A pathway-based graphical interface for navigating the glycoenzyme database. The goal of the project is to define the paradigms by which carbohydrate binding proteins function in cellular communication. These pages are divided into six categories: -Glycosphingolipid: Sub-categories are Isogloboseries, Globoseries, Neo-lactoseries, Lactoseries and Ganglioseries - N-linked: Sub-categories are High-mannose, Hybrid and Complex -Mucin -Terminal Core 1 -Other O-linked -Terminal All: Includes all potential terminal structures for each glycan category

A pathway-based graphical interface for navigating the glycoenzyme database. The goal of the project is to define the paradigms by which carbohydrate binding proteins function in cellular communication. These pages are divided into six categories: -Glycosphingolipid: Sub-categories are Isogloboseries, Globoseries, Neo-lactoseries, Lactoseries and Ganglioseries - N-linked: Sub-categories are High-mannose, Hybrid and Complex -Mucin -Terminal Core 1 -Other O-linked -Terminal All: Includes all potential terminal structures for each glycan category

Revised database of O- and C-glycosylated proteins. The criteria for inclusion are at least one experimentally verified O- or C-glycosylation site. Each entry contains information about the glycan involved, the species, sequence, a literature reference and http-linked cross-references to other databases. Version 6.00 has 242 glycoprotein entries. The terminal sugar linked to serine or threonine is cited when known. The database is non-redundant in the sense that it contains no identical sequences, unless there is conflicting glycosylation data. Mucins have tandem repeat sequences, which are O-glycosylated. This result in some redundancy of the O-glycosylation sites. For prediction purposes they have also included a version of the database which contains no identical O-glycosylation sites (window=9) called O-Unique.seq. Data can no longer be retrieved by anonymous ftp. Only http is supported. New data, comments and suggestions are welcome.

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

Glycoproteins, representing more than 50% of human proteins and most biopharmaceuticals, are crucial for regulating various biological processes. The complexity of multiple glycosylation sites often leads to incomplete sequence coverage and ambiguous glycan modification profiles. Here, we developed an integrative mass spectrometry-based approach for decoding unknown glycoproteins, which is featured with the combination of deglycosylation-mediated de novo sequencing with glycosylation site characterization. We utilized the enzymatic deglycosylation of N-/ O-glycans to achieve comprehensive sequence coverage. Additionally, EThcD fragmentation enables the identification of high-quality long peptides, facilitating precise protein assembly. We subsequently applied this method to de novo sequencing of the highly glycosylated therapeutic fusion protein Etanercept (Enbrel). We also sequenced three new tumor necrosis factor receptor:Fc-fusion biologics with largely unknown sequences, unveiling subtle distinctions in the primary sequences. Furthermore, we characterized N- and O-glycosylation modifications of these proteins at subunit, glycopeptide, and glycan levels. This strategy bridges the gap between the de novo sequencing and glycosylation modification, providing comprehensive information on the primary structure and glycosylation modifications for glycoproteins. Notably, our method could be a robust solution for accurate sequencing of the glycoproteins and has practical value not only in basic research but also in the biopharmaceutical industry.

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H218O, IGOT) and finally LC–MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H218O, IGOT) and finally LC–MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.

Diseases of glycosylation, usually referred to as congenital disorders of glycosylation (CDG), are rare inherited disorders ascribing defects of nucleotide-sugar biosynthesis and transport, glycosyl transfer events and vesicular transport. Most CDGs cause neurological impairment ranging from severe psychomotor retardation to mild intellectual disability. Defects in N-glycosylation are the main cause of CDGs (Marquardt & Denecke 2003, Grunewald et al. 2002, Hennet 2012, Goreta et al. 2012) and can be identified by a characteristic abnormal isoelectric focusing profile of plasma transferrin (Jaeken et al. 1984, Stibler & Jaeken 1990). Disorders of O-glycosylation, glycosaminoglycan and glycolipid metabolism have recently been discovered and, together with N-glycosylation, represent the major pathways affected by glycan biosynthetic disorders (Freeze 2006, Jaeken 2011). In addition, glycosylation diseases associated with the enzymes that mediate the biosynthesis of glycosylation precursors are described in this section. As the number of these disorders has increased, nomenclature has been simplified so that now, the name of the mutant gene is followed by the abbreviation CDG (Jaeken et al. 2009). Effective therapies for most types of CDGs are so far not available (Thiel & Korner 2013).

