant for subsequent injections and administered subcutaneously three times at 3-weeks intervals. Two weeks after the third injection, the mouse received an intraperitoneal injection of aqueous hK13, and 3 days later it was sacrificed and its spleen removed. To generate monoclonal anti-hK13 antibodies, the splenocytes were fused with the Sp2/0 myeloma cells with use of polyethylene glycol 1500. The fused cells were cultured in 96-well plates in DMEM (Invitrogen) containing 200 mL/L fetal calf serum, 200 mmol/L glutamine, 10 g/L OPI (oxaloacetic acid, pyruvic acid, insulin), and 20 g/L HAT (hypoxanthine, aminopterin, thymidine; Sigma) at 37 °C in a 5% CO2 atmosphere for 10–14 days. The supernatants were collected and screened for positive clones by the following immunoassay. Sheep anti-mouse IgG, Fc fragment-specific antibody (Jackson ImmunoResearch) was immobilized on 96-well white ELISA plates. Tissue culture supernatants diluted 10-fold in a general diluent [containing 60 g/L BSA, 50 mmol/L Tris (pH 7.80), and 0.5 g/L sodium azide] were applied to the plates and incubated for 1 h; the wells were then washed six times. Biotinylated recombinant hK13 was then added (5–10 ng/well) and incubated for 1 h, and the plates were washed. Finally, alkaline phosphatase-conjugated streptavidin was added and incubated for 30 min, the plates were washed, and the alkaline phosphatase activity was detected with time-resolved fluorescence, as described elsewhere (15). The positive clones were expanded sequentially in 24-well and 6-well plates in complete medium (reducing the fetal calf serum to 150 mL/L and changing the HAT to HT). Supernatants were further tested by performing IgG isotyping, and clones were subjected to limiting dilution. The clones were then expanded in flasks to generate large amounts of supernatants in serum free-medium (CD-1 medium; Invitrogen) containing 200 mmol/L glutamine.
purification of monoclonal anti-HK13 antibodies
Tissue culture supernatants containing monoclonal antibodies were diluted twofold in 20 mmol/L sodium phosphate buffer (pH 7.0) and injected into a HiTrap Protein G column (Pharmacia). After the column was washed with 20 mmol/L sodium phosphate, the antibodies were eluted with 0.1 mol/L glycine buffer (pH 2.7). The eluted antibody solutions were neutralized and then dialyzed overnight in 0.1 mol/L sodium bicarbonate solution.
immunofluorometric assay for hK13
Standard assay procedure.
For the polyclonal assay (mouse polyclonal/rabbit polyclonal), white polystyrene microtiter plates were coated with sheep anti-mouse IgG, Fc fragment-specific antibody (100 µL of coating antibody solution containing 500 ng of antibody diluted in 50 mmol/L Tris buffer, pH 7.80 in each well) by incubation overnight. The plates were then washed six times with the washing buffer (9 g/L NaCl and 0.5 g/L Tween 20 in 10 mmol/L Tris buffer, pH 7.40). Mouse anti-hK13 polyclonal antiserum was diluted 500-fold in a general diluent [60 g/L BSA, 50 mmol/L Tris (pH 7.80), 0.5 g/L sodium azide], and 100 µL was applied to each well. After a 1-h incubation, the plates were washed six times with washing buffer.
hK13 calibrators or samples were then pipetted into each well (50 µL/well along with 50 µL of the general diluent) and incubated for 1 h with shaking; the plates were then washed with washing buffer six times. Subsequently, 100 µL of rabbit anti-hK13 antiserum diluted 1000-fold in buffer A (containing the components of the general diluent plus 25 mL/L normal mouse serum, 100 mL/L normal goat serum, and 10 g/L bovine IgG) was applied to each well and incubated for 30 min; plates were then washed as described above. Finally, 100 µL/well alkaline phosphatase-conjugated goat anti-rabbit IgG (Fc fragment-specific), diluted 2000-fold in buffer A, was added to each well and incubated for 30 min, and plates were washed as above.
