Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System
2008; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês
10.1074/mcp.m800516-mcp200
ISSN1535-9484
AutoresUroš Kuzmanov, Nianxin Jiang, Christopher R. Smith, Antoninus Soosaipillai, Eleftherios P. Diamandis,
Tópico(s)Complement system in diseases
ResumoOvarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with α2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma. Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with α2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma. Disturbed glycosylation patterns have been observed in the majority of human cancers. Over the past 40 years, a number of physiologically expressed proteins containing abnormal glycan structures have been shown to be tumor-associated antigens (1Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10231-10233Crossref PubMed Scopus (809) Google Scholar). For example, prostate-specific antigen (PSA) 1The abbreviations used are: PSA, prostate-specific antigen; ACN, acetonitrile; CA125, cancer antigen 125; CNS, central nervous system; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; KLK6, kallikrein 6; MES, 4-morpholineethanesulfonic acid; SNA, Sambucus nigra agglutinin; TBS, Tris-buffered saline; MS/MS, tandem mass spectrometry; NHS, N-hydroxysuccinimide; BGH, bovine growth hormone. and ribonuclease 1 were found to be differentially glycosylated in prostate and pancreatic cancers, respectively (2Peracaula R. Tabares G. Royle L. Harvey D.J. Dwek R.A. Rudd P.M. de Llorens R. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins.Glycobiology. 2003; 13: 457-470Crossref PubMed Scopus (254) Google Scholar, 3Peracaula R. Royle L. Tabares G. Mallorqui-Fernandez G. Barrabes S. Harvey D.J. Dwek R.A. Rudd P.M. de Llorens R. Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states.Glycobiology. 2003; 13: 227-244Crossref PubMed Scopus (62) Google Scholar). It has been suggested that the disturbed glycosylation of proteins is an early event of oncogenic transformation, aiding in the invasion and metastasis of tumor cells (1Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10231-10233Crossref PubMed Scopus (809) Google Scholar, 4Aubert M. Panicot L. Crotte C. Gibier P. Lombardo D. Sadoulet M.O. Mas E. Restoration of alpha(1,2) fucosyltransferase activity decreases adhesive and metastatic properties of human pancreatic cancer cells.Cancer Res. 2000; 60: 1449-1456PubMed Google Scholar, 5Hsu C.C. Lin T.W. Chang W.W. Wu C.Y. Lo W.H. Wang P.H. Tsai Y.C. Soyasaponin-I-modified invasive behavior of cancer by changing cell surface sialic acids.Gynecol. Oncol. 2005; 96: 415-422Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 6Kannagi R. Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer.Glycoconj. J. 1997; 14: 577-584Crossref PubMed Scopus (301) Google Scholar, 7Kannagi R. Izawa M. Koike T. Miyazaki K. Kimura N. Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis.Cancer Sci. 2004; 95: 377-384Crossref PubMed Scopus (478) Google Scholar, 8Phillips M.L. Nudelman E. Gaeta F.C. Perez M. Singhal A.K. Hakomori S. Paulson J.C. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex.Science. 1990; 250: 1130-1132Crossref PubMed Scopus (1301) Google Scholar, 9Takada A. Ohmori K. Yoneda T. Tsuyuoka K. Hasegawa A. Kiso M. Kannagi R. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium.Cancer Res. 1993; 53: 354-361PubMed Google Scholar, 10Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells.Science. 1990; 250: 1132-1135Crossref PubMed Scopus (887) Google Scholar, 11Zhu Y. Srivatana U. Ullah A. Gagneja H. Berenson C.S. Lance P. Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro.Biochim. Biophys. Acta. 2001; 1536: 148-160Crossref PubMed Scopus (88) Google Scholar). As such, a selective advantage might be conferred on tumor cells with increased glycan structures, allowing them to evade immune response during the invasion and metastasis processes (12Gorelik E. Galili U. Raz A. On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis.Cancer Metastasis Rev. 2001; 20: 245-277Crossref PubMed Scopus (252) Google Scholar). In ovarian cancer, a number of proteins are found to be aberrantly glycosylated, including CA125 (13Jankovic M.M. Milutinovic B.S. Glycoforms of CA125 antigen as a possible cancer marker.Cancer Biomark. 2008. 2008; 4: 35-42Google Scholar), α1-proteinase inhibitor (14Turner G.A. Goodarzi M.T. Thompson S. Glycosylation of alpha-1-proteinase inhibitor and haptoglobin in ovarian cancer: evidence for two different mechanisms.Glycoconj. J. 1995; 12: 211-218Crossref PubMed Scopus (49) Google Scholar), haptoglobin (14Turner G.A. Goodarzi M.T. Thompson S. Glycosylation of alpha-1-proteinase inhibitor and haptoglobin in ovarian cancer: evidence for two different mechanisms.Glycoconj. J. 1995; 12: 211-218Crossref PubMed Scopus (49) Google Scholar), other acute phase proteins (15Saldova R. Royle L. Radcliffe C.M. Abd Hamid U.M. Evans R. Arnold J.N. Banks R.E. Hutson R. Harvey D.J. Antrobus R. Petrescu S.M. Dwek R.A. Rudd P.M. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.Glycobiology. 2007; 17: 1344-1356Crossref PubMed Scopus (333) Google Scholar), and IgGs (15Saldova R. Royle L. Radcliffe C.M. Abd Hamid U.M. Evans R. Arnold J.N. Banks R.E. Hutson R. Harvey D.J. Antrobus R. Petrescu S.M. Dwek R.A. Rudd P.M. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.Glycobiology. 2007; 17: 1344-1356Crossref PubMed Scopus (333) Google Scholar). In particular, there is mounting evidence of increased sialylation of proteins and deregulated sialylation pathways in ovarian cancer (16Aranganathan S. Senthil K. Nalini N. A case control study of glycoprotein status in ovarian carcinoma.Clin. Biochem. 2005; 38: 535-539Crossref PubMed Scopus (30) Google Scholar). Altered sialylation of proteins in this disease is indicated by increased levels of the sialyl LewisX and sialyl-Tn antigens in ovarian carcinoma, even at early stages of progression (15Saldova R. Royle L. Radcliffe C.M. Abd Hamid U.M. Evans R. Arnold J.N. Banks R.E. Hutson R. Harvey D.J. Antrobus R. Petrescu S.M. Dwek R.A. Rudd P.M. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.Glycobiology. 2007; 17: 1344-1356Crossref PubMed Scopus (333) Google Scholar, 17Inoue M. Ton S.M. Ogawa H. Tanizawa O. Expression of Tn and sialyl-Tn antigens in tumor tissues of the ovary.Am. J. Clin. Pathol. 1991; 96: 711-716Crossref PubMed Scopus (59) Google Scholar, 18Inoue M. Fujita M. Nakazawa A. Ogawa H. Tanizawa O. Sialyl-Tn, sialyl-Lewis Xi, CA 19-9, CA 125, carcinoembryonic antigen, and tissue polypeptide antigen in differentiating ovarian cancer from benign tumors.Obstet. Gynecol. 1992; 79: 434-440Crossref PubMed Scopus (34) Google Scholar). This coincides with the findings showing disrupted sialyltransferase protein expression (19Wang P.H. Li Y.F. Juang C.M. Lee Y.R. Chao H.T. Tsai Y.C. Yuan C.C. Altered mRNA expression of sialyltransferase in squamous cell carcinomas of the cervix.Gynecol. Oncol. 2001; 83: 121-127Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 20Wang P.H. Lo W.L. Hsu C.C. Lin T.W. Lee W.L. Wu C.Y. Yuan C.C. Tasi Y.C. Different enzyme activities of sialyltransferases in gynecological cancer cell lines.Eur. J. Gynaecol. Oncol. 2002; 23: 221-226PubMed Google Scholar, 21Wang P.H. Li Y.F. Juang C.M. Lee Y.R. Chao H.T. Ng H.T. Tsai Y.C. Yuan C.C. Expression of sialyltransferase family members in cervix squamous cell carcinoma correlates with lymph node metastasis.Gynecol. Oncol. 