Abstract
Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.
Highlights
From the ‡Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria, the §Klinische Forschergruppe der Frauenklinik, Klinikum Rechts der Isar der TU München, 81675 Munich, Germany, the ¶Institute of Molecular Medicine and Cell Research and ʈBIOSS Centre for Biological Signaling Studies, University of Freiburg, 79104 Freiburg, Germany, the **German Cancer Consortium (DKTK), 69120 Heidelberg, Germany, the ‡‡German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany, and the §§Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany
Pro-KLK1 and -2 harbor one (KLK2) was secreted by Leishmania cells with an average protein yield of about 0.9 mg/liter of culture, and its identity was confirmed by mass spectrometry
The propeptide was removed with enterokinase, generating mature KLK2, which was subjected to gel filtration on a Superdex 75 column
Summary
The genes encoding mature KLK2 (Ile-16 –Pro-245a) and the dead mutant KLK2S195A were cloned into the pQE-30 vector (Qiagen) with a His tag preceding the optimized enterokinase recognition sequence SGDR [9]. 50 g of purified human serum fibronectin (Innovative Research, Novi, MI) were incubated with 0.5 g of KLK2 from E. coli or LEXSY cells for different periods of time (0, 1, 2, 4, and 24 h) at 37 °C in 50 mM Tris-HCl, pH 8.0, containing 0.1% Nonidet P-40, 150 mM NaCl. As a control, corresponding amounts of fibronectin or protease alone were monitored at the identical condition. The cleavage was terminated by mixing with SDS loading buffer and boiling for 5 min immediately after a given time point
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
