Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G
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...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2015-08-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | http://www.mdpi.com/2218-273X/5/3/1832 |
| _version_ | 1818570560235372544 |
|---|---|
| author | Ian Loke Nicolle H. Packer Morten Thaysen-Andersen |
| author_facet | Ian Loke Nicolle H. Packer Morten Thaysen-Andersen |
| author_sort | Ian Loke |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T13:43:21Z |
| format | Article |
| id | doaj.art-675cd22e06a64fb7b0aea9e722b608ee |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-12-14T13:43:21Z |
| publishDate | 2015-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-675cd22e06a64fb7b0aea9e722b608ee2022-12-21T22:59:23ZengMDPI AGBiomolecules2218-273X2015-08-01531832185410.3390/biom5031832biom5031832Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin GIan Loke0Nicolle H. Packer1Morten Thaysen-Andersen2Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Sydney 2109, AustraliaDepartment of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Sydney 2109, AustraliaDepartment of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Sydney 2109, AustraliaNeutrophil 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.http://www.mdpi.com/2218-273X/5/3/1832neutrophilcathepsin GN-glycanglycopeptideglycoproteinchitobiosepaucimannoseN-acetylglucosamineglycomicsazurophilic granule |
| spellingShingle | Ian Loke Nicolle H. Packer Morten Thaysen-Andersen Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G Biomolecules neutrophil cathepsin G N-glycan glycopeptide glycoprotein chitobiose paucimannose N-acetylglucosamine glycomics azurophilic granule |
| title | Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G |
| title_full | Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G |
| title_fullStr | Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G |
| title_full_unstemmed | Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G |
| title_short | Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G |
| title_sort | complementary lc ms ms based n glycan n glycopeptide and intact n glycoprotein profiling reveals unconventional asn71 glycosylation of human neutrophil cathepsin g |
| topic | neutrophil cathepsin G N-glycan glycopeptide glycoprotein chitobiose paucimannose N-acetylglucosamine glycomics azurophilic granule |
| url | http://www.mdpi.com/2218-273X/5/3/1832 |
| work_keys_str_mv | AT ianloke complementarylcmsmsbasednglycannglycopeptideandintactnglycoproteinprofilingrevealsunconventionalasn71glycosylationofhumanneutrophilcathepsing AT nicollehpacker complementarylcmsmsbasednglycannglycopeptideandintactnglycoproteinprofilingrevealsunconventionalasn71glycosylationofhumanneutrophilcathepsing AT mortenthaysenandersen complementarylcmsmsbasednglycannglycopeptideandintactnglycoproteinprofilingrevealsunconventionalasn71glycosylationofhumanneutrophilcathepsing |