Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14
2006; Elsevier BV; Volume: 282; Issue: 4 Linguagem: Inglês
10.1074/jbc.m608348200
ISSN1083-351X
AutoresCarla A. Borgoño, Iacovos P. Michael, Julie Shaw, Liu‐Ying Luo, Manik C. Ghosh, Antoninus Soosaipillai, Linda Grass, Dionyssios Katsaros, Eleftherios P. Diamandis,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoHuman tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins α1-antitrypsin, α2-antiplasmin, antithrombin III, and α1-antichymotrypsin with second order rate constants (k+2/Ki) of 49.8, 23.8, 1.48, and 0.224 μm–1 min–1, respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I–IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated. Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins α1-antitrypsin, α2-antiplasmin, antithrombin III, and α1-antichymotrypsin with second order rate constants (k+2/Ki) of 49.8, 23.8, 1.48, and 0.224 μm–1 min–1, respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I–IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated. Proteases include a group of enzymes that catalyze peptide bond hydrolysis. Serine proteases (SP), 2The abbreviations used are: SP, serine proteases; ACT, α1-antichymotrypsin; AP, α2-antiplasmin; AS4.5, angiostatin4.5; ATIII, antithrombin III; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ECM, extracellular matrix; IGFBP, insulin-like growth factor-binding protein; HPLC, high performance liquid chromatography; KLK, kallikrein; LMWK, low molecular weight kininogen; MMP, matrix-metalloprotease; PAI-1, plasminogen activator inhibitor; RSL, reactive site loop; serpin, serine protease inhibitor; MES, 4-morpholineethanesulfonic acid; PSA, prostate-specific antigen; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AMC, 7-amino-4-methylcoumarin; ACN, acetonitrile; IGF, insulin-like growth factor; Tos, tosyl; Boc, t-butoxycarbonyl; Suc, succinyl; AT, α1-antitrypsin. characterized by the presence of a nucleophilic serine residue at the active site, account for 30% of all proteases within the human degradome (1Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (761) Google Scholar). Based on structural homology, SP are categorized into 12 clans, the most highly populated being clan PA(S), and are further classified into families that share high sequence similarity (2Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (470) Google Scholar). Human tissue kallikreins (KLK, EC 3.4.21) form a subgroup of 15 secreted (chymo)tryptic-like SP within the S1A family of clan PA(S) and are encoded by the largest contiguous cluster of protease genes in the entire genome, located on chromosome 19q13.4 (1Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (761) Google Scholar, 3Yousef G.M. Diamandis E.P. Endocr. Rev. 2001; 22: 184-204Crossref PubMed Scopus (595) Google Scholar, 4Borgono C.A. Michael I.P. Diamandis E.P. Mol. Cancer Res. 2004; 2: 257-280PubMed Google Scholar). Because KLKs are widely expressed, KLK activity is implicated in an assorted array of normal physiological processes (e.g. blood pressure regulation, skin homeostasis, and semen liquefaction) and in the pathobiology of several diseases (e.g. cancer, neurodegenerative disorders, and dermatoses) (3Yousef G.M. Diamandis E.P. Endocr. Rev. 2001; 22: 184-204Crossref PubMed Scopus (595) Google Scholar, 4Borgono C.A. Michael I.P. Diamandis E.P. Mol. Cancer Res. 2004; 2: 257-280PubMed Google Scholar, 5Borgono C.A. Diamandis E.P. Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (560) Google Scholar). Because of the frequent dysregulation of KLK expression in malignancy, KLKs have been intensely studied in terms of their clinical applicability as cancer biomarkers. In addition to human kallikrein 3 (KLK3, also known as prostate-specific antigen (PSA)), the premier biomarker in clinical medicine for prostate cancer (6Diamandis E.P. Trends Endocrinol. Metab. 1998; 9: 310-316Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), most other KLKs have emerged as promising markers of diagnosis, prognosis, prediction of therapeutic response, and monitoring for several cancer types, particularly ovarian carcinoma (5Borgono C.A. Diamandis E.P. Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (560) Google Scholar). Along with seven other KLK genes, human tissue kallikrein gene 14 (KLK14) was independently identified in our laboratory by positional candidate cloning in 2001 (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 8Hooper J.D. Bui L.T. Rae F.K. Harvey T.J. Myers S.A. Ashworth L.K. Clements J.A. Genomics. 2001; 73: 117-122Crossref PubMed Scopus (56) Google Scholar). Subsequent studies demonstrated that the KLK14 gene is under steroid-hormone regulation (9Yousef G.M. Fracchioli S. Scorilas A. Borgono C.A. Iskander L. Puopolo M. Massobrio M. Diamandis E.P. Katsaros D. Am. J. Clin. Pathol. 2003; 119: 346-355Crossref PubMed Scopus (61) Google Scholar, 10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar) and is most highly expressed in the glandular epithelia of the breast (10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar, 11Fritzsche F. Gansukh T. Borgono C.A. Burkhardt M. Pahl S. Mayordomo E. Winzer K.J. Weichert W. Denkert C. Jung K. Stephan C. Dietel M. Diamandis E.P. Dahl E. Kristiansen G. Br. J. Cancer. 2006; 94: 540-547Crossref PubMed Scopus (22) Google Scholar) and prostate (8Hooper J.D. Bui L.T. Rae F.K. Harvey T.J. Myers S.A. Ashworth L.K. Clements J.A. Genomics. 2001; 73: 117-122Crossref PubMed Scopus (56) Google Scholar, 10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar) and in the epidermis (i.e. stratum granulosum) and appendages (i.e. hair follicular epithelium and eccrine sweat glands) of the skin (10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar, 12Komatsu N. Takata M. Otsuki N. Toyama T. Ohka R. Takehara K. Saijoh K. J. Investig. Dermatol. 2003; 121: 542-549Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Stefansson K. Brattsand M. Ny A. Glas B. Egelrud T. Biol. Chem. 2006; 387: 761-768Crossref PubMed Scopus (61) Google Scholar). Consequent to its secretion, KLK14 forms a constituent of seminal plasma (10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar) and sweat (14Komatsu N. Tsai B. Sidiropoulos M. Saijoh K. Levesque M.A. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2006; 126: 925-929Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and is present in its catalytically active form in the stratum corneum of the skin (13Stefansson K. Brattsand M. Ny A. Glas B. Egelrud T. Biol. Chem. 2006; 387: 761-768Crossref PubMed Scopus (61) Google Scholar, 14Komatsu N. Tsai B. Sidiropoulos M. Saijoh K. Levesque M.A. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2006; 126: 925-929Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 15Komatsu N. Saijoh K. Sidiropoulos M. Tsai B. Levesque M.A. Elliott M.B. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2005; 125: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Furthermore, aberrant KLK14 expression has been detected in patients with breast (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar, 11Fritzsche F. Gansukh T. Borgono C.A. Burkhardt M. Pahl S. Mayordomo E. Winzer K.J. Weichert W. Denkert C. Jung K. Stephan C. Dietel M. Diamandis E.P. Dahl E. Kristiansen G. Br. J. Cancer. 2006; 94: 540-547Crossref PubMed Scopus (22) Google Scholar, 16Yousef G.M. Borgono C.A. Scorilas A. Ponzone R. Biglia N. Iskander L. Polymeris M.E. Roagna R. Sismondi P. Diamandis E.P. Br. J. Cancer. 2002; 87: 1287-1293Crossref PubMed Scopus (42) Google Scholar), ovarian (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 9Yousef G.M. Fracchioli S. Scorilas A. Borgono C.A. Iskander L. Puopolo M. Massobrio M. Diamandis E.P. Katsaros D. Am. J. Clin. Pathol. 2003; 119: 346-355Crossref PubMed Scopus (61) Google Scholar, 10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar), prostate (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 17Yousef G.M. Stephan C. Scorilas A. Ellatif M.A. Jung K. Kristiansen G. Jung M. Polymeris M.E. Diamandis E.P. Prostate. 2003; 56: 287-292Crossref PubMed Scopus (56) Google Scholar), and testicular (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar) cancers and peeling skin syndrome (18Komatsu N. Suga Y. Saijoh K. Liu A.C. Khan S. Mizuno Y. Ikeda S. Wu H.K. Jayakumar A. Clayman G.L. Shirasaki F. