A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity
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 wi...
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| Published in: | The Journal of biological chemistry Vol. 291; no. 2; pp. 593 - 604 |
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| Main Authors: | , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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United States
08.01.2016
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| ISSN: | 1083-351X |
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| 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. |
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| AbstractList | 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. |
| Author | Kellermann, Josef Skala, Wolfgang Brandstetter, Hans Biniossek, Martin L Magdolen, Viktor Schilling, Oliver Goettig, Peter Briza, Peter Guo, Shihui |
| Author_xml | – sequence: 1 givenname: Shihui surname: Guo fullname: Guo, Shihui organization: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria – sequence: 2 givenname: Wolfgang surname: Skala fullname: Skala, Wolfgang organization: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria – sequence: 3 givenname: Viktor surname: Magdolen fullname: Magdolen, Viktor organization: the Klinische Forschergruppe der Frauenklinik, Klinikum Rechts der Isar der TU München, 81675 Munich, Germany – sequence: 4 givenname: Peter surname: Briza fullname: Briza, Peter organization: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria – sequence: 5 givenname: Martin L surname: Biniossek fullname: Biniossek, Martin L organization: the Institute of Molecular Medicine and Cell Research and – sequence: 6 givenname: Oliver surname: Schilling fullname: Schilling, Oliver organization: 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 – sequence: 7 givenname: Josef surname: Kellermann fullname: Kellermann, Josef organization: the Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany – sequence: 8 givenname: Hans surname: Brandstetter fullname: Brandstetter, Hans organization: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria – sequence: 9 givenname: Peter surname: Goettig fullname: Goettig, Peter email: peter.goettig@sbg.ac.at organization: From the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria, peter.goettig@sbg.ac.at |
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| Keywords | prostate cancer kallikrein zymogen activation substrate specificity N-linked glycosylation serine protease autolytic inactivation enzyme kinetics |
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| SubjectTerms | Amino Acid Sequence Autolysis Enzyme Activation Fibronectins - metabolism Glycosylation Humans Kinetics Models, Molecular Molecular Sequence Data Polysaccharides - metabolism Protein Structure, Secondary Proteolysis Recombinant Proteins - isolation & purification Structure-Activity Relationship Substrate Specificity Time Factors Tissue Kallikreins - chemistry Tissue Kallikreins - metabolism |
| Title | A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity |
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