2002; 86: 45-52Abstract Full Text PDF PubMed Scopus (55) Google Scholar) and altered mRNA expression of several sialyltransferases in ovarian cancer cells (22Wang P.H. Lee W.L. Juang C.M. Yang Y.H. Lo W.H. Lai C.R. Hsieh S.L. Yuan C.C. Altered mRNA expressions of sialyltransferases in ovarian cancers.Gynecol. Oncol. 2005; 99: 631-639Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Human tissue kallikreins are a family of 15 secreted serine proteases with trypsin or chymotrypsin-like activities. Through the use of RT-PCR, ELISA, immunohistochemical, and bioinformatic techniques, most kallikreins have been shown to be deregulated in a number of malignancies including breast, ovarian, prostate, and testicular cancer (23Borgono C.A. Diamandis E.P. The emerging roles of human tissue kallikreins in cancer.Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (553) Google Scholar, 24Borgono C.A. Michael I.P. Diamandis E.P. Human tissue kallikreins: physiologic roles and applications in cancer.Mol. Cancer Res. 2004; 2: 257-280Crossref PubMed Google Scholar, 25Obiezu C.V. Diamandis E.P. Human tissue kallikrein gene family: applications in cancer.Cancer Lett. 2005; 224: 1-22Crossref PubMed Scopus (120) Google Scholar). Elevated levels of kallikrein 6 (KLK6), a trypsin-like protease, in serum and tissue extracts have been shown to forecast for poor prognosis in ovarian cancer (26Hoffman B.R. Katsaros D. Scorilas A. Diamandis P. Fracchioli S. Rigault de la Longrais IA Colgan T. Puopolo M. Giardina G. Massobrio M. Diamandis E.P. Immunofluorometric quantitation and histochemical localization of kallikrein 6 protein in ovarian cancer tissue: a new independent unfavourable prognostic biomarker.Br. J. Cancer. 2002; 87: 763-771Crossref PubMed Scopus (68) Google Scholar, 27Kim H. Scorilas A. Katsaros D. Yousef G.M. Massobrio M. Fracchioli S. Piccinno R. Gordini G. Diamandis E.P. Human kallikrein gene 5 (KLK5) expression is an indicator of poor prognosis in ovarian cancer.Br. J. Cancer. 2001; 84: 643-650Crossref PubMed Scopus (131) Google Scholar, 28Kyriakopoulou L.G. Yousef G.M. Scorilas A. Katsaros D. Massobrio M. Fracchioli S. Diamandis E.P. Prognostic value of quantitatively assessed KLK7 expression in ovarian cancer.Clin. Biochem. 2003; 36: 135-143Crossref PubMed Scopus (64) Google Scholar, 29Luo L.Y. Katsaros D. Scorilas A. Fracchioli S. Piccinno R. Rigault de la Longrais IA Howarth D.J. Diamandis E.P. Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma.Clin. Cancer Res. 2001; 7: 2372-2379PubMed Google Scholar, 30Obiezu C.V. Scorilas A. Katsaros D. Massobrio M. Yousef G.M. Fracchioli S. Rigault de la Longrais IA Arisio R. Diamandis E.P. Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients.Clin. Cancer Res. 2001; 7: 2380-2386PubMed Google Scholar, 31Tanimoto H. Underwood L.J. Shigemasa K. Parmley T.H. O'Brien T.J. Increased expression of protease M in ovarian tumors.Tumour Biol. 2001; 22: 11-18Crossref PubMed Scopus (73) Google Scholar, 32Yousef G.M. Diamandis E.P. The new human tissue kallikrein gene family: structure, function, and association to disease.Endocr. Rev. 2001; 22: 184-204PubMed Scopus (0) Google Scholar). KLK6 has a wide expression pattern at both the mRNA and protein levels. However, immunohistochemical and ELISA studies have shown that the major site of KLK6 expression is the central nervous system (CNS), with very high (mg/liter) levels of the protein detected in cerebrospinal fluid (CSF) (33Shan S.J. Scorilas A. Katsaros D. Diamandis E.P. Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects.Br. J. Cancer. 