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...
Saved in:
| Published in: | The Journal of biological chemistry Vol. 291; no. 2; pp. 593 - 604 |
|---|---|
| Main Authors: | , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
08.01.2016
|
| Subjects: | |
| ISSN: | 1083-351X |
| Online Access: | Get more information |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| 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. |
|---|---|
| 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 |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/26582203$$D View this record in MEDLINE/PubMed |
| BookMark | eNo1UDtPwzAYtBCI0sLMhjyypNhObCdjVZUWUQTiIbFFjvO5uDhJiR2k8OsJarnldA_dcGN0XDc1IHRJyZQSmdxsCz19oJRPRUZJJo_QGSVpHMWcvo_Q2PstGZBk9BSNmOApYyQ-Q-0Mv9h64wAvXa9VjVXA4QNwlkXrptnhxuBVVw3-vXLOfrZg66gFpwKU-Al2wZbKA2b4GTbdn-vxTAf7rYJthq26xIv6p68GqfeBDf05OjHKebg48AS93S5e56to_bi8m8_WkeYkC5GhXIlCEJJxrlLCmDGiILwgShohkxRIzGKaEEiV0IXRQoHkLDFKSpkYlrAJut7v7trmqwMf8sp6Dc6pGprO51QKkvKUxmSoXh2qXVFBme9aW6m2z_9_Yr_V_WmL |
| CitedBy_id | crossref_primary_10_1016_j_jprot_2017_05_024 crossref_primary_10_1038_s41598_018_29058_6 crossref_primary_10_3390_ijms241814035 crossref_primary_10_1016_j_semcancer_2017_03_002 crossref_primary_10_3390_toxins9080242 crossref_primary_10_1016_j_procbio_2019_08_019 crossref_primary_10_1038_s41568_021_00436_z crossref_primary_10_3390_ijms17121969 crossref_primary_10_1002_btpr_2752 crossref_primary_10_1016_j_actatropica_2016_12_018 crossref_primary_10_1016_j_jbc_2022_102403 crossref_primary_10_1093_evolut_qpaf012 crossref_primary_10_1016_j_abb_2016_05_017 crossref_primary_10_1515_hsz_2016_0205 crossref_primary_10_1007_s00253_016_7712_4 crossref_primary_10_1515_hsz_2024_0127 crossref_primary_10_1016_j_biochi_2019_09_004 crossref_primary_10_1016_j_jbc_2021_101227 crossref_primary_10_1515_hsz_2018_0148 |
| ContentType | Journal Article |
| Copyright | 2016 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Copyright_xml | – notice: 2016 by The American Society for Biochemistry and Molecular Biology, Inc. |
| DBID | CGR CUY CVF ECM EIF NPM 7X8 |
| DOI | 10.1074/jbc.M115.691097 |
| DatabaseName | Medline MEDLINE MEDLINE (Ovid) MEDLINE MEDLINE PubMed MEDLINE - Academic |
| DatabaseTitle | MEDLINE Medline Complete MEDLINE with Full Text PubMed MEDLINE (Ovid) MEDLINE - Academic |
| DatabaseTitleList | MEDLINE MEDLINE - Academic |
| Database_xml | – sequence: 1 dbid: NPM name: PubMed url: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed sourceTypes: Index Database – sequence: 2 dbid: 7X8 name: MEDLINE - Academic url: https://search.proquest.com/medline sourceTypes: Aggregation Database |
| DeliveryMethod | no_fulltext_linktorsrc |
| Discipline | Anatomy & Physiology Chemistry |
| EISSN | 1083-351X |
| EndPage | 604 |
| ExternalDocumentID | 26582203 |
| Genre | Research Support, Non-U.S. Gov't Journal Article |
| GroupedDBID | --- -DZ -ET -~X 0R~ 18M 29J 2WC 34G 39C 4.4 53G 5BI 5GY 5RE 5VS 79B 85S AAEDW AAFWJ AALRI AARDX AAXUO ABDNZ ABOCM ABPPZ ABRJW ACGFO ACNCT ADBBV ADIYS ADNWM ADVLN AENEX AEXQZ AFOSN AFPKN AITUG AKRWK ALMA_UNASSIGNED_HOLDINGS AMRAJ AOIJS BAWUL BTFSW CGR CJ0 CS3 CUY CVF DIK DU5 E3Z EBS ECM EIF EJD F5P FDB FRP GROUPED_DOAJ GX1 H13 HH5 HYE IH2 KQ8 L7B N9A NPM OK1 P0W P2P R.V RHF RHI RNS ROL RPM SJN TBC TN5 TR2 UHB UKR UPT VQA W8F WH7 WOQ XSW YQT YSK YWH YZZ Z5M ~02 ~KM .7T 7X8 AAYWO ACVFH ADCNI AEUPX AFPUW AIGII AKBMS AKYEP |
| ID | FETCH-LOGICAL-c509t-f15a6b600955a8022ff6b05b0a7f6748e0323140e8a6cbfc6ae7524fa7774f242 |