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2006; 126: 2338-2342Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), at the tissue and/or serum level. Correlative clinical data have also linked elevated KLK14 mRNA expression with aggressive forms of breast (11Fritzsche F. Gansukh T. Borgono C.A. Burkhardt M. Pahl S. Mayordomo E. Winzer K.J. Weichert W. Denkert C. Jung K. Stephan C. Dietel M. Diamandis E.P. Dahl E. Kristiansen G. Br. J. Cancer. 2006; 94: 540-547Crossref PubMed Scopus (22) Google Scholar, 16Yousef G.M. Borgono C.A. Scorilas A. Ponzone R. Biglia N. Iskander L. Polymeris M.E. Roagna R. Sismondi P. Diamandis E.P. Br. J. Cancer. 2002; 87: 1287-1293Crossref PubMed Scopus (42) Google Scholar) and prostate cancer (17Yousef G.M. Stephan C. Scorilas A. Ellatif M.A. Jung K. Kristiansen G. Jung M. Polymeris M.E. Diamandis E.P. Prostate. 2003; 56: 287-292Crossref PubMed Scopus (56) Google Scholar) and with the prognosis of breast (16Yousef G.M. Borgono C.A. Scorilas A. Ponzone R. Biglia N. Iskander L. Polymeris M.E. Roagna R. Sismondi P. Diamandis E.P. Br. J. Cancer. 2002; 87: 1287-1293Crossref PubMed Scopus (42) Google Scholar) and ovarian (9Yousef G.M. Fracchioli S. Scorilas A. Borgono C.A. Iskander L. Puopolo M. Massobrio M. Diamandis E.P. Katsaros D. Am. J. Clin. Pathol. 2003; 119: 346-355Crossref PubMed Scopus (61) Google Scholar) cancer patients. Thus, KLK14 represents a potential biomarker and therapeutic target for several pathologic conditions. As with all S1A family SP, KLK14 is synthesized as an inactive precursor or zymogen (herein denoted pro-KLK14), containing a 6-amino acid N-terminal pro-peptide that maintains its latency (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 8Hooper J.D. Bui L.T. Rae F.K. Harvey T.J. Myers S.A. Ashworth L.K. Clements J.A. Genomics. 2001; 73: 117-122Crossref PubMed Scopus (56) Google Scholar). Proteolytic removal of the pro-peptide at Lys24–Ile25 is required to generate active KLK14, a process that may be performed by KLK5 (19Brattsand M. Stefansson K. Lundh C. Haasum Y. Egelrud T. J. Investig. Dermatol. 2005; 124: 198-203Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Because of the presence of Asp198 in its S1 binding pocket (7Yousef G.M. Magklara A. Chang A. Jung K. Katsaros D. Diamandis E.P. Cancer Res. 2001; 61: 3425-3431PubMed Google Scholar, 8Hooper J.D. Bui L.T. Rae F.K. Harvey T.J. Myers S.A. Ashworth L.K. Clements J.A. Genomics. 2001; 73: 117-122Crossref PubMed Scopus (56) Google Scholar), KLK14 is predicted to exert trypsin-like substrate specificity with a preference for basic P1 residues (Schechter and Berger nomenclature (20Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4793) Google Scholar) is used to describe the interaction between protease subsites (Sn-S1;S1′–Sn′) and corresponding substrate residues (Pn-P1;P1′–Pn′), where P1–P1′ denotes the scissile bond). However, we (21Felber L.M. Borgono C.A. Cloutier S.M. Kundig C. Kishi T. Ribeiro C.J. Jichlinski P. Gygi C.M. Leisinger H.J. Diamandis E.P. Deperthes D. Biol. Chem. 2005; 386: 291-298Crossref PubMed Scopus (54) Google Scholar) and others (19Brattsand M. Stefansson K. Lundh C. Haasum Y. Egelrud T. J. Investig. Dermatol. 2005; 124: 198-203Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), through the use of phage-display technology and chromogenic substrates, respectively, have recently demonstrated that KLK14 can accommodate both basic (Arg and Lys) and hydrophobic (Tyr) P1 residues at the S1 subsite with a preference for Arg. Hence, KLK14 manifests dual, trypsin-like and chymotrypsin-like, substrate specificity. To date, putative biological substrates and (patho)physiological roles for KLK14 have been inferred from its expression pattern, substrate specificity, and the known function of co-localized KLKs. Phage-displayed pentapeptide motifs preferred by KLK14 were identified within several intact proteins, including the extracellular matrix (ECM) molecules laminin, collagen type IV, and matrilin-4 (21Felber L.M. Borgono C.A. Cloutier S.M. Kundig C. Kishi T. Ribeiro C.J. Jichlinski P. Gygi C.M. Leisinger H.J. Diamandis E.P. Deperthes D. Biol. Chem. 2005; 386: 291-298Crossref PubMed Scopus (54) Google Scholar). The latter may support a role for KLK14 in cancer progression via ECM digestion, particularly in breast, ovarian, and prostate tumors that bear elevated KLK14 levels. Because of its presence and prominent activity in the stratum corneum of the skin (13Stefansson K. Brattsand M. Ny A. Glas B. Egelrud T. Biol. Chem. 2006; 387: 761-768Crossref PubMed Scopus (61) Google Scholar, 14Komatsu N. Tsai B. Sidiropoulos M. Saijoh K. Levesque M.A. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2006; 126: 925-929Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 15Komatsu N. Saijoh K. Sidiropoulos M. Tsai B. Levesque M.A. Elliott M.B. Takehara K. Diamandis E.P. J. Investig. Dermatol. 2005; 125: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), KLK14 is also implicated in epidermal desquamation (i.e. shedding) via degradation of intercellular (corneo)desmosomal adhesion molecules that link adjacent corneocytes (19Brattsand M. Stefansson K. Lundh C. Haasum Y. Egelrud T. J. Investig. Dermatol. 2005; 124: 198-203Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). More recently, our group has reported that KLK14 may also mediate pleiotropic effects by signaling through proteinase-activated receptors 1, 2, and 4 (22Oikonomopoulou K. Hansen K.K. Saifeddine M. Tea I. Blaber M. Blaber S.I. Scarisbrick I. Andrade-Gordon P. Cottrell G.S. Bunnett N.W. Diamandis E.P. Hollenberg M.D. J. Biol. Chem. 2006; 281: 32095-32112Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). KLK14 may exercise its functions alone or in a proteolytic cascade pathway, as KLK5 may activate pro-KLK14 and vice versa (19Brattsand M. Stefansson K. Lundh C. Haasum Y. Egelrud T. J. Investig. Dermatol. 2005; 124: 198-203Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Collectively, studies on KLK14 reveal that this novel SP may play important (patho)physiological roles at the main sites of its expression and bears clinical utility. To gain further insights into the biological significance of KLK14 action, this study examines KLK14 expression, specificity, activity against candidate substrates, and several modes of regulation. Mature KLK14—Mature recombinant KLK14 was produced in the Easyselect™ Pichia pastoris expression system (Invitrogen), as described previously (21Felber L.M. Borgono C.A. Cloutier S.M. Kundig C. Kishi T. Ribeiro C.J. Jichlinski P. Gygi C.M. Leisinger H.J. Diamandis E.P. Deperthes D. Biol. Chem. 2005; 386: 291-298Crossref PubMed Scopus (54) Google Scholar). Briefly, PCR-amplified KLK14 cDNA encoding the mature enzyme form of KLK14 (amino acids 25–251 based on GenBank™ accession number AAK48524) was cloned into P. pastoris expression vector pPICZαA (Invitrogen) at EcoRI and XbaI restriction enzyme sites between the 5′ promoter and the 3′ terminator of the AOX1 gene, in-frame with the yeast α-mating factor (for secretion), using standard techniques. The PmeI-linearized pPICZαA-KLK14 construct was transformed into chemically competent P. pastoris yeast strains X-33, GS115, and KM71. A stable X-33 transformant was selected, and recombinant KLK14 expression was induced with 1% methanol/day for 6 days at 30 °C in a shaking incubator (250 rpm). Mature recombinant KLK14 was purified to homogeneity from culture supernatant by a two-step procedure consisting of cation-exchange and affinity chromatography. Typically, 1 liter of culture supernatant was clarified by centrifugation and concentrated 20-fold by positive pressure ultrafiltration in an Amicon™ stirring chamber (Millipore Corporation, Bedford, MA) with a 10-kDa cutoff nitrocellulose membrane (Millipore). The concentrated supernatant was diluted 1:4 in 10 mm MES (pH 5.3) and fractionated on a pre-equilibrated 5-ml cation-exchange HiTrap™ SP HP column (GE Healthcare), with the automatedÁKTA FPLC system (GE Healthcare). Adsorbed KLK14 was eluted with a multistep salt gradient using 1 m KCl in 10 mm MES (pH 5.3) at a flow rate of 3 ml/min as follows: (a) continuous linear gradient of 0–0.3 m KCl for 17 min, (b) maintenance at 0.3 m KCl for 20 min, (c) followed by a continuous linear gradient from 0.3–1 m KCl for 50 min. Elution fractions (3 ml) containing KLK14 were identified (as described below), pooled, and concentrated using Biomax-10 Ultrafree®-15 centrifugal filter device (Millipore Corp., Bedford, MA). The concentrated fractions were diluted 1:4 in 100 mm Tris-HCl (pH 7.8) binding buffer and incubated with 1 ml of soybean trypsin inhibitor-agarose beads (Sigma) overnight at 4 °C. The