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  • Complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact N-glycoprotein profiling reveals unconventional Asn71-glycosylation of human neutrophil cathepsin G

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    Author(s)
    Loke, Ian
    Packer, Nicolle H.
    Thaysen-Andersen, Morten
    Griffith University Author(s)
    Packer, Nicki
    Year published
    2015
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    Abstract
    Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%), paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ: ...
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    Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%), paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ: 7.9%; Manα1,6Manβ1,4GlcNAcβ1,4GlcNAcβ: 3.7%, trace level of Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5–17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation.
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    Journal Title
    Biomolecules
    Volume
    5
    Issue
    3
    DOI
    https://doi.org/10.3390/biom5031832
    Copyright Statement
    © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    Subject
    Biochemistry and cell biology
    Biochemistry and cell biology not elsewhere classified
    Publication URI
    http://hdl.handle.net/10072/336669
    Collection
    • Journal articles

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