| IEDL.DBID | 7X8 |
| ISICitedReferencesCount | 22 |
| ISICitedReferencesURI | http://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=Summon&SrcAuth=ProQuest&DestLinkType=CitingArticles&DestApp=WOS_CPL&KeyUT=000367830500008&url=https%3A%2F%2Fcvtisr.summon.serialssolutions.com%2F%23%21%2Fsearch%3Fho%3Df%26include.ft.matches%3Dt%26l%3Dnull%26q%3D |
| IngestDate | Fri Sep 05 06:14:50 EDT 2025 Wed Feb 19 02:29:42 EST 2025 |
| IsDoiOpenAccess | false |
| IsOpenAccess | true |
| IsPeerReviewed | true |
| IsScholarly | true |
| Issue | 2 |
| Keywords | prostate cancer kallikrein zymogen activation substrate specificity N-linked glycosylation serine protease autolytic inactivation enzyme kinetics |
| Language | English |
| License | 2016 by The American Society for Biochemistry and Molecular Biology, Inc. |
| LinkModel | DirectLink |
| MergedId | FETCHMERGED-LOGICAL-c509t-f15a6b600955a8022ff6b05b0a7f6748e0323140e8a6cbfc6ae7524fa7774f242 |
| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| OpenAccessLink | http://www.jbc.org/article/S0021925820361937/pdf |
| PMID | 26582203 |
| PQID | 1760858130 |
| PQPubID | 23479 |
| PageCount | 12 |
| ParticipantIDs | proquest_miscellaneous_1760858130 pubmed_primary_26582203 |
| PublicationCentury | 2000 |
| PublicationDate | 2016-01-08 |
| PublicationDateYYYYMMDD | 2016-01-08 |
| PublicationDate_xml | – month: 01 year: 2016 text: 2016-01-08 day: 08 |
| PublicationDecade | 2010 |
| PublicationPlace | United States |
| PublicationPlace_xml | – name: United States |
| PublicationTitle | The Journal of biological chemistry |
| PublicationTitleAlternate | J Biol Chem |
| PublicationYear | 2016 |
| SSID | ssj0000491 |
| Score | 2.3063812 |
| Snippet | Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In... |
| SourceID | proquest pubmed |
| SourceType | Aggregation Database Index Database |
| StartPage | 593 |
| 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 |
| URI | https://www.ncbi.nlm.nih.gov/pubmed/26582203 https://www.proquest.com/docview/1760858130 |
| Volume | 291 |
| WOSCitedRecordID | wos000367830500008&url=https%3A%2F%2Fcvtisr.summon.serialssolutions.com%2F%23%21%2Fsearch%3Fho%3Df%26include.ft.matches%3Dt%26l%3Dnull%26q%3D |
| hasFullText | |
| inHoldings | 1 |
| isFullTextHit | |
| isPrint | |
| link | http://cvtisr.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwpZ3bS8MwFMaDN9AXL_M2bxxBfIu2Tds0TzLEC-jG8AJ7G2mawHS2c5vC_Os9SVt8EgRf-lIC5TSXX05Ovo-QE8OMn2nGqMpiSa3xNhVMBjT0dZIKw5RhqTOb4J1O0uuJbpVwm1RllfWc6CbqrFA2R37u8xjpIMEp92L0Tq1rlD1drSw05skiQ5SxJV2896MWjvTr13I-PDx_SdVZGwnoLBa-U3n6jSnd2nK99t-vWierFVVCq-wGG2RO5w2y2cpxR_02g1NwdZ4ugd4gy5e1x9smGbfgEdeuoYab4QyDDHIKSIQgBL0vihEUBlyWH-7kcDh4HetBTt3tF51B19bDZLgIQgAPpaO9nkBL1X5pIPMMrvKvmROFLV8g8m-R5-urp8tbWrkwUIUwMaXGj2Scxk6rTtqLucbEqRelnuTGOpVojyEjhp5OZKxSo2KpeRSERnIkS4MEsE0W8iLXuwQynohMJyIKtY8gGAlsYcV1QtzFeVyoJjmuo9zHQNijC5nr4mPS_4lzk-yUv6o_KuU4-gFCVBB4bO8PrffJChJPmUNJDsiiwTGuD8mS-pwOJuMj133w2em2vwELxdBG |
| linkProvider | ProQuest |
| openUrl | ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=A+Single+Glycan+at+the+99-Loop+of+Human+Kallikrein-related+Peptidase+2+Regulates+Activation+and+Enzymatic+Activity&rft.jtitle=The+Journal+of+biological+chemistry&rft.au=Guo%2C+Shihui&rft.au=Skala%2C+Wolfgang&rft.au=Magdolen%2C+Viktor&rft.au=Briza%2C+Peter&rft.date=2016-01-08&rft.eissn=1083-351X&rft.volume=291&rft.issue=2&rft.spage=593&rft_id=info:doi/10.1074%2Fjbc.M115.691097&rft_id=info%3Apmid%2F26582203&rft_id=info%3Apmid%2F26582203&rft.externalDocID=26582203 |