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Biotechnology Advances 2024A key aspect of successful viral vaccine design is the elicitation of neutralizing antibodies targeting viral attachment and fusion glycoproteins that embellish viral... (Review)
Review
A key aspect of successful viral vaccine design is the elicitation of neutralizing antibodies targeting viral attachment and fusion glycoproteins that embellish viral particles. This observation has catalyzed the development of numerous viral glycoprotein mimetics as vaccines. Glycans can dominate the surface of viral glycoproteins and as such, the viral glycome can influence the antigenicity and immunogenicity of a candidate vaccine. In one extreme, glycans can form an integral part of epitopes targeted by neutralizing antibodies and are therefore considered to be an important feature of key immunogens within an immunization regimen. In the other extreme, the existence of peptide and bacterially expressed protein vaccines shows that viral glycosylation can be dispensable in some cases. However, native-like glycosylation can indicate native-like protein folding and the presence of conformational epitopes. Furthermore, going beyond native glycan mimicry, in either occupancy of glycosylation sites or the glycan processing state, may offer opportunities for enhancing the immunogenicity and associated protection elicited by an immunogen. Here, we review key determinants of viral glycosylation and how recombinant immunogens can recapitulate these signatures across a range of enveloped viruses, including HIV-1, Ebola virus, SARS-CoV-2, Influenza and Lassa virus. The emerging understanding of immunogen glycosylation and its control will help guide the development of future vaccines in both recombinant protein- and nucleic acid-based vaccine technologies.
Topics: Glycosylation; Glycoproteins; Antibodies, Neutralizing; Epitopes; Vaccines; Polysaccharides
PubMed: 37972669
DOI: 10.1016/j.biotechadv.2023.108283 -
Targeted protein posttranslational modifications by chemically induced proximity for cancer therapy.The Journal of Biological Chemistry Apr 2023Post-translational modifications (PTMs) regulate all aspects of protein function. Therefore, upstream regulators of PTMs, such as kinases, acetyltransferases, or... (Review)
Review
Post-translational modifications (PTMs) regulate all aspects of protein function. Therefore, upstream regulators of PTMs, such as kinases, acetyltransferases, or methyltransferases, are potential therapeutic targets for human diseases, including cancer. To date, multiple inhibitors and/or agonists of these PTM upstream regulators are in clinical use, while others are still in development. However, these upstream regulators control not only the PTMs of disease-related target proteins but also other disease-irrelevant substrate proteins. Thus, nontargeted perturbing activities may introduce unwanted off-target toxicity issues that limit the use of these drugs in successful clinical applications. Therefore, alternative drugs that solely regulate a specific PTM of the disease-relevant protein target may provide a more precise effect in treating disease with relatively low side effects. To this end, chemically induced proximity has recently emerged as a powerful research tool, and several chemical inducers of proximity (CIPs) have been used to target and regulate protein ubiquitination, phosphorylation, acetylation, and glycosylation. These CIPs have a high potential to be translated into clinical drugs and several examples such as PROTACs and MGDs are now in clinical trials. Hence, more CIPs need to be developed to cover all types of PTMs, such as methylation and palmitoylation, thus providing a full spectrum of tools to regulate protein PTM in basic research and also in clinical application for effective cancer treatment.
Topics: Humans; Protein Processing, Post-Translational; Proteins; Ubiquitination; Phosphorylation; Glycosylation; Acetylation; Neoplasms
PubMed: 36870680
DOI: 10.1016/j.jbc.2023.104572 -
The Journal of Biological Chemistry 2021Structures deposited in the Protein Data Bank (PDB) facilitate our understanding of many biological processes including those that fall under the general category of... (Review)
Review
Structures deposited in the Protein Data Bank (PDB) facilitate our understanding of many biological processes including those that fall under the general category of glycobiology. However, structure-based studies of how glycans affect protein structure, how they are synthesized, and how they regulate other biological processes remain challenging. Despite the abundant presence of glycans on proteins and the dense layers of glycans that surround most of our cells, structures containing glycans are underrepresented in the PDB. There are sound reasons for this, including difficulties in producing proteins with well-defined glycosylation and the tendency of mobile and heterogeneous glycans to inhibit crystallization. Nevertheless, the structures we do find in the PDB, even some of the earliest deposited structures, have had an impact on our understanding of function. I highlight a few examples in this review and point to some promises for the future. Promises include new structures from methodologies, such as cryo-EM, that are less affected by the presence of glycans and experiment-aided computational methods that build on existing structures to provide insight into the many ways glycans affect biological function.
Topics: Databases, Protein; Glycomics; Glycosylation; Polysaccharides; Protein Conformation; Proteins
PubMed: 33744289
DOI: 10.1016/j.jbc.2021.100556 -
Journal of Industrial Microbiology &... Nov 2020Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and... (Review)
Review
Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.
