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Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation.

ABSTRACT: We used lectin microarray and mass spectrometric analysis to identify the N-linked glycosylation patterns of hepatitis C virus (HCV) particles. HCV J6/JFH-1 chimeric cell culture (HCVcc) in the culture supernatant was concentrated and purified by ultrafiltration and sucrose gradient ultracentrifugation. Twelve fractions were collected from the top and analyzed for viral infectivity and HCV RNA content after sucrose gradient separation. HCV RNA and proteins were separated by ultracentrifugation in a continuous 10% to 60% sucrose gradient to purify viral particles based on their sedimentation velocities. HCVcc particles were found mainly in fractions 6 to 8, as determined by quantitative polymerase chain reaction (qPCR) analysis for HCV RNA and ELISA of the HCV core protein. The N-glycans on HCV proteins were analyzed by lectin microarray and mass spectrometry. We identified that 32 of 37 lectins displayed the positive binding signals and 16 types of N-glycoforms of which the major HCV glycoforms were high mannose-type N-linked oligosaccharides, hybrid N-glycans, and fucosylated N-glycans. Our study provided new detailed information regarding the majority of the glycan-protein profile, complementing to previous findings of glycan-HCV protein interactions.

SUBMITTER: Guo Y

PROVIDER: S-EPMC5908620 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation.

Guo Yonghong Y Yu Hanjie H Zhong Yaogang Y He Yu Y Qin Xinmin X Qin Yuan Y Zhou Yun Y Zhang Peixin P Zhang Ying Y Li Zheng Z Jia Zhansheng Z

Medicine 20180401 15

We used lectin microarray and mass spectrometric analysis to identify the N-linked glycosylation patterns of hepatitis C virus (HCV) particles. HCV J6/JFH-1 chimeric cell culture (HCVcc) in the culture supernatant was concentrated and purified by ultrafiltration and sucrose gradient ultracentrifugation. Twelve fractions were collected from the top and analyzed for viral infectivity and HCV RNA content after sucrose gradient separation. HCV RNA and proteins were separated by ultracentrifugation i ...[more]

PMID: 29642144

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Project description:Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3-6 monosaccharides attached to specific serine residues can be extended by 20-200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-022-00092-3.

Dataset Information

Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation.

Publications

Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation.

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