Study Of Protein Glycosylation Research Articles

Decoding Extracellular Protein Glycosylation in Human Health and Disease.

Protein glycosylation, the covalent attachment of carbohydrate, or glycan, structures onto the protein backbone, is one of the most complex and heterogeneous post-translational modifications (PTMs). Extracellular protein glycosylation, in particular N- and mucin-type O-glycosylation, plays pivotal roles in a number of biophysical and biochemical processes, such as protein folding and stability, cell adhesion, signaling, and protection. As such, aberrant glycosylation is implicated in a variety of diseases, including cancer. However, the nontemplated nature and structural heterogeneity of protein glycosylation hinder glycoprotein characterization with traditional methods. Recent advances in analytical techniques have improved capabilities for decoding glycan complexity, a promising step toward understanding the role of glycosylation in human health and disease. In this review, we highlight key and emerging techniques to study protein glycosylation, and we emphasize how these techniques have improved our understanding of glycosylation in a biologically relevant context.

Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

Molecular conjugation to antibodies has emerged as a growing strategy to combine the mechanistic activities of the attached molecule with the specificity of antibodies. A variety of technologies have been applied for molecular conjugation; however, these approaches face several limitations, including disruption of antibody structure, destabilization of the antibody, and/or heterogeneous conjugation patterns. Collectively, these challenges lead to reduced yield, purity, and function of conjugated antibodies. While glycoengineering strategies have largely been applied to study protein glycosylation and manipulate cellular metabolism, these approaches also harbor great potential to enhance the production and performance of protein therapeutics. Here, we devise a novel glycoengineering workflow for the development of site-specific antibody conjugates. This approach combines metabolic glycoengineering using azido-sugar analogs with newly installed N-linked glycosylation sites in the antibody constant domain to achieve specific conjugation to the antibody via the introduced N-glycans. Our technique allows facile and efficient manufacturing of well-defined antibody conjugates without need for complex or destructive chemistries. Moreover, introduction of conjugation sites in the antibody fragment crystallizable (Fc) domain renders this approach widely applicable and target agnostic. Our platform can accommodate up to 3 conjugation sites in tandem, and the extent of conjugation can be tuned through use of different sugar analogs or production in different cell lines. We demonstrated that our platform is compatible with various use-cases, including fluorescent labeling, antibody-drug conjugation, and targeted gene delivery. Overall, this study introduces a versatile and effective yet strikingly simple approach to produce antibody conjugates for research, industrial, and medical applications.

MS-PyCloud: A Cloud Computing-Based Pipeline for Proteomic and Glycoproteomic Data Analyses.

Rapid development and wide adoption of mass spectrometry-based glycoproteomic technologies have empowered scientists to study proteins and protein glycosylation in complex samples on a large scale. This progress has also created unprecedented challenges for individual laboratories to store, manage, and analyze proteomic and glycoproteomic data, both in the cost for proprietary software and high-performance computing and in the long processing time that discourages on-the-fly changes of data processing settings required in explorative and discovery analysis. We developed an open-source, cloud computing-based pipeline, MS-PyCloud, with graphical user interface (GUI), for proteomic and glycoproteomic data analysis. The major components of this pipeline include data file integrity validation, MS/MS database search for spectral assignments to peptide sequences, false discovery rate estimation, protein inference, quantitation of global protein levels, and specific glycan-modified glycopeptides as well as other modification-specific peptides such as phosphorylation, acetylation, and ubiquitination. To ensure the transparency and reproducibility of data analysis, MS-PyCloud includes open-source software tools with comprehensive testing and versioning for spectrum assignments. Leveraging public cloud computing infrastructure via Amazon Web Services (AWS), MS-PyCloud scales seamlessly based on analysis demand to achieve fast and efficient performance. Application of the pipeline to the analysis of large-scale LC-MS/MS data sets demonstrated the effectiveness and high performance of MS-PyCloud. The software can be downloaded at https://github.com/huizhanglab-jhu/ms-pycloud.

Construction of dual-hydrophilic metal-organic framework with hierarchical porous structure for efficient glycopeptide enrichment

As an important role in life activities, it is necessary and important to study protein glycosylation. The pre-enrichment of N-glycopeptides is a significant step in glycoproteomics research. According to the inherent size, hydrophilicity and other properties of N-glycopeptides, affinity materials designed to match them will be able to separate N-glycopeptides from complex samples. In this work, we designed and prepared dual-hydrophilic hierarchical porous metal-organic frameworks (MOFs) nanospheres by metal-organic assembly (MOA) based template method and post-synthesis modification strategy. The hierarchical porous structure significantly improved the diffusion rate and binding sites for N-glycopeptide enrichment. Furthermore, the combination of hydrophilic MOFs and small molecules endowed the as-prepared MOFs nanospheres excellent hydrophilicity, which is conducive to the enrichment of N-glycopeptides based on hydrophilic interaction liquid chromatography (HILIC). Therefore, the nanospheres showed surprising enrichment ability for N-glycopeptides such as excellent selectivity (1/500, human serum immunoglobulin G/bovine serum albumin, m/m) and extremely low detective limitation (0.5 fmol). Meanwhile, 550 N-glycopeptides were identified from rat liver samples, proving its application potential in glycoproteomics research and providing design idea for porous affinity materials.