2007; 96: 362-372Crossref PubMed Scopus (39) Google Scholar, 34Shaw J.L. Diamandis E.P. Distribution of 15 human kallikreins in tissues and biological fluids.Clin. Chem. 2007; 53: 1423-1432Crossref PubMed Scopus (311) Google Scholar, 35Shih I. Salani R. Fiegl M. Wang T.L. Soosaipillai A. Marth C. Muller-Holzner E. Gastl G. Zhang Z. Diamandis E.P. Ovarian cancer specific kallikrein profile in effusions.Gynecol. Oncol. 2007; 105: 501-507Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). As such, the major source of KLK6 in the circulation of normal individuals is the CNS. The up-regulation of KLK6 in ovarian cancer and its unfavorable prognostic value have been well-established (33Shan S.J. Scorilas A. Katsaros D. Diamandis E.P. Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects.Br. J. Cancer. 2007; 96: 362-372Crossref PubMed Scopus (39) Google Scholar, 36Diamandis E.P. Scorilas A. Fracchioli S. Van Gramberen M. De Bruijn H. Henrik A. Soosaipillai A. Grass L. Yousef G.M. Stenman U.H. Massobrio M. Van Der Zee A.G. Vergote I. Katsaros D. Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.J. Clin. Oncol. 2003; 21: 1035-1043Crossref PubMed Scopus (178) Google Scholar, 37Rosen D.G. Wang L. Atkinson J.N. Yu Y. Lu K.H. Diamandis E.P. Hellstrom I. Mok S.C. Liu J. Bast Jr., R.C. Potential markers that complement expression of CA125 in epithelial ovarian cancer.Gynecol. Oncol. 2005; 99: 267-277Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). It has been previously shown that virtually all ovarian tumors express KLK6, some of them at extremely high levels (33Shan S.J. Scorilas A. Katsaros D. Diamandis E.P. Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects.Br. J. Cancer. 2007; 96: 362-372Crossref PubMed Scopus (39) Google Scholar, 37Rosen D.G. Wang L. Atkinson J.N. Yu Y. Lu K.H. Diamandis E.P. Hellstrom I. Mok S.C. Liu J. Bast Jr., R.C. Potential markers that complement expression of CA125 in epithelial ovarian cancer.Gynecol. Oncol. 2005; 99: 267-277Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). During ovarian cancer development and progression, tumor-derived KLK6 diffuses into the general circulation (33Shan S.J. Scorilas A. Katsaros D. Diamandis E.P. Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects.Br. J. Cancer. 2007; 96: 362-372Crossref PubMed Scopus (39) Google Scholar, 36Diamandis E.P. Scorilas A. Fracchioli S. Van Gramberen M. De Bruijn H. Henrik A. Soosaipillai A. Grass L. Yousef G.M. Stenman U.H. Massobrio M. Van Der Zee A.G. Vergote I. Katsaros D. Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.J. Clin. Oncol. 2003; 21: 1035-1043Crossref PubMed Scopus (178) Google Scholar). Despite these highly favorable characteristics of KLK6 as an ovarian cancer biomarker, the sensitivity of the test performed in serum (for both early and late stage disease) has been shown not to exceed that of the classical ovarian cancer biomarker, CA125 (36Diamandis E.P. Scorilas A. Fracchioli S. Van Gramberen M. De Bruijn H. Henrik A. Soosaipillai A. Grass L. Yousef G.M. Stenman U.H. Massobrio M. Van Der Zee A.G. Vergote I. Katsaros D. Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.J. Clin. Oncol. 2003; 21: 1035-1043Crossref PubMed Scopus (178) Google Scholar). The combination of KLK6 and CA125 resulted in modest increases in sensitivity (10–30% over and above CA125 alone) for both early and late stage disease (36Diamandis E.P. Scorilas A. Fracchioli S. Van Gramberen M. De Bruijn H. Henrik A. Soosaipillai A. Grass L. Yousef G.M. Stenman U.H. Massobrio M. Van Der Zee A.G. Vergote I. Katsaros D. Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.J. Clin. Oncol. 