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

Revised database of O- and C-glycosylated proteins. The criteria for inclusion are at least one experimentally verified O- or C-glycosylation site. Each entry contains information about the glycan involved, the species, sequence, a literature reference and http-linked cross-references to other databases. Version 6.00 has 242 glycoprotein entries. The terminal sugar linked to serine or threonine is cited when known. The database is non-redundant in the sense that it contains no identical sequences, unless there is conflicting glycosylation data. Mucins have tandem repeat sequences, which are O-glycosylated. This result in some redundancy of the O-glycosylation sites. For prediction purposes they have also included a version of the database which contains no identical O-glycosylation sites (window=9) called O-Unique.seq. Data can no longer be retrieved by anonymous ftp. Only http is supported. New data, comments and suggestions are welcome.

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

Congenital disorders of glycosylation (CDGs) are a group of autosomal recessive disorders caused by enzymatic defects in the synthesis and processing of asparagine (N)-linked glycans or oligosaccharides on glycoproteins. These glycoconjugates play critical roles in processes such as metabolism, cell recognition and adhesion, cell migration, protease resistance, host defense, and antigenicity. CDGs are divided into 2 main groups: type I CDGs comprise defects in the assembly of the dolichol lipid-linked oligosaccharide (LLO) chain and its transfer to the nascent protein, whereas type II CDGs comprise defects in the trimming and processing of protein-bound glycans (Marquardt & Denecke 2003, Grunewald et al. 2002, Hennet 2012, Cylwik et al. 2013).

Bovine milk-derived exosomes (BMDEs) have potential applications in the pharmaceutical industry as drug delivery carriers. A comprehensive analysis of protein glycosylation in exosomes is necessary to elucidate the process of targeted delivery. In this work, free oligosaccharides (FOSs), O-glycans, and N-glycans in BMDEs and whey were first analyzed through multiple derivation strategies. In summary, 13 FOSs, 44 O-glycans, and 94 N-glycans were identified in bovine milk. To analyze site-specific glycosylation of glycoproteins, a one-step method was used to enrich and characterize intact glycopeptides. A total of 1359 proteins including 114 glycoproteins were identified and most of these were located in the exosomes. Approximately 95 glycopeptides were initially discovered and 5 predicted glycosites were confirmed in BMDEs. Collectively, these findings revealed the characterization and distribution of glycans and glycoproteins in BMDEs, providing insight into the potential applications of BMDEs in drug delivery and food science.

Revised database of O- and C-glycosylated proteins. The criteria for inclusion are at least one experimentally verified O- or C-glycosylation site. Each entry contains information about the glycan involved, the species, sequence, a literature reference and http-linked cross-references to other databases. Version 6.00 has 242 glycoprotein entries. The terminal sugar linked to serine or threonine is cited when known. The database is non-redundant in the sense that it contains no identical sequences, unless there is conflicting glycosylation data. Mucins have tandem repeat sequences, which are O-glycosylated. This result in some redundancy of the O-glycosylation sites. For prediction purposes they have also included a version of the database which contains no identical O-glycosylation sites (window=9) called O-Unique.seq. Data can no longer be retrieved by anonymous ftp. Only http is supported. New data, comments and suggestions are welcome.

A pathway-based graphical interface for navigating the glycoenzyme database. The goal of the project is to define the paradigms by which carbohydrate binding proteins function in cellular communication. These pages are divided into six categories: -Glycosphingolipid: Sub-categories are Isogloboseries, Globoseries, Neo-lactoseries, Lactoseries and Ganglioseries - N-linked: Sub-categories are High-mannose, Hybrid and Complex -Mucin -Terminal Core 1 -Other O-linked -Terminal All: Includes all potential terminal structures for each glycan category