Diflunisal phosphate (100 µL of a 1 mmol/L solution) in substrate buffer (0.1 mol/L Tris, pH 9.1, 0.1 mol/L NaCl, and 1 mmol/L MgCl2) was added to each well and incubated for 10 min. Developing solution (100 µL, containing 1 mol/L Tris base, 0.4 mol/L NaOH, 2 mmol/L TbCl3, and 3 mmol/L EDTA) was pipetted into each well and mixed for 1 min. The fluorescence was measured with a time-resolved fluorometer, the Cyberfluor 615 Immunoanalyzer (MDS Nordion). The calibration and data reduction were performed automatically, as described elsewhere (15). For the monoclonal antibody assay (two monoclonal antibodies), white polystyrene microtiter plates were coated directly with one monoclonal antibody specific for hK13 (code 2-17; 500 ng/well). The plates were then washed six times with the washing buffer. Subsequently, hK13 calibrators were added along with a second biotinylated monoclonal antibody (code 11C1; 50 ng/well). Detection was achieved with alkaline phosphatase-conjugated streptavidin. Diflunisal phosphate and developing solution were added as stated above. For the monoclonal/polyclonal immunoassay configuration, mouse monoclonal 11C1 was used for directly coating microtiter plates and the rabbit polyclonal antibody was used for detection, with the procedures described above. More details about such procedures have been described elsewhere for similar immunoassays for hK6 (8) and hK10 (9).
Determination of the sensitivity of the hK13 immunoassay.
Recombinant hK13 was used to generate the calibration curve. hK13 calibrators were prepared by diluting the purified recombinant hK13 in the general diluent. These calibrators were then used to define the detection limit of the assay.
Determination of the specificity of the hK13 immunoassay.
Biological fluids and recombinant hK13 were used to determine the specificity of the developed immunoassay. These samples were first measured by the standard assay procedure described above. The mouse and rabbit anti-hK13 antisera were then successively replaced with sera from the same animals, obtained before immunization (preimmune sera). The samples were measured again, and the fluorescence counts were compared with the counts obtained by the standard assay. The cross-reactivities of other homologous proteins were investigated using purified recombinant hK1–hK15 (available in house), all diluted in the general diluent.
human tissue cytosolic extracts and biological fluids
Human tissue cytosolic extracts were prepared as follows: Various frozen human tissues (0.2 g) were pulverized on dry ice to fine powders. Extraction buffer [1 mL, containing 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, 10 g/L NP-40 surfactant, 1 mmol/L phenylmethylsulfonyl fluoride, 1 g/L aprotinin, and 1 g/L leupeptin] was added to the tissue powders, and the mixture was incubated on ice for 30 min with repeated shaking and vortex-mixing every 10 min. Mixtures were then centrifuged at 14 000g at 4 °C for 30 min. The supernatants (cytosolic extracts) were then collected. The biological fluids were leftovers of samples submitted for routine biochemical testing. All tissue cytosolic extracts and biological fluids were stored at -80 °C until use.
recovery
Recombinant hK13 was added to the general diluent (control), to male and female normal sera, and to various biological fluids at different concentrations; the samples with added hK13 were measured with the monoclonal/monoclonal, monoclonal/polyclonal, and polyclonal/polyclonal hK13 immunoassays. Recoveries were then calculated after subtraction of the endogenous concentrations.
fractionation of biological fluids with size-exclusion hplc
To determine the molecular mass of the protein detected in the biological fluids and tissue extracts, various samples were fractionated with gel-filtration chromatography, as described elsewhere (8)(9). The fractions were collected and analyzed for hK13 by the developed immunoassay.
immunohistochemistry
Rabbit polyclonal antibody was raised against full-length hK13, as described above. Immunohistochemical staining for hK13 was performed according to a standard immunoperoxidase method. Briefly, paraffin-embedded tissue sections (4 µm) were fixed and dewaxed. Endogenous peroxidase activity was blocked with 30 mL/L aqueous hydrogen peroxide for 15 min. Sections were then treated with 4 g/L pepsin at pH 2.0 for 5 min at 42 °C and blocked with 20 mL/L protein blocker (Signet Laboratories) for 10 min. The primary antibody was then added at a 1:1000 dilution for 1 h at room temperature. After the plates were washed, biotinylated anti-rabbit antibody (Signet), diluted fourfold in antibody dilution buffer (Dako), was added. After incubation and washing, streptavidin-tagged horseradish peroxidase was added for 30 min at room temperature. After washing, detection was achieved with diaminobenzidine for 5–10 min. The slides were then counterstained with hematoxylin and then mounted with coverslips.
