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N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Sophia Schedin Weiss' group)
Division of Molecular Biosciences, Imperial College London. (Anne Dell's group)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12, e29011- p.Article in journal (Refereed) Published
Abstract [en]

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH(2)-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc(3)Hex(5)HexNAc(4), consistent with a core fucosylated bi-antennary glycan with terminal Lewis x. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH(2)-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3 +/- 0.2 and 4.1 +/- 0.5 M(-1) s(-1) for the natural full-length PCI and a form lacking six amino acids at the NH(2)-terminus, respectively, whereas these constants were 4.8 +/- 0.1 and 29 +/- 7 M(-1) s(-1) for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH(2-)terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

Place, publisher, year, edition, pages
2011. Vol. 6, no 12, e29011- p.
Keyword [en]
N-glycosylation, Prostate specific antigen, Protein C inhibitor, Seminal plasma, Serpin
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-131131DOI: 10.1371/journal.pone.0029011ISI: 000298665600021OAI: oai:DiVA.org:uu-131131DiVA: diva2:353014
Available from: 2010-09-23 Created: 2010-09-23 Last updated: 2017-12-12Bibliographically approved
In thesis
1. A New Look into Protein C Inhibitor: Posttranslational Modifications and their Functions
Open this publication in new window or tab >>A New Look into Protein C Inhibitor: Posttranslational Modifications and their Functions
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The influences of posttranslational modifications on the functions of the versatile serpin protein C inhibitor (PCI) were studied. PCI is a serine protease inhibitor that is expressed in many tissues and secreted to various fluids in human, including blood plasma, seminal plasma, and urine. PCI in blood can act both as an anticoagulant and a procoagulant and is believed to play a role in pathogen defence. PCI in reproductive tissues is believed to regulate human reproduction at several steps, including the fertilization process. Due to the broad protease specificity and the contradictory activities, the physiological role of PCI is elusive. In this work the inhibitor was purified from blood and seminal plasma by immunoaffinity chromatography. Blood-derived PCI was found to be highly heterogeneous, due to variations in posttranslational modifications. The occupancy and structures of N- and O-glycans attached to blood plasma PCI and N-glycans of seminal plasma PCI were determined by mass spectrometry. An O-glycosylation site at Thr 20 was identified in PCI derived from blood. N-glycan structures of PCI isolated from blood and seminal plasma differed markedly, demonstrating that they are expressed in a tissue-specific manner. Proteolytic processing also appeared to be tissue-specific, since N-terminally cleaved PCI was found in PCI isolated both from blood and seminal plasma, but the length of the lacking segment differed. The effects of the N-linked glycans and the N-terminus of PCI on protease inhibition were determined using enzymatic measurements with chromogenic substrates. The N-glycans and the N-terminus had different effects on the inhibition of thrombin, factor Xa and prostate specific antigen, demonstrating that posttranslational modifications of PCI affect its functional specificity. These findings enhance the understanding of the regulation of the various functions of PCI and may potentially be used for the production of specialized PCI variants for medical purposes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 601
Keyword
Protein C inhibitor, posttranslational modifications, N-glycan, O-glycan, mass spectrometry, factor Xa, thrombin, prostate specific antigen
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-131126 (URN)978-91-554-7901-5 (ISBN)
Public defence
2010-11-05, C10:301, BMC, Husarg. 3, Uppsala, 09:15 (English)
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Available from: 2010-10-15 Created: 2010-09-23 Last updated: 2010-10-22Bibliographically approved

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Engström, ÅkeHreinsson, JuliusWånggren, Kjell

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