Proceedings of workshop: "Neuroglycoproteins in health and disease", INNOGLY cost action.

The Cost Action “Innovation with glycans: new frontiers from synthesis to new biological targets” (INNOGLY) hosted the Workshop “Neuroglycoproteins in health and disease”, in Alicante, Spain, on March 2022. This event brought together an european group of scientists that presented novel insights into changes in glycosylation in diseases of the central nervous system and cancer, as well as new techniques to study protein glycosylation. Herein we provide the abstracts of all the presentations. Graphical

Progress in Monolithic Column-based Separation and Enrichment of Glycoproteins

Abstract As a post-translational modification, protein glycosylation not only reflects on body health, but also serves as the target of drug treatment. Therefore, it is of great significance to study protein glycosylation for the diagnosis and treatment of diseases. However, many glycoproteins of biological importance exhibit relatively low abundance, high complexity, and wide dynamic range in biological samples. It is indispensable to explore highly efficient sample pretreatment methods. In recent years, monolithic columns have attracted extensive attention in the field of separation and enrichment of glycoproteins due to their rapid separation, high efficiency, and good biological compatibility. In this paper, recent progress of monolithic columns in the field of separation and enrichment of glycoproteins is surveyed, and the future development is prospected.

Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However, current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here, we show that by merely increasing the tandem mass spectrometry m/z range from 2000 to 4000 during electron transfer higher energy collisional dissociation (EThcD) fragmentation, a wealth of highly informative c and z ion fragment ions are additionally detected, facilitating improved identification of glycopeptides. We demonstrate the benefit of this extended mass range on various classes of glycopeptides containing phosphorylated, fucosylated, and/or sialylated N-glycans. We conclude that the current software solutions for glycopeptide identification also require further improvements to realize the full potential of extended mass range glycoproteomics. To stimulate further developments, we provide data sets containing all classes of glycopeptides (high mannose, hybrid, and complex) measured with standard (2000) and extended (4000) m/z range that can be used as test cases for future development of software solutions enhancing automated glycopeptide analysis.

Biomimetic polymer material for glycopeptide high selective enrichment

The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.

Advances in mass spectrometry-based glycoproteomics.

Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018).

Synthesis of Lipid-Linked Oligosaccharides (LLOs) and Their Phosphonate Analogues as Probes To Study Protein Glycosylation Enzymes

Here we review chemical and chemoenzymatic methods for the synthesis of lipid-linked oligosaccharides (LLOs) and their phosphonate analogues, which serve as substrates and inhibitors to investigate the structure and mechanism of protein N-glycosylation enzymes. We emphasize how to overcome the challenges pertaining to the instability and difficult physicochemical properties of this class of compounds.1 Introduction2 LLO Syntheses2.1 Glycosyl Phosphate Syntheses2.2 Glycosyl Phosphonates2.3 Lipid Elongation2.4 Lipid Phosphates2.5 Coupling Reaction Strategies3 Chemoenzymatic Synthesis of Elongated LLOs4 Biological Properties of Synthetic LLOs5 Conclusion

Using Chemical Synthesis To Study and Apply Protein Glycosylation.

Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.

Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae

Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in Saccharomyces cerevisiae by coupling stable isotope labeling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly.

Evaluation of ion mobility for the separation of glycoconjugate isomers due to different types of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level

The study of protein glycosylation can be regarded as an intricate but very important task, making glycomics one of the most challenging and interesting, albeit under-researched, type of “omics” science. Complexity escalates remarkably when considering that carbohydrates can form severely branched structures with many different constituents, which often leads to the formation of multiple isomers. In this regard, ion mobility (IM) spectrometry has recently demonstrated its power for the separation of isomeric compounds. In the present work, the potential of traveling wave IM (TWIMS) for the separation of isomeric glycoconjugates was evaluated, using mouse transferrin (mTf) as model glycoprotein. Particularly, we aim to assess the performance of this platform for the separation of isomeric glycoconjugates due to the type of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level. Straightforward separation of isomers was achieved with the analysis of released glycans, as opposed to the glycopeptides which showed a more complex pattern. Finally, the developed methodology was applied to serum samples of mice, to investigate its robustness when analyzing real complex samples. Biological significanceIon mobility mass spectrometry is a promising analytical technique for the separation of glycoconjugate isomers due to type of sialic acid linkage. The impact of such a small modification in the glycan structure is more evident in smaller analytes, reason why the analysis of free glycans was easier compared to the intact protein or the glycopeptides. The established methodology could be regarded as starting point in the separation of highly decorated glycoconjugates. This is an important topic nowadays, as differences in the abundance of some glycan isomers could be the key for the early diagnosis, control or differentiation of certain diseases, such as inflammation or cancer.

