OGP: A Repository of Experimentally Characterized O-Glycoproteins to Facilitate Studies on O-Glycosylation

AbstractNumerous studies on cancer, biopharmaceuticals, and clinical trials have necessitated comprehensive and precise analysis of protein O-glycosylation. However, the lack of updated and convenient databases deters the storage and utilization of emerging O-glycoprotein data. To resolve this issue, an O-glycoprotein repository named OGP was established in this work. It was constructed with a collection of O-glycoprotein data from different sources. OGP contains 9354 O-glycosylation sites and 11,633 site-specific O-glycans mapping to 2133 O-glycoproteins, and it is the largest O-glycoprotein repository thus far. Based on the recorded O-glycosites, an O-glycosylation site prediction tool was developed. Moreover, an OGP-backed website is already available (http://www.oglyp.org/). The website comprises four specially designed and user-friendly modules: Statistic Analysis, Database Search, Site Prediction, and Data Submit. The first version of OGP repository and the website allow users to obtain vast O-glycoprotein related information, such as protein accession numbers, glycopeptides, site-specific glycan structures, experimental methods, and potential glycosylation sites. O-glycosylation data mining can be performed efficiently on this website, which can greatly facilitates O-glycosylation studies.

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Intelligent Techniques Analysis for Glycosylation Site Prediction

Current Bioinformatics ◽

10.2174/1574893615666210108094847 ◽

2021 ◽

Vol 15 ◽

Author(s):

Alhassan Alkuhlani ◽

Walaa Gad ◽

Mohamed Roushdy ◽

Abdel-Badeeh M. Salem

Keyword(s):

Machine Learning ◽

Prediction Models ◽

Cell Interaction ◽

Glycosylation Site ◽

Machine Learning Classification ◽

Site Prediction ◽

Glycosylation Sites ◽

Wide Range ◽

Feature Extraction And Selection ◽

Computational Intelligent

Background: Glycosylation is one of the most common post-translation modifications (PTMs) in organism cells. It plays important roles in several biological processes including cell-cell interaction, protein folding, antigen’s recognition, and immune response. In addition, glycosylation is associated with many human diseases such as cancer, diabetes and coronaviruses. The experimental techniques for identifying glycosylation sites are time-consuming, extensive laboratory work, and expensive. Therefore, computational intelligence techniques are becoming very important for glycosylation site prediction. Objective: This paper is a theoretical discussion of the technical aspects of the biotechnological (e.g., using artificial intelligence and machine learning) to digital bioinformatics research and intelligent biocomputing. The computational intelligent techniques have shown efficient results for predicting N-linked, O-linked and C-linked glycosylation sites. In the last two decades, many studies have been conducted for glycosylation site prediction using these techniques. In this paper, we analyze and compare a wide range of intelligent techniques of these studies from multiple aspects. The current challenges and difficulties facing the software developers and knowledge engineers for predicting glycosylation sites are also included. Method: The comparison between these different studies is introduced including many criteria such as databases, feature extraction and selection, machine learning classification methods, evaluation measures and the performance results. Results and conclusions: Many challenges and problems are presented. Consequently, more efforts are needed to get more accurate prediction models for the three basic types of glycosylation sites.

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Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

The Journal of Cell Biology ◽

10.1083/jcb.132.6.1199 ◽

1996 ◽

Vol 132 (6) ◽

pp. 1199-1208 ◽

Cited By ~ 120

Author(s):

A Bartolazzi ◽

A Nocks ◽

A Aruffo ◽

F Spring ◽

I Stamenkovic

Keyword(s):

Cell Adhesion ◽

Regulatory Mechanism ◽

Cell Attachment ◽

Glycosylation Site ◽

Side Chain ◽

Site Specific ◽

Coated Substrate ◽

Glycosylation Sites ◽

Coated Surfaces

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

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Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme

Biochemical Journal ◽

10.1042/bj3040917 ◽

1994 ◽

Vol 304 (3) ◽

pp. 917-922 ◽

Cited By ~ 10

Author(s):

R D Basco ◽

L M Hernández ◽

M D Muñox ◽

I Olivero ◽

E Andaluz ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Thermal Inactivation ◽

Glycosylation Site ◽

Translation Product ◽

Site Specific ◽

Potential Glycosylation Site ◽

Glycosylation Sites ◽

Conformational Constraints ◽

Specific Elongation ◽

Protein Portion

Three exoglucanases (Exgs), ExgIa, ExgIb and Exg325, are secreted by Saccharomyces cerevisiae cells. They share a common protein portion with two potential glycosylation sites (sequons) but differ in the amount of N-linked carbohydrate [Basco, R.D., Muñoz, M.D., Hernández, L.M., Váquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. ExgIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernández, L.M., Olivero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-9831]. Exg325 carries a single, short oligosaccharide bound to Asn325 whereas ExgIa has at least one large oligosaccharide, since it has not been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gln for Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg165 but not by mutant Exg325. Additional studies on sequential deglycosylation of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the susceptibility of both oligosaccharides to the endoglycosidase, and analysis of the presence of GlcNAc at both asparagine residues after total deglycosylation with endo H, indicated that ExgIa contained two oligosaccharides, a short one bound to Asn165 and a large one bound to Asn325, and, accordingly, originated from ExgIb. The elongation of the second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to proteases as compared with ExgIb; however, the affinity of the enzyme towards laminarin decreased by 50%. This site-specific elongation occurred in the oligosaccharide that was less susceptible to endo H, indicating that these properties are determined by different conformational constraints.