beads were then packed into an Econo-Pac open column (Bio-Rad) and washed three times with binding buffer. KLK14 was eluted with 0.1 m glycine buffer (pH 3.0). Pro-KLK14—First-strand cDNA synthesis was performed by reverse transcriptase using the Superscript™ preamplification system (Invitrogen) with 2 μg of total human cerebellum RNA (Clontech) as a template. The cDNA encoding prepro-KLK14 (amino acids 1–251 based on GenBank™ accession number AAK48524) was PCR-amplified in a 50-μl reaction mixture containing 1 μl of cerebellum cDNA as a template, 100 ng of primers (forward, 5′ CACC ATG TTC CTC CTG CTG ACA GCA CTT; reverse, 5′ AGA CCA TCA TTT GTC CCG CAT CGT TTC CT, containing CACC sequence required for TOPO® cloning (underlined) and the native KLK14 stop codon (italics)), 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 200 μm deoxynucleoside triphosphates (dNTPs), and 0.75 μl (2.6 units) of Expand Long Template PCR polymerase mix (Roche Diagnostics) on an Eppendorf master cycler (Eppendorf, Westbury, NY). PCR conditions were 94 °C for 2 min, followed by 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s for 40 cycles, and a final extension at 68 °C for 7 min. The PCR product was cloned into the mammalian expression vector pcDNA3.1™D/V5-His-TOPO® (Invitrogen) using the pcDNA3.1™ Directional TOPO® expression kit (Invitrogen), according to the manufacturer's protocol. The KLK14 sequence within the construct, denoted pcDNA3.1-KLK14, was confirmed with an automated DNA sequencer using vector-specific primers in both directions. Human embryonic kidney (HEK)-293 cells (American Type Culture Collection (ATCC), Manassas, VA) were stably transfected with 3 μg of PmeI-linearized pcDNA3.1-KLK14 by lipofection using PolyFect transfection reagent (Qiagen, Valencia, CA), according to the manufacturer's instructions. Transfected HEK-293 cells were incubated in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS), in a humidified atmosphere containing 5% CO2. After 48 h, cells were re-plated into 35-mm dishes, and the selection agent Geneticin (G418, 300 μg/ml final concentration; Invitrogen) was added. Resistant clones were chosen over 14 days under continuous G418 selection and cultured separately in 24 wells followed by 6-well plates. One clone, denoted P1–4, was selected for large scale expression of pro-KLK14 and grown in DMEM with 10% FBS until 80% confluency. At this stage, the media were replaced with serum-free CD Chinese hamster ovary media supplemented with GlutaMax™I (8 mm final concentration; Invitrogen). Conditioned media was collected by centrifugation after 10 days of incubation. Recombinant pro-KLK14 was purified to homogeneity from the conditioned serum-free media of HEK-293 cells stably transfected with pcDNA3.1-KLK14 sequentially by cation-exchange and reversed-phase chromatography. Prior to purification, 1 liter of media was prepared as described above for yeast culture supernatant. Concentrated CM was diluted 1:4 in 10 mm MES (pH 5.3) and applied to a previously equilibrated 5-ml SP HP-Sepharose column (GE Healthcare), previously equilibrated with 10 mm MES (pH 5.3), on theÁKTA fast performance liquid chromatography system at a flow rate of 1 ml/min. Pro-KLK14 was eluted with a multistep salt gradient of 1 m KCl in 10 mm MES (pH 5.3) at a flow rate of 3 ml/min as follows: (a) continuous linear gradient of 0–0.25 m KCl for 17 min, (b) maintenance at 0.25 m KCl for 33 min, (c) linear gradient of 0.25–0.35 m KCl for 8.3 min, (d) maintenance at 0.35 m KCl for 33 min, and (e) followed by a linear gradient of 0.35-0.8 M KCl for 33 min. Elution fractions (3 ml) were analyzed by our newly developed enzyme-linked immunosorbent assay (ELISA; described herein) and by detection methods mentioned below. Fractions containing pro-KLK14 were then pooled, concentrated, and supplemented with a final concentration of 1% trifluoroacetic acid and loaded in 0.1% trifluoroacetic acid in H2O onto a 1-ml Vydac C4 reversed-phase column (The Separations Group, Inc., Hesperia, CA) connected to an Agilent series 1100 HPLC system equipped with a diode array detector (Agilent Technologies, Inc, Palo Alto, CA). Elution was performed with a multistep acetonitrile (ACN) gradient at a flow rate of 0.8 ml/min consisting