2003; 21: 1035-1043Crossref PubMed Scopus (178) Google Scholar). At early stages, the increase of serum KLK6 contributed by ovarian cancer cells is usually not sufficient to raise KLK6 above the normal serum levels. Therefore, the ability to differentiate KLK6 originating from the CNS (normally found in the serum of healthy individuals) and KLK6 originating from ovarian tumors could potentially increase the diagnostic value of KLK6 as an ovarian cancer biomarker. Toward this purpose, the differential N-glycosylation patterns of KLK6 from ascites fluid of ovarian cancer patients and CSF of healthy individuals were examined. Initially, anion-exchange chromatography with the two biological fluids resulted in different elution patterns, indicative of differential post-translational modifications or processing. Different N-glycosylation patterns of the two isoforms of KLK6 were confirmed by glycosidase digestion followed by gel shift mobility assays. Additionally, the presence of sialylation on the two isoforms was determined by lectin-antibody sandwich ELISA methodology. The composition and structure of the glycans present on the two subpopulations of KLK6 were elucidated by monitoring KLK6 glycopeptides by electrospray ionization-Orbitrap tandem mass spectrometry (MS/MS). Our main finding is that KLK6 from ovarian cancer ascites (but not CSF) is extensively sialylated. This difference in sialylation may be exploited in the future for developing a specific biomarker for ovarian carcinoma. Anion-exchange chromatography was performed using a Mono Q 4.6/100 PE Tricorn high performance column (GE Healthcare) attached to an Agilent 1100 series high performance liquid chromatography system. The running buffer used was 20 mm Tris solution at pH 8.6. Biological fluids (100 μl) were diluted 1:1 in running buffer and loaded onto the column for 5 min with a 0.5 ml/min flow rate. Maintaining the same flow rate, bound proteins were eluted with a linear gradient of increasing NaCl concentration in running buffer (0–400 mm) over the next 35 min. Fractions were collected every min. KLK6 levels in each fraction were measured using a previously described sandwich-type ELISA method (38Diamandis E.P. Yousef G.M. Soosaipillai A.R. Grass L. Porter A. Little S. Sotiropoulou G. Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.Clin. Biochem. 2000; 33: 369-375Crossref PubMed Scopus (122) Google Scholar), utilizing two mouse monoclonal antibodies. Monoclonal mouse antibody against KLK6 (developed in-house; code 27-4) was coated on the NHS-activated-Sepharose 4 Fast Flow beads as per manufacturer's instructions (1 mg of antibody per 1 ml of beads). Immunoisolation was performed by incubating 10 ml of biological fluid with 1 ml of NHS beads for 2 h at room temperature with slow end-to-end rotation. The beads were then washed with 20 ml of a TBS solution with 1 m urea at pH 7.5. The antibody-bound KLK6 was eluted using 10 ml of a 1 m glycine solution at a pH of 2.5. The isolated KLK6 was subjected to buffer exchange with TBS, pH 7.5, and concentrated down to 150 μl using the Millipore Amicon Ultracel spin column with a molecular weight of 10 cutoff. The biological fluids were leftovers of samples submitted for routine biochemical testing or collected with informed consent and institutional review board approval and stored at −80 °C until use. The CSF samples were clear in appearance, without any visible blood contamination and were pools from ∼100 male and female patients. Ovarian cancer ascites used were pools from three late stage ovarian cancer patients. A C-terminally his-tagged KLK6 genomic clone construct in a pcDNA5/FRT/V5-HIS-TOPO backbone (Invitrogen) was generously