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50 kDa) and much smaller amounts of a 28-kDa protein. Both proteins were visible on Western blots (data not shown). The bands representing the 50- and 28-kDa proteins were excised from Coomassie-stained gels, and selected trypsin digested fragments were sequenced by MS/MS. The partial sequence VSGWGTTTSPQVNYPK was identified from the peptide fragments and precisely matched the fragment expected for amino acids 159–174 of hK13 (7). Four other fragments also matched the sequence (data not shown).
We postulated that the higher molecular mass recombinant hK13 protein is highly glycosylated. To explore this possibility, we separated this protein by SDS-PAGE and stained the gels with Coomassie blue before and after digestion with the deglycosylating enzyme PNGaseF (Fig. 1A
). On treatment with PNGaseF, the molecular weight of the recombinant hK13 protein was reduced to 25–28 kDa, consistent with the molecular mass of the nonglycosylated hK13 protein (7). Staining for glycoproteins with acidic fuchsin sulfite was abolished by treatment with PNGaseF (Fig. 1B
).
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The N-terminal sequence of the purified recombinant hK13 was VLNTNG, confirming that the protein represented the active form of the enzyme because activation occurs after cleavage of a K 
V bond between amino acids 25 and 26 of the pre-proenzyme (6)(7).
hK13 cleaved the two trypsin substrates, FSR-AMC and VPR-AMC, in a concentration-dependent manner. For example, under the experimental conditions outlined, hK13 activity produced an increase in the fluorescence of the substrate FSR-AMC, from 1.1 to 1.9 arbitrary units with 10 ng of hK13, to 6.9 arbitrary units with 100 ng of hK13, and to 14.5 arbitrary units with 160 ng of hK13 in 30 min at 25 °C. For the substrate VPR-AMC, the enzyme increased the fluorescence from 0.5 arbitrary units to 1.9 arbitrary units with 10 ng of enzyme, to 13.9 arbitrary units with 100 ng of enzyme, and to 35.5 arbitrary units with 160 ng of enzyme.
production of antibodies and development of the immunofluorometric assay
Recombinant hK13 protein was used as an immunogen in mice and rabbits to generate polyclonal and monoclonal antibodies. These antibodies were then used for immunohistochemical localization of hK13 (see below) and for the development of three different versions of an ELISA-type immunofluorometric assay. Among the three assay configurations (polyclonal mouse coating antibody/polyclonal rabbit detection antibody, monoclonal mouse coating antibody/polyclonal rabbit detection antibody, and monoclonal mouse coating antibody/biotinylated monoclonal mouse detection antibody), the most sensitive was based on a monoclonal/polyclonal configuration. In selected cases, all three research configurations were used, as indicated in the text. Below, we will describe the characteristics of the developed ELISA-type immunofluorometric procedure.
sensitivity, detection limit, specificity, linearity, and precision of the HK13 immunofluorometric assay
Sensitivity.
A typical calibration curve for the hK13 immunofluorometric assay is shown in Fig. 2
. The detection limit, defined as the concentration of hK13 that can be distinguished from zero with 95% confidence, was 0.05 µg/L. These data were obtained with the mouse monoclonal (11CI)/rabbit polyclonal immunoassay configuration, which was more sensitive than the other two assay configurations (monoclonal/monoclonal and polyclonal/polyclonal immunoassays).
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Specificity.
For all assay configurations, we confirmed that the assay specifically measures hK13. When we replaced mouse and rabbit antisera with preimmune mouse and rabbit sera, the fluorescence signals of hK13-positive samples were reduced to nearly zero (data not shown). Recombinant hK1, hK2, hK3, hK4, hK5, hK6, hK7, hK8, hK9, hK10, hK11, hK12, hK14, and hK15 proteins did not produce measurable readings, even at concentrations 1000-fold higher than that of hK13.
Linearity and precision.
Samples of milk, seminal plasma, and amniotic fluid were linear on dilution. Within- and between-run imprecision was assessed with various hK13 calibrators and clinical samples. In all cases, the CVs were 3–8% (n = 12).
