Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System

Abstract

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 enzymelinked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with ␣2-6-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. Molecular & Cellular Proteomics 8:791-798, 2009.

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

“…An in-gel digestion protocol was followed, as described elsewhere (10). In all cases, peptides were extracted from solution using C18 OMIX tips (Varian Inc., Lake Forest, CA) and eluted in 5 l of elution buffer B (65% acetonitrile, 0.1% formic acid).…”

Potential Tumor Biomarkers for Ovarian Cancer

“…This protein is a trypsin-like serine protease consisting of a single Nglycosylation site. When juxtaposed against the same protein derived from a non-malignant site, only KLK6 taken from ovarian cancer ascitic fluid displayed 2,6 branched sialylation (Kuzmanov et al, 2009). KLK6 is also a serum marker and these results may translate to this less invasive approach.…”

“…Glycosylation, the formation of a linkage of a glycan with a protein, a lipid, or other organic molecule, greatly increases the complexity of those latter molecules and, resultantly, the potential for information storage. Altered protein glycosylation is believed to be an early event in tumorigenesis which contributes to invasion and metastasis of tumor cells (Chiang et al, 2010;Dall'Olio and Chiricolo, 2001;Hakomori, 1996;Hakomori, 2002;Varki et al, 1969;Kuzmanov et al, 2009;Meezan et al, 1969;Saldova et al, 2008;Yogeeswaran and Salk, 1981). Many prominent proteins in OEC pathophysiology, such as integrins and the receptor for epidermal growth factor (EGFR), are found to be heavily glycosylated (Stroop et al, 2000;Gu and Taniguchi, 2004).…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…KLK6 has been found to be upregulated in ovarian cancer (Seiz et al, 2012). Interestingly, glycosylation patterns of KLK6 from cerebrospinal fluid and ovarian cancer ascites differ significantly, in particular, by an increased heterogeneity and higher degree of terminal sialylation in the cancer samples, as shown by mass spectrometry, raising hopes for a new diagnostic tool in oncology (Kuzmanov et al, 2009(Kuzmanov et al, , 2012. Insect cell-expressed human KLK6 was highly active against tryptic fluorogenic substrates, similar to the E. coli expressed variant (Angelo et al, 2006;Mekdes Debela et al, 2006b).…”