This dataset is an outcome of a series of experiments conducted to investigate the significance of glycosylation on the function of the human calcium-sensing receptor (CaSR). The CaSR is a family C G protein-coupled receptor that plays an important role in regulating whole body extracellular calcium (Ca2+o) homeostasis. The exact location of the binding sites for Ca2+o (and other cations) is still unknown, but experimental and in silico analysis has provided evidence that they may be in the extracellular domain and the transmembrane domain. Post translational modifications leads to the covalent attachment of a number of negatively charged sugars, which may provide a negatively charged surface for polyvalent cations to interact with. There are 11 potential N-linked glycosylation sites in the CaSR’s extracellular domain. A single conservative asparagine to glutatmine mutation was undertaken to disrupt individual N-linked glycosylation consensus sequences. Alanine scanning mutations were also used to assess the significance of potential O-linked glycosylation sites (small clusters of Ser/The residues near the membrane anchor points of the exo-loops in the transmembrane domain). These mutant receptors were then transiently expressed in HEK293 cells. The impact of each mutation on cell expression and function was assessed by the following techniques:Western blotting: Hand cast SDS-PAGE gel and Membrane transfer was undertaken using the BioRad mini-protean system, Precast gradient gels (4 – 15%) were TGX precast gels from Biorad. Western blotting films were scanned in .JPG and .PNG format. Quantification of bands was undertaken using GNU Imaging Manipulation Program (GIMP) imaging software.Cell surface expression ELISAs: Antibody based enzyme-linked immunosorbent assays (ELISAs) were used. The chromogenic substrate for horseradish peroxidase (HRP) was 3,3’,5,5’-Tetramethylbenzidine (TMB). Absorbance at 450 nM was read using an EnVision Multilabel Reader and exported using .XLS files. Data were represented and analysed using GraphPad Prism 5.Microfluorimetry with fura-2 AM (a calcium-sensitive fluorophore): Fluorescent images were taken on a Zeiss Axiovert 200M microscope with a Carl Zeiss monochrome HSm digital camera using a 63x Zeiss long-distance objective. Excitation filters, emission filters and dichroic mirrors were all obtained from Zeiss. The fluorescent light source was provided by a Lambda Sutter DG-4 wavelength switcher. The ratiometric (340/380 nm) region-of-interest data were exported using Stallion software (.SLD files) and subsequently processed using GraphPad Prism 5.0.The creation of the mutant cells and expression/function experiments were carried out by Dr Sarah Brennan and Karolina Windloch. Data analysis was undertaken by Dr Sarah Brennan. For further information, refer to the associated thesis.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Protein glycosylation plays a key role in regulating protein stability, aggregation, and synaptic function, and emerging evidence strongly suggests that glycosylation is related to this disease. However, the extent and functional consequences of site-specific N-glycosylation alterations in AD remain to be further explored. In this study, we employed a dendrimer boronic acid (DBA)-based enrichment strategy combined with multiplexes proteomics to systematically analyze protein N-glycosylation in postmortem human brain tissues. We identified 3,105 N-glycosylation sites from 1,299 glycoproteins and performed a systematic assessment of site-specific glycosylation alterations in AD. Glycoproteins involved in cholesterol efflux were upregulated, whereas those associated with chemical synaptic transmission and ion transmembrane transport were significantly downregulated in AD. We observed widespread dysregulation of N-glycosylation across multiple protein domains, particularly in the ConA-like lectins/glucanases and Zn-dependent exopeptidases domains. Notably, we identified 161 N-glycosylation sites located within aggregation-prone regions (APRs), suggesting a potential protective role of glycosylation against protein aggregation. N-glycosylation sites located within APRs on plaque-associated glycoproteins were significantly reduced in AD, highlighting their potential involvement in protein aggregation and plaque formation. Additionally, downregulated N-glycosylation sites were enriched in synaptic membrane proteins, such as Ca2+ ion channels, GABA-gated chloride channels, and glutamate receptors, implicating glycosylation loss in synaptic dysfunction. Comprehensive and site-specific characterization of N-glycosylation alterations in the AD brain tissues reveals its potential role in protein aggregation and synaptic dysfunction. Our findings suggest that the loss of N-glycosylation may contribute to the pathogenesis of AD by impairing synaptic transmission and promoting protein aggregation. This study provides novel insights into glycosylation-dependent mechanisms of neurodegeneration, highlighting N-glycosylation as a potential therapeutic target for AD.

Analysis of gp120 N-glycosylation. N-linked glycosylation sites of gp120 were interrogated for the presence of 55 glycoforms from chronic vs acute HIV virions.

Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host’s immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs.

Recombinant proteins are of great interest in glycobiology and proteomics, known especially for their reproducibility and accessibility. However, variation in glycosylation among recombinant glycoproteins is not well understood and may depend on numerous conditions in the biomanufacturing process. In order to confidently assess variation in glycosylation measurements, it is vital to both optimize the measurement of, and determine the degree of variation between, distributions of glycosylation on specific sites of glycoproteins. This is especially important for glycoproteins that are known to have rapid sequence changes, such as with different influenza strains. In this study, eight strains of recombinant influenza hemagglutinin and neuraminidase produced from HEK293 cell line were obtained from four vendors and digestion was conducted using a series of complex multi-enzymatic methods designed to isolate glycopeptide sequons. Site-specific glycosylation profiles of intact glycopeptides were produced using mass spectrometric evaluation on an orbitrap system and compared using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 1000), whereas replicates resulted in low variation (similarity score = 957 out of 1000). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans in all of these influenza glycoproteins were monofucosylated and complex, as reported by other laboratories. However, it was found that different vendors can produce very different glycan distributions for the same glycosylation site. Notably, it is demonstrated that glycan distributions are similar for conserved regions of influenza glycoproteins. Overall, these methods present a potential use in developing reproducible measurements of glycosylated biologics for quality control or making more informed decisions in biomanufacturing.