Characterization of Site-Specific N-Glycosylation

Hevér, Helga; Darula, Zsuzsanna; Medzihradszky, Katalin F.

doi:10.1007/978-1-4939-9055-9_8

Cited by 11 publications

(9 citation statements)

References 79 publications

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“…Moreover, our opinion is that the band with an electrophoretic migration of 17-kDa is due to Ageritin micro-heterogeneity given its cross-reactivity with anti-Ageritin antibodies. One possibility could be the presence of posttranslational modifications such as N -linked glycosylation considering that Ageritin sequence contains two potential N -glycosylation consensus motifs at N79 and N118 ( Figure S2 ) [ 22 , 23 ].…”

Section: Resultsmentioning

confidence: 99%

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita

Baglivo

Ragucci

D’Incecco

et al. 2020

IJMS

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The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5′ region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

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Section: Resultsmentioning

confidence: 99%

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita

Baglivo

Ragucci

D’Incecco

et al. 2020

IJMS

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“…It ranges from glycopeptide analysis, which provides information about site-specific glycosylation, to complete intact glycoprotein analysis, which provides intuitive information on the degree of glycosylation of the whole protein [ 31 , 32 , 49 , 50 , 51 ]. In particular, glycoproteomic analysis at the glycopeptide level is an effective method that can simultaneously acquire information on glycans and glycosylation sites [ 52 , 53 , 54 ], but it is not utilized in clinics due to the time required for the sample preparation step, difficulties of tandem MS analysis, and complex data interpretation. Therefore, there is a need for an easy and fast glycoproteomic analysis tool that can be used in the clinical field [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Aberrant Glycosylation of Serum Haptoglobin for Gastric Cancer Diagnosis Using a Middle-Up-Down Glycoproteome Platform

Jeong

Kim²,

et al. 2021

JPM

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Gastric cancer is a frequently occurring cancer and is the leading cause of cancer-related deaths. Recent studies have shown that aberrant glycosylation of serum haptoglobin is closely related to gastric cancer and has enormous potential for use in diagnosis. However, there is no platform with high reliability and high reproducibility to comprehensively analyze haptoglobin glycosylation covering microheterogeneity to macroheterogeneity for clinical applications. In this study, we developed a middle-up-down glycoproteome platform for fast and accurate monitoring of haptoglobin glycosylation. This platform utilizes an online purification of LC for sample desalting, and an in silico haptoglobin glycopeptide library constructed by combining peptides and N-glycans to readily identify glycopeptides. In addition, site-specific glycosylation with glycan heterogeneity can be obtained through only a single MS analysis. Haptoglobin glycosylation in clinical samples consisting of healthy controls (n = 47) and gastric cancer patients (n = 43) was extensively investigated using three groups of tryptic glycopeptides: GP1 (including Asn184), GP2 (including Asn207 and Asn211), and GP3 (including Asn241). A total of 23 individual glycopeptides were determined as potential biomarkers (p < 0.00001). In addition, to improve diagnostic efficacy, we derived representative group biomarkers with high AUC values (0.929 to 0.977) through logistic regression analysis for each GP group. It has been found that glycosylation of haptoglobin is highly associated with gastric cancer, especially the glycosite Asn241. Our assay not only allows to quickly and easily obtain information on glycosylation heterogeneity of a target glycoprotein but also makes it an efficient tool for biomarker discovery and clinical diagnosis.

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“…20 The rapid and precise isolation of glycoprotein from mixtures, for example by lectins, is becoming increasingly relevant, especially since most biological therapies currently used are glycoproteins. 21 © 2021 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License…”

Section: Introductionmentioning

confidence: 99%

“…* This result was % w/w of protein in the crude extract determined using Bradford assay21 . All results represent 4 replicates.…”

mentioning

confidence: 99%

Glycoprotein Isolated from Eurycoma longifolia (Tongkat Ali) is Capable of Boosting Testosterone Levels in Leydig cells

2021

TJNPR

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Eurycoma longifolia (Tongkat Ali) is renowned for its aphrodisiac potential, and its active constituent has been presumed to be a protein and more likely a glycosylated protein. In this study, the ability of the glycoprotein to increase testosterone hormone levels was investigated. The dried root powder of the plant was extracted using water under reflux. The protein fraction was separated using size-exclusion chromatography and subjected to SDS-PAGE analysis. Thereafter the protein fraction was isolated from its glycoprotein using a lectin column. Finally, TM-3 Leydig cells were treated with the isolated glycoprotein fraction (50 µg/mL). The extraction yielded 14.3% w/w protein and the SDS PAGE analysis showed a single band at approximately 20 kDa. Treatment of TM-3 Leydig cells with the glycoprotein fraction for 72 hours demonstrated an increase in testosterone levels by almost 100% (0.36 ± 0.03 nmol/L) in comparison to the untreated cells (0.18 ± 0.05 nmol/L). The findings suggested that the glycoprotein in Eurycoma longifolia root can be easily isolated because its sugar moiety can bind to a lectin affinity column. Moreover, this glycoprotein was shown to have testosteroneboosting activity. These findings identified the glycoprotein as the bioactive constituent associated with its aphrodisiac properties.

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Characterization of Site-Specific N-Glycosylation

Cited by 11 publications

References 79 publications

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita

Detection of Aberrant Glycosylation of Serum Haptoglobin for Gastric Cancer Diagnosis Using a Middle-Up-Down Glycoproteome Platform

Glycoprotein Isolated from Eurycoma longifolia (Tongkat Ali) is Capable of Boosting Testosterone Levels in Leydig cells

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