title: A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity.
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.
authors.affiliation: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Shihui
authors.initials: S
authors.lastname: Guo
authors.affiliation: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Skala
authors.affiliation: the Klinische Forschergruppe der Frauenklinik, Klinikum Rechts der Isar der TU München, 81675 Munich, Germany.
authors.forename: Viktor
authors.initials: V
authors.lastname: Magdolen
authors.affiliation: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.affiliation: the Institute of Molecular Medicine and Cell Research and.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: the Institute of Molecular Medicine and Cell Research and BIOSS 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.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.affiliation: the Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Kellermann
authors.affiliation: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
authors.affiliation: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria, peter.goettig@sbg.ac.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Goettig
mesh_terms: D000595:Amino Acid Sequence; D001329:Autolysis; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors; D020840:Tissue Kallikreins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005353:Fibronectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D020840:Tissue Kallikreins
keywords: N-linked glycosylation; autolytic inactivation; enzyme kinetics; kallikrein; prostate cancer; serine protease; substrate specificity; zymogen activation