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Glycosylation of GD4 and Thy-1

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0133 ◽

1993 ◽

Vol 342 (1299) ◽

pp. 43-50 ◽

Cited By ~ 11

Keyword(s):

Cho Cells ◽

Chinese Hamster ◽

Glycosylation Site ◽

Truncated Form ◽

Site Specific ◽

Glycosylation Sites ◽

Oligosaccharide Processing ◽

Domain 3 ◽

Protein Cell ◽

Oligosaccharide Structures

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with m utant molecules with only single glycosylation sites and with a truncated form containing only the two NH 2 -terminal domains, indicate that independent processing occurs at each glycosylation site and that dom ain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

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Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

Molecules ◽

10.3390/molecules24213924 ◽

2019 ◽

Vol 24 (21) ◽

pp. 3924

Author(s):

Hahm ◽

Lee ◽

Ahn

Keyword(s):

Fusion Protein ◽

Glycosylation Site ◽

Tandem Mass ◽

Vascular Endothelial ◽

Glycosylation Pattern ◽

Site Specific ◽

Glycosylation Sites ◽

Human Igg1 ◽

Endothelial Growth Factor ◽

Ionization Tandem Mass Spectrometry

A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

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Nglyc: A Random Forest Method for Prediction of N-Glycosylation Sites in Eukaryotic Protein Sequence

Protein and Peptide Letters ◽

10.2174/0929866526666191002111404 ◽

2020 ◽

Vol 27 (3) ◽

pp. 178-186 ◽

Cited By ~ 2

Author(s):

Ganesan Pugalenthi ◽

Varadharaju Nithya ◽

Kuo-Chen Chou ◽

Govindaraju Archunan

Keyword(s):

Random Forest ◽

Protein Sequence ◽

Glycosylation Site ◽

Computational Method ◽

The Other ◽

Eukaryotic Protein ◽

Random Forest Method ◽

Glycosylation Sites ◽

Human And Mouse ◽

Better Than

Background: N-Glycosylation is one of the most important post-translational mechanisms in eukaryotes. N-glycosylation predominantly occurs in N-X-[S/T] sequon where X is any amino acid other than proline. However, not all N-X-[S/T] sequons in proteins are glycosylated. Therefore, accurate prediction of N-glycosylation sites is essential to understand Nglycosylation mechanism. Objective: In this article, our motivation is to develop a computational method to predict Nglycosylation sites in eukaryotic protein sequences. Methods: In this article, we report a random forest method, Nglyc, to predict N-glycosylation site from protein sequence, using 315 sequence features. The method was trained using a dataset of 600 N-glycosylation sites and 600 non-glycosylation sites and tested on the dataset containing 295 Nglycosylation sites and 253 non-glycosylation sites. Nglyc prediction was compared with NetNGlyc, EnsembleGly and GPP methods. Further, the performance of Nglyc was evaluated using human and mouse N-glycosylation sites. Results: Nglyc method achieved an overall training accuracy of 0.8033 with all 315 features. Performance comparison with NetNGlyc, EnsembleGly and GPP methods shows that Nglyc performs better than the other methods with high sensitivity and specificity rate. Conclusion: Our method achieved an overall accuracy of 0.8248 with 0.8305 sensitivity and 0.8182 specificity. Comparison study shows that our method performs better than the other methods. Applicability and success of our method was further evaluated using human and mouse N-glycosylation sites. Nglyc method is freely available at https://github.com/bioinformaticsML/ Ngly.

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Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

International Journal of Molecular Sciences ◽

10.3390/ijms22105369 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5369

Author(s):

Martina Pirro ◽

Yassene Mohammed ◽

Arnoud H. de Ru ◽

George M. C. Janssen ◽

Rayman T. N. Tjokrodirijo ◽

...

Keyword(s):

Cell Lines ◽

Quantitative Proteomics ◽

Glycosylation Site ◽

Total Cell ◽

Tandem Mass ◽

Proteomics Data ◽

Site Specific ◽

Oxonium Ions ◽

Tandem Mass Tag ◽

Colorectal Cancer Cell Lines

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

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Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1.

The EMBO Journal ◽

10.1002/j.1460-2075.1987.tb02359.x ◽

1987 ◽

Vol 6 (5) ◽

pp. 1233-1244 ◽

Cited By ~ 158

Author(s):

R. B. Parekh ◽

A. G. Tse ◽

R. A. Dwek ◽

A. F. Williams ◽

T. W. Rademacher

Keyword(s):

Glycosylation Site ◽

Site Specific ◽

Tissue Specific ◽

Lentil Lectin ◽

Lectin Recognition

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Defining HIV-1 Envelope N-Glycan Microdomains through Site-Specific Heterogeneity Profiles

Journal of Virology ◽

10.1128/jvi.01177-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 5

Author(s):

Audra A. Hargett ◽

Qing Wei ◽

Barbora Knoppova ◽

Stacy Hall ◽

Zhi-Qiang Huang ◽

...

Keyword(s):

Immune System ◽

Immune Evasion ◽

Immune Escape ◽

High Resolution Mass Spectrometry ◽

National Laboratory ◽

Site Specific ◽

Los Alamos National Laboratory ◽

Glycosylation Sites ◽

Env Protein ◽

Hiv 1

ABSTRACT The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants. IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

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