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein.

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff’s staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Chemical Glycoproteomics.

Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

Label-free glycoprofiling with multiplex surface plasmon resonance: a tool to quantify sialylation of erythropoietin.

Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.

Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels.

Cellulose in plant cell walls is the largest reservoir of renewable carbon on Earth. The saccharification of cellulose from plant biomass into soluble sugars can be achieved using fungal and bacterial cellulolytic enzymes, cellulases, and further converted into fuels and chemicals. Most fungal cellulases are both N- and O-glycosylated in their native form, yet the consequences of glycosylation on activity and structure are not fully understood. Studying protein glycosylation is challenging as glycans are extremely heterogeneous, stereochemically complex, and glycosylation is not under direct genetic control. Despite these limitations, many studies have begun to unveil the role of cellulase glycosylation, especially in the industrially relevant cellobiohydrolase from Trichoderma reesei, Cel7A. Glycosylation confers many beneficial properties to cellulases including enhanced activity, thermal and proteolytic stability, and structural stabilization. However, glycosylation must be controlled carefully as such positive effects can be dampened or reversed. Encouragingly, methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

New trends in the study of protein glycosylation in oncological diseases

Glycomics and glycoproteomics represent relatively new directions in detail analyses of complex bio-logical media. These areas of increasing importance to cancer research complement the more established genomic profiling and proteomics. Glycoproteins are being increasingly recognized as important in cellular interactions and adhesion. Structural alterations of their glycan moieties seem to occur in different cancer conditions. We review current directions in glycomic profiling and glycoproteomic investigations of bio-logical fluids and tissues pertaining to cancer. The used methods rely on capillary separation techniques, mass spectrometry, and the glycan and lectin arrays. They all show considerable promise for new diagnostic and prognostic measurements.

Glycomic analysis by glycoprotein immobilization for glycan extraction and liquid chromatography on microfluidic chip.

Glycosylation is one of the most common protein modifications and profoundly regulates many biological processes. Aberrant glycosylation is reported to associate with diseases such as cancers, human immunodeficiency virus, and immune disorders. It is considerably important to study protein glycosylation and the associated glycans for diagnostics and disease prognostics. Unlike other protein modifications, glycans attached to proteins are enormously complex. Therefore, the comprehensive analysis of glycans from biological or clinical samples is an unmet technical challenge. Development of the high-throughput method will facilitate the glycomics analysis. In this study, we developed a novel method for the high-throughput analysis of N-glycans from glycoproteins using glycoprotein immobilization for glycan extraction (GIG) coupled with liquid chromatography (LC) in an integrated microfluidic platform (chipLC). The separated glycans were then analyzed by mass spectrometry. Briefly, proteins were first immobilized on a solid support. Glycans on immobilized glycoproteins were modified on solid phase to increase the detection and structure analysis. N-Glycans were then enzymatically released and subsequentially separated by porous graphitized carbon particles packed in the same device. By applying the GIG-chipLC for glycomic analysis of human sera, we identified N-glycans with 148 distinct N-glycan masses. The platform was used to analyze N-glycans from mouse heart tissue and serum. The extracted N-glycans from tissues indicated that unique unsialylated N-glycans were detected in tissues that were missing from the proximal or distal serum, whereas common N-glycans from tissues and serum have mature and sialylated structures. The GIG-chipLC provides a simple and robust platform for glycomic analysis of complex biological and clinical samples.

The sweet and sour of serological glycoprotein tumor biomarker quantification

Aberrant and dysregulated protein glycosylation is a well-established event in the process of oncogenesis and cancer progression. Years of study on the glycobiology of cancer have been focused on the development of clinically viable diagnostic applications of this knowledge. However, for a number of reasons, there has been only sparse and varied success. The causes of this range from technical to biological issues that arise when studying protein glycosylation and attempting to apply it to practical applications. This review focuses on the pitfalls, advances, and future directions to be taken in the development of clinically applicable quantitative assays using glycan moieties from serum-based proteins as analytes. Topics covered include the development and progress of applications of lectins, mass spectrometry, and other technologies towards this purpose. Slowly but surely, novel applications of established and development of new technologies will eventually provide us with the tools to reach the ultimate goal of quantification of the full scope of heterogeneity associated with the glycosylation of biomarker candidate glycoproteins in a clinically applicable fashion.