期刊
BIOMOLECULES
卷 5, 期 3, 页码 1832-1854出版社
MDPI
DOI: 10.3390/biom5031832
关键词
neutrophil; cathepsin G; N-glycan; glycopeptide; glycoprotein; chitobiose; paucimannose; N-acetylglucosamine; glycomics; azurophilic granule
资金
- Macquarie University Research Scholarship (iMQRES)
- Australian Cystic Fibrosis postgraduate studentship
- Cancer Institute, NSW, Australia
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 (GlcNAc: 55.2%; Fuc1,6GlcNAc: 22.7%), paucimannosidic N-glycans (Man1,4GlcNAc1,4GlcNAc: 10.6%; Man1,4GlcNAc1,4(Fuc1,6)GlcNAc: 7.9%; Man1,6Man1,4GlcNAc1,4GlcNAc: 3.7%, trace level of Man1,6Man1,4GlcNAc1,4(Fuc1,6)GlcNAc), and trace levels of monoantennary 2,6- and 2,3-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 (similar to 19 angstrom) to the active site of nCG, but due to the truncated nature of Asn71-glycans (similar to 5-17 angstrom) 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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