provided by Dr. Yves Courty (Faculte de Medicine, F3700 Tours, France). The asparagine residue at position 134 of KLK6 in this construct was mutated to glycine by site-directed mutagenesis using standard T7 forward and BGH reverse as terminal primers and 5′-GAC TGC TCA GCC GGC ACC ACC AGC TGC-3′ and 5′-GCA GCT GGT GGT GCC GGC TGA GCA GTC-3′ as mutagenic internal primers. Human embryonic kidney cells (HEK 293) at 80% confluency were transiently transfected with wild-type and mutant constructs in T175 tissue culture flasks with 70 μg of plasmid DNA and 170 μl of the Lipofectamine 2000 transfection reagent as per manufacturer's instructions (Invitrogen). Following transfection and a 72 h growth period, the supernatant was collected, concentrated 10-fold as described above, and the his-tagged KLK6 protein was purified using agarose-bound nickel-nitrilotriacetic acid in batch, as per the manufacturer's protocol (Qiagen). All samples were run on pre-cast NuPAGE 12% Bis-Tris gels in MES/SDS running buffer as per manufacturer's protocol (Invitrogen). The gels were run for 2 h at 200 V. The resolved proteins were transferred onto Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 V for 1 h. Membrane blocking was performed by incubation with TBS-T (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 for 1 h at room temperature. The membrane was then probed with anti-KLK6 polyclonal rabbit antibody (produced in-house; diluted 1:2000 in TBS-T with 5% nonfat dry milk) for 1 h at room temperature. The membrane was washed three times for 15 min with TBS-T and incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody (1:2000 in TBS-T with 5% nonfat dry milk; Jackson ImmunoResearch) for 1 h at room temperature. Finally, the membranes were washed again as above, and the signal was detected on x-ray film using a chemiluminescent substrate (Diagnostic Products Corp.). Immunoisolated KLK6 was treated with N-glycosidase F (PNGase F) from Flavobacterium meningosepticum and acetyl-neuraminyl hydrolase (Neuraminidase) from Cloistridium perfringens as per manufacturer's instructions (New England Biolabs), where the enzymes were used in 10-fold excess for 2 h at 37 °C. Sambucus nigra agglutinin (SNA, Vector Labs) in 50 mm Tris-HCl, pH 7.8 was coated on a 96-well white polystyrene microtiter plate (100 μl of 5 ng/μl SNA per well) by overnight incubation at room temperature and washed twice in wash buffer (10 mmol/liter Tris-HCl, pH 7.4, containing 150 mmol/liter NaCl and 0.5 ml/L Tween 20). Different dilutions of immunoisolated KLK6 in 100 μl of 50 mm Tris-HCl, pH 7.8 were incubated on the plate for 2 h at room temperature with continuous shaking followed by 6 wash steps, as described above. To detect the presence of SNA-bound KLK6, 100 μl/well of biotinylated mouse monoclonal detection antibody E24 (50 ng) diluted in 1% bovine serum albumin were added to each well, incubated at room temperature for 1 h and washed 6 times. Subsequently, 100 μl (5 ng) of alkaline phosphatase-conjugated streptavidin diluted in 1% bovine serum albumin was added to each well, incubated for 15 min with continuous shaking, and washed 6 times. 