Activation and activity of glycosylated KLKs 3, 4 and 11

Abstract

Human kallikrein-related peptidases 3, 4, 11, and KLK2, the activator of KLK3/PSA, belong to the prostatic group of the KLKs, whose major physiological function is semen liquefaction during the fertilization process. Notably, these KLKs are upregulated in prostate cancer and are used as clinical biomarkers or have been proposed as therapeutic targets. However, this potential awaits a detailed characterization of these proteases. In order to study glycosylated prostatic KLKs resembling the natural proteases, we used Leishmania (LEXSY) and HEK293 cells for secretory expression. Both systems allowed the subsequent purification of soluble pro-KLK zymogens with correct propeptides and of the mature forms. Periodic acid-Schiff reaction, enzymatic deglycosylation assays, and mass spectrometry confirmed the glycosylation of these KLKs. Activation of glycosylated pro-KLKs 4 and 11 turned out to be most efficient by glycosylated KLK2 and KLK4, respectively. By comparing the glycosylated prostatic KLKs with their non-glycosylated counterparts from Escherichia coli, it was observed that the N-glycans stabilize the KLK proteases and change their activation profiles and their enzymatic activity to some extent. The functional role of glycosylation in prostate-specific KLKs could pave the way to a deeper understanding of their biology and to medical applications.

Prostate-specific antigen glycoprofiling as diagnostic and prognostic biomarker of prostate cancer

“…1.2). N-glycan on PSA stabilizes the structure of the enzyme while to some extent changing the activation profile and enzymatic activity [17]. A detailed description of the PSA's glycan structure can be found elsewhere [18,19].…”

“…There is another very important issue to consider while implementing glycan assays for PCa diagnostics-the assay [17]. Green box (middle) image: typical glycan structures present at Asn61 on PSA from healthy men or men with BPH.…”

“…Red box (right) image: typical glycan structures present at Asn61 of PSA from PCa patients. Glycans present at position Asn61 were compiled from various sources [17,[19][20][21][22]. A typical glycan structure present at Asn102 on PSA as a result of an unusual missense mutation (the rs61752561 in KLK3 genes) resulting in the conversion of Asp102 to Asn102 is positioned between the green and red boxes.…”

“…At the opposite side to the active site, an O-glycan is linked to Thr125, which consists of GalNAc-Gal (insert). The figure is taken from an open access publication[17]. Green box (middle) image: typical glycan structures present at Asn61 on PSA from healthy men or men with BPH.…”

Expression und Untersuchung von Pflanzenproteinen mit Leishmania tarentolae

“…Neben Legumain konnten mit LEXSY zahlreiche weitere, in anderen Systemen schwer oder nicht exprimierbare Proteine gewonnen werden, wie z. B. Antikörper [10], das Hepatitis-C-Virus-Hüllprotein [11], das Transmembranprotein Channelrhodopsin 2 [12] oder humane Kallikreine (KLKs) [13,14]. Damit stellt LEXSY einen wichtigen Vertreter im stetig wachsenden Arsenal von Expressionssystemen dar.…”