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N-glycans and the N terminus of protein C inhibitor affect the cofactor-enhanced rates of thrombin inhibition
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 27, 18601-11 p.Article in journal (Refereed) Published
Abstract [en]

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

Place, publisher, year, edition, pages
2008. Vol. 283, no 27, 18601-11 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-99782DOI: 10.1074/jbc.M800608200ISI: 000257165600016PubMedID: 18467335OAI: oai:DiVA.org:uu-99782DiVA: diva2:208737
Available from: 2009-03-19 Created: 2009-03-19 Last updated: 2011-06-28Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 601
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
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)
Available from: 2010-10-15 Created: 2010-09-23 Last updated: 2010-10-22Bibliographically approved

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Engström, Åke
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