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Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

ABSTRACT: Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.

SUBMITTER: Cao X

PROVIDER: S-EPMC9092021 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

Cao Xinyi X Meng Peiyi P Shao Yuyin Y Yan Guoquan G Yao Jun J Zhou Xinwen X Liu Chao C Zhang Lei L Shu Hong H Lu Haojie H

Frontiers in molecular biosciences 20220427

Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) c ...[more]

PMID: 35573732

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Project description:UnlabelledBroad-spectrum O-linked protein glycosylation is well characterized in the major Neisseria species of importance to human health and disease. Within strains of Neisseria gonorrhoeae, N. meningitidis, and N. lactamica, protein glycosylation (pgl) gene content and the corresponding oligosaccharide structure are fairly well conserved, although intra- and interstrain variability occurs. The status of such systems in distantly related commensal species, however, remains largely unexplored. Using a strain of deeply branching Neisseria elongata subsp. glycolytica, a heretofore unrecognized tetrasaccharide glycoform consisting of di-N-acetylbacillosamine-glucose-di-N-acetyl hexuronic acid-N-acetylhexosamine (diNAcBac-Glc-diNAcHexA-HexNAc) was identified. Directed mutagenesis, mass spectrometric analysis, and glycan serotyping confirmed that the oligosaccharide is an extended version of the diNAcBac-Glc-based structure seen in N. gonorrhoeae and N. meningitidis generated by the successive actions of PglB, PglC, and PglD and glucosyltransferase PglH orthologues. In addition, a null mutation in the orthologue of the broadly conserved but enigmatic pglG gene precluded expression of the extended glycoform, providing the first evidence that its product is a functional glycosyltransferase. Despite clear evidence for a substantial number of glycoprotein substrates, the major pilin subunit of the endogenous type IV pilus was not glycosylated. The latter finding raises obvious questions as to the relative distribution of pilin glycosylation within the genus, how protein glycosylation substrates are selected, and the overall structure-function relationships of broad-spectrum protein glycosylation. Together, the results of this study provide a foundation upon which to assess neisserial O-linked protein glycosylation diversity at the genus level.ImportanceBroad-spectrum protein glycosylation systems are well characterized in the pathogenic Neisseria species N. gonorrhoeae and N. meningitidis. A number of lines of evidence indicate that the glycan components in these systems are subject to diversifying selection and suggest that glycan variation may be driven in the context of glycosylation of the abundant and surface-localized pilin protein PilE, the major subunit of type IV pili. Here, we examined protein glycosylation in a distantly related, nonpathogenic neisserial species, Neisseria elongata subsp. glycolytica. This system has clear similarities to the systems found in pathogenic species but makes novel glycoforms utilizing a glycosyltransferase that is widely conserved at the genus level but whose function until now remained unknown. Remarkably, PilE pilin is not glycosylated in this species, a finding that raises important questions about the evolutionary trajectories and overall structure-function relationships of broad-spectrum protein glycosylation systems in bacteria.

Dataset Information

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

Publications

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

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