of 5-min linear gradients of 20–25, 25–30, 30–32, 32–35, 35–40, and 40–100% ACN intervened by 15-min steps at 25, 30, 32, and 35% ACN. ACN was removed from each fraction by evaporation with nitrogen gas at a pressure of 15 p.s.i. For both recombinant KLK14 proteins, purity was assessed on silver-stained SDS-polyacrylamide gels (described below). Concentration was determined by the BCA method (Pierce), and protein identity was confirmed by tandem mass spectrometry, as described previously (23Luo L.Y. Grass L. Howarth D.J. Thibault P. Ong H. Diamandis E.P. Clin. Chem. 2001; 47: 237-246Crossref PubMed Scopus (87) Google Scholar). Proteins were aliquoted and stored at –80 °C in 0.1 m sodium acetate buffer (pH 5.0). To monitor recombinant KLK14 production, purification, and/or activity, samples were analyzed by SDS-PAGE, Western blot, and zymography. SDS-PAGE—SDS-PAGE was performed using the NuPAGE BisTris electrophoresis system and precast 4–12% gradient polyacrylamide gels at 200 V for 30 min (Invitrogen). Proteins were visualized with a Coomassie G-250 staining solution, SimplyBlue™ SafeStain (Invitrogen), and/or by silver staining with the Silver Xpress™ kit (Invitrogen), according to the manufacturer's instructions. Western Blots—For immunodetection of KLK14, proteins resolved by SDS-PAGE were subsequently transferred onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 V for 1 h. The membrane was blocked with Tris-buffered saline/Tween (0.1 mol/liter Tris-HCl buffer (pH 7.5) containing 0.15 mol/liter NaCl and 0.1% Tween 20) supplemented with 5% nonfat dry milk overnight at 4 °C and probed with a KLK14 polyclonal rabbit antibody (produced in-house; diluted 1:2000 in Tris-buffered saline/Tween) for 1 h at room temperature. The membrane was washed three times for 15 min with Tris-buffered saline/Tween and treated with alkaline phosphatase-conjugated goat anti-rabbit antibody (1:10,000 in Tris-buffered saline/Tween; Jackson ImmunoResearch) for 1 h at room temperature. Finally, the membranes were washed again as above, and fluorescence was detected on x-ray film using chemiluminescent substrate (Diagnostic Products Corp., Los Angeles). Zymography—The proteolytic activity of recombinant KLK14 proteins was visualized by gelatin zymography (Invitrogen). Recombinant KLK14 was diluted 1:1 in Tris-glycine SDS sample buffer and electrophoresed on precast Novex® 10% gelatin zymogram gels (Invitrogen) at 125 V for 2.5 h at 4 °C. The gels were subsequently incubated in zymogram renaturing buffer (Invitrogen) for two 30-min intervals at room temperature, followed by incubation in zymogram developing buffer (Invitrogen) for 3 h at 37°C. Gels were stained with SimplyBlue™ SafeStain and destained until the white lytic bands corresponding to areas of protease activity were visible. New Zealand White female rabbits and BALB/c female mice were repeatedly immunized with purified recombinant mature KLK14 (100 μg) for polyclonal and monoclonal antibody development, respectively. KLK14 was diluted 1:1 in complete Freund's adjuvant for the first injection and in incomplete Freund's adjuvant for subsequent injections. Injections were repeated three times for mice and six times for rabbits at 3-week intervals. The polyclonal sera were tested every 2 weeks by an immunofluorometric method described in detail elsewhere (10Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar), until the highest antibody titers against KLK14 were detected. Monoclonal antibodies against KLK14 were produced by standard hybridoma technology. The splenocytes from preselected immunized mice were fused with the Sp2/0 myeloma cells using polyethylene glycol 1500. The resultant hybridoma cells were cultured in 96-well plates in DMEM (Invitrogen) containing 20% FBS, 200 mm glutamine, 1% OPI (oxaloacetic acid, pyruvic acid, insulin), and 2% HAT (hypoxanthine, aminopterin, thymidine; Sigma) for selection at 37 °C, 5% CO2 for 10–14 days. Hybridoma supernatants were collected and screened by an immunofluorometric technique described previously (24Obiezu C.V. Shan S.J. Soosaipillai A. Luo L.Y. Grass L. Sotiropoulou G. Petraki C.D. Papanastasiou P.A. Levesque M.A. Diamandis E.P. Clin. Chem. 2005; 51: 1432
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