100 μl of diflunisal phosphate solution (0.1 mol/liter Tris-HCl, pH 9.1, containing 1 mmol/liter diflunisal phosphate, 0.1 mol/liter NaCl, and 1 mmol/liter MgCl2) was then added to each well, and incubated for 10 min with continuous shaking followed by the addition of 100 μl of developing solution (1 mmol/liter Tris base, 0.4 mol/L NaOH, 2 mmol/L TbCl3, and 3 mmol/L EDTA) to each well and mixed for 1 min. Fluorescence was measured with the PerkinElmer EnVision 2103 Multilabel Reader. Immunoisolated KLK6 (1 μg) was resolved on a pre-cast NuPAGE 12% Bis-Tris as described above. The gel was stained with SimplyBlue SafeStain (Invitrogen) and destained in water, per manufacturer's protocol. The KLK6 bands were excised from the gel and dehydrated with acetonitrile (ACN) for 10 min at room temperature. ACN was aspirated, and the bands were reduced in 300 μl of 10 mm dithiothreitol (Sigma-Aldrich) in a 50 mm NH4HCO3 solution for 30 min at 60 °C and allowed to cool to room temperature for 10 min. Following the removal of the reducing solution, the reduced protein in the gel bands was alkylated by addition of 300 μl of a 100 mm iodoacetamide in 50 mm NH4HCO3 solution for 1 h at 37 °C in the dark. Upon removal of the alkylating solution, the gel bands were shrunk with ACN and rehydrated with 50 mm NH4HCO3. This was repeated 3 times. After the last ACN dehydration step, the gel bands were resuspended in 100 μl of 50 mm NH4HCO3 solution containing 1 μg of sequencing grade modified trypsin (Promega) and left overnight at 37 °C for digestion. 40 μl of this solution was used for each MS/MS run. KLK6-derived tryptic peptides were initially bound to a 2 cm C18 pre-column with a 200 μm diameter and eluted onto a resolving 5 cm analytical C18 column (75 μm diameter) with a 15 mm tip (New Objective). The liquid chromatography setup was connected to a Thermo LTQ Orbitrap XL mass spectrometer with a nanoelectrospray ionization source (Proxeon). Analysis of the eluted peptides was done by tandem mass spectrometry in positive-ion mode. A two buffer system was utilized where Buffer A (running) contained 0.1% formic acid, 5% ACN, and 0.02% trifluoroacetic acid in water and Buffer B (elution) contained 90% ACN, 0.1% formic acid, and 0.02% trifluoroacetic acid in water. For structure determination a parent mass list was created for the glycopeptides of interest, and each glycopeptide was fragmented with 25, 30, and 35% normalized collision energy in HCD mode and 35% normalized collision energy in CID mode. Charge state rejection was enabled to reject charge states 1+, 2+, and unassigned charge states. HCD collision energy was optimized in the calibration procedure according to manufacturer's instructions. All data-dependent scan events had isolation width set to 3.0. The glycan structure of KLK6 was determined by MS/MS analysis of the DCSANTTSCHILGWGK glycopeptide. The retention time of the KLK6 glycopeptides was determined by observing the presence of common diagnostic oxonium ions in MS2 spectra (i.e. 204.08 for N-acetylglucosamine or 366.13 for a hexose-linked N-acetylglucosamine). Once this was determined, MS1 spectra over that period of time were combined in a single spectrum using QualBrowser on Xcalibur software (Version 2.0), and individual peaks (corresponding to visually chosen monoisotopic masses of each ion) were inspected as indicators for the presence of glycosylation on the KLK6 glycopeptide. Only triply charged ions were inspected. Corresponding monoisotopic masses were referenced against the Glycomod tool, which provided the output of glycan composition on the given glycopeptide within 5 parts per million mass tolerances. The glycan composition allowed for the inference of the glycan structures, which were further confirmed (where available) against glycan structure databases. For most of the observed ions, analysis of MS2 data for the presence of fragment glycopeptides and glycans was used to further confirm that these were indeed the suspected molecules (data not shown). Anion-exchange chromatography was used to examine the differential elution patterns of KLK6 from CSF and ovarian cancer ascites fluid. Following the chromatography step, eluted fractions w
Referência(s)