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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Vydáno v:The Journal of biological chemistry Ročník 291; číslo 2; s. 593 - 604
Hlavní autoři: Guo, Shihui, Skala, Wolfgang, Magdolen, Viktor, Briza, Peter, Biniossek, Martin L, Schilling, Oliver, Kellermann, Josef, Brandstetter, Hans, Goettig, Peter
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 08.01.2016
Témata:
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
prostate cancer
kallikrein
zymogen activation
substrate specificity
N-linked glycosylation
serine protease
autolytic inactivation
enzyme kinetics
ISSN:1083-351X
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Shrnutí: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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ISSN:1083-351X
DOI:10.1074/jbc.M115.691097