HK13 in selected human tissue extracts and biological fluids
The highest tissue extract concentrations of hK13 were in esophagus and tonsil, followed by salivary gland, prostate, kidney, skin, trachea, ureter, testis, breast, lung, and thyroid (Fig. 3
). A similar pattern was also seen in fetal tissues (Fig. 3
).
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In biological fluids, the highest concentrations were seen in seminal plasma, followed by amniotic fluid, breast milk, and follicular fluid (Table 1
). Cerebrospinal fluid contained only traces of hK13. hK13 was below the detection limit of the assay in all male and female serum samples from healthy individuals.
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ovarian cancer cytosols and ascites fluids
Among 20 ovarian carcinoma extracts, 10 were positive for hK13, with concentrations ranging from 0.2 to 15 µg/L. None of the 10 extracts from either healthy ovarian tissue or benign ovarian disease were highly positive (Fig. 4
). Among 44 ascites fluid samples from women with advanced ovarian carcinoma, all were positive for hK13, with values ranging from 0.1 to 20 µg/L. The mean (SD) and median values were 2.3 (4.2) and 0.8 µg/L, respectively.
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recovery of HK13 from biological fluids
Recoveries of recombinant hK13 added to amniotic fluid and milk were 70–98% (range) but were 5–10% with male and female sera. This low recovery was verified with all three versions of the developed immunoassay. Recoveries were similar (5–10%) for serum (from men and women) when we used hK13 from amniotic fluid and seminal plasma (data not shown).
fractionation of biological fluids with size-exclusion hplc
In gel-filtration column chromatography, immunoassayable hK13 of tissue extracts consistently eluted in a peak corresponding to a molecular mass of 30 kDa, consistent with the molecular mass of free (noncomplexed) hK13. This single peak was present in an esophageal extract, seminal plasma, amniotic fluid, and follicular fluid. By contrast, in ascites fluid from ovarian cancer patients, we also detected a small peak corresponding to a molecular mass of 100 kDa (Fig. 5
).
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immunohistochemistry
We localized hK13 immunohistochemically in both nonmalignant and malignant prostatic tissues (Fig. 6
). Staining was mostly cytoplasmic in epithelial cells, whereas stroma was negative. Some nuclear staining of epithelial cells was also evident.
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28 kDa) and heavily glycosylated (50 kDa), the latter being the predominant species. This protein represents the active form of the enzyme and has trypsin-like cleavage specificity, as demonstrated by synthetic substrates.
Our most sensitive assay was based on a mouse monoclonal coating/rabbit polyclonal detection antibody configuration. This assay is highly sensitive and specific, detecting hK13 at concentrations 
0.05µg/L. Size-exclusion HPLC of biological fluids indicated that this assay detects only one immunoreactive peak of the expected molecular mass (30 kDa). These data suggest that our immunoassay detects the free fraction of hK13, although in ascites fluid from an ovarian cancer patient, there is another peak, likely representing hK13 bound to a proteinase inhibitor or another interacting protein. These possibilities are currently under investigation.
We identified hK13 in seminal plasma, amniotic fluid, follicular fluid, and breast milk, suggesting that this enzyme is secreted by cells of male and female reproductive organs. The enzyme was immunohistochemically localized in prostatic tissues (Fig. 6
). Unlike other kallikreins (e.g., hK6 and hK10) (8)(9), hK13 is not present at appreciable amounts in cerebrospinal fluid. Similarly, the concentration of hK13 in serum of healthy men and women appears to be very low, below the detection limit of our assay (<0.05 µg/L). The availability of this highly specific and sensitive immunoassay will facilitate further studies to examine whether the hK13 concentration in serum is altered in disease states, including cancer. It is already known that at least four kallikreins (hK2, hK3, hK6, and hK10) are serologic cancer biomarkers (4)(10)(11)(12).
Our immunoassay revealed that the recovery of recombinant hK13 from serum was incomplete, with values ranging from 5% to 10%. We did not observe differences among the monoclonal/monoclonal, monoclonal/polyclonal, and polyclonal/polyclonal immunoassay configurations. We postulated that serum contains proteinase inhibitors or other interacting proteins that bind to free hK13. Our recombinant protein is enzymatically active and may be sequestered by excess proteinase inhibitors such as 2-macroglobulin. It is already well known that two other kallikreins, hK3 (PSA) and hK2, form complexes with many proteinase inhibitors in serum (16)(17)(18)(19)(20)(21)(22)(23)(24)(25). This proposal merits further investigation.
It has already been demonstrated that multiple kallikreins are overexpressed in ovarian carcinoma [reviewed in Ref. (26)]. There is experimental evidence that these kallikreins may form a cascade enzymatic pathway similar to the pathways of coagulation and fibrinolysis. In this report, we present the first evidence that hK13 is also overexpressed in a subset of patients with ovarian carcinoma (Fig. 4
). We also report relatively high concentrations of hK13 in ascites fluid of women with ovarian cancer. These data suggest that hK13 should be added to the growing number of kallikreins that are overexpressed in ovarian carcinoma.
In conclusion, we describe for the first time production of recombinant hK13 proteins, production of antibodies, and development of highly sensitive and specific immunologic assays for hK13 quantification. We further demonstrate the presence of hK13 in human tissues and biological fluids. In the future, it may be worthwhile to study hK13 expression in disease states and examine whether hK13 concentration in serum can serve as a disease biomarker similar to other kallikreins (hK2, hK3, hK6, and hK10).
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Footnotes |
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References |
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1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.1-antichymotrypsin. Clin Chem 1991;37:1618-1625.2-macroglobulin. Clin Chem 1998;44:2471-2479.The following articles in journals at HighWire Press have cited this article:
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  J. L.V. Shaw and E. P. Diamandis Distribution of 15 Human Kallikreins in Tissues and Biological Fluids Clin. Chem., August 1, 2007; 53(8): 1423 - 1432. [Abstract] [Full Text] [PDF]
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 J. Dorn, M. Schmitt, R. Kates, B. Schmalfeldt, M. Kiechle, A. Scorilas, E. P. Diamandis, and N. Harbeck Primary Tumor Levels of Human Tissue Kallikreins Affect Surgical Success and Survival in Ovarian Cancer Patients Clin. Cancer Res., March 15, 2007; 13(6): 1742 - 1748. [Abstract] [Full Text] [PDF]
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M. H. Slagter, A. Scorilas, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, E. J. Giltay, M. Paliouras, and E. P. Diamandis Effect of Testosterone Administration on Serum and Urine Kallikrein Concentrations in Female-to-Male Transsexuals Clin. Chem., August 1, 2006; 52(8): 1546 - 1551. [Abstract] [Full Text] [PDF]
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M. H. Slagter, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, A. Scorilas, E. J. Giltay, M. Paliouras, and E. P. Diamandis Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens Clin. Chem., July 1, 2006; 52(7): 1356 - 1365. [Abstract] [Full Text] [PDF]
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 Z Dlamini and K D Bhoola Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma J. Clin. Pathol., September 1, 2005; 58(9): 915 - 922. [Abstract] [Full Text] [PDF]
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C. V. Obiezu, S. J.C. Shan, A. Soosaipillai, L.-Y. Luo, L. Grass, G. Sotiropoulou, C. D. Petraki, P. A. Papanastasiou, M. A. Levesque, and E. P. Diamandis Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids Clin. Chem., August 1, 2005; 51(8): 1432 - 1442. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, I. P. Michael, and E. P. Diamandis Human Tissue Kallikreins: Physiologic Roles and Applications in Cancer Mol. Cancer Res., May 1, 2004; 2(5): 257 - 280. [Abstract] [Full Text] [PDF]
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 A. Scorilas, C. A. Borgono, N. Harbeck, J. Dorn, B. Schmalfeldt, M. Schmitt, and E. P. Diamandis Human Kallikrein 13 Protein in Ovarian Cancer Cytosols: A New Favorable Prognostic Marker J. Clin. Oncol., February 15, 2004; 22(4): 678 - 685. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, L. Grass, A. Soosaipillai, G. M. Yousef, C. D. Petraki, D. H. C. Howarth, S. Fracchioli, D. Katsaros, and E. P. Diamandis Human Kallikrein 14: A New Potential Biomarker for Ovarian and Breast Cancer Cancer Res., December 15, 2003; 63(24): 9032 - 9041. [Abstract] [Full Text] [PDF]
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