Topics: Biotechnology; Cell-Free System; Glycoproteins; Glycosylation; Humans; Prokaryotic Cells; Protein Biosynthesis
PubMed: 33090335
DOI: 10.1007/s10295-020-02321-4 -
Technology in Cancer Research &... 2023Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated... (Review)
Review
Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated with cancer. Common findings include that aggressive cancers tend to have higher expression levels of enzymes that regulate glycosylation as well as glycoproteins with greater levels of complexity, increased branching, and enhanced chain length. Research in cancer glycoproteomics over the last 50-plus years has mainly focused on technology development used to observe global changes in glycosylation. Efforts have also been made to connect glycans to their protein carriers as well as to delineate the role of these modifications in intracellular signaling and subsequent cell function. This review discusses currently available techniques utilizing mass spectrometry-based technologies used to study glycosylation and highlights areas for future advancement.
Topics: Humans; Biomarkers, Tumor; Workflow; Glycosylation; Mass Spectrometry; Neoplasms; Polysaccharides
PubMed: 36740994
DOI: 10.1177/15330338221148811 -
Experimental & Molecular Medicine May 2023The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1), primarily produced by hepatocytes and neutrophils, is a multifunctional protein that modulates various...
The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1), primarily produced by hepatocytes and neutrophils, is a multifunctional protein that modulates various signaling cascades, mainly TGFβ signaling. Serum LRG1 and neutrophil-derived LRG1 have different molecular weights due to differences in glycosylation, but the impact of the differential glycan composition in LRG1 on its cellular function is largely unknown. We previously reported that LRG1 can promote both angiogenic and neurotrophic processes under hyperglycemic conditions by interacting with LPHN2. Here, we determined the crystal structure of LRG1, identifying the horseshoe-like solenoid structure of LRG1 and its four N-glycosylation sites. In addition, our biochemical and cell-biological analyses found that the deglycosylation of LRG1, particularly the removal of glycans on N325, is critical for the high-affinity binding of LRG1 to LPHN2 and thus promotes LRG1/LPHN2-mediated angiogenic and neurotrophic processes in mouse tissue explants, even under normal glucose conditions. Moreover, the intracavernous administration of deglycosylated LRG1 in a diabetic mouse model ameliorated vascular and neurological abnormalities and restored erectile function. Collectively, these data indicate a novel role of LRG1 glycans as molecular switches that can tune the range of LRG1's cellular functions, particularly the LRG1/LPHN2 signaling axis.
Topics: Animals; Male; Mice; Disease Models, Animal; Glycoproteins; Glycosylation; Signal Transduction
PubMed: 37121976
DOI: 10.1038/s12276-023-00992-4 -
Biomolecules Feb 2024Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to... (Review)
Review
Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations characterized by distinct interactomes-namely, protein assemblies-under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation's influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly, and disease.
Topics: Glycosylation; Protein Processing, Post-Translational; Polysaccharides; Protein Conformation; Prions
PubMed: 38540703
DOI: 10.3390/biom14030282 -
Mass Spectrometry Reviews Aug 2019Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site-specific analysis of glycoproteins in... (Review)
Review
Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site-specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)-based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS-based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS-based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX-XX, 2019.
Topics: Animals; Glycomics; Glycopeptides; Glycoproteins; Glycosylation; Humans; Mass Spectrometry; Polysaccharides; Proteomics
PubMed: 30605224
DOI: 10.1002/mas.21586 -
Journal of Bacteriology Jun 2023Flavobacterium johnsoniae is a free-living member of the phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding...
Flavobacterium johnsoniae is a free-living member of the phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding motility dependent on the type IX secretion system (T9SS). -Glycosylation has been reported in several species, and the -glycosylation of S-layer proteins in Tannerella forsythia was shown to be important for certain virulence features. In this study, we characterized the -glycoproteome of F. johnsoniae and identified 325 -glycosylation sites within 226 glycoproteins. The structure of the major glycan was found to be a hexasaccharide with the sequence Hex-(Me-dHex)-Me-HexA-Pent-HexA-Me-HexNAcA. Bioinformatic localization of the glycoproteins predicted 68 inner membrane proteins, 60 periplasmic proteins, 26 outer membrane proteins, 57 lipoproteins, and 9 proteins secreted by the T9SS. The glycosylated sites were predominantly located in the periplasm, where they are postulated to be beneficial for protein folding/stability. Six proteins associated with gliding motility or the T9SS were demonstrated to be -glycosylated. Flavobacterium johnsoniae is a Gram-negative bacterium that is found in soil and water. It is frequently used as a model species for studying gliding motility and the T9SS. In this study, we characterized the -glycoproteome of F. johnsoniae and identified 325 -glycosylation sites within 226 glycoproteins. The glycosylated domains were mainly localized to the periplasm. The function of -glycosylation is likely related to protein folding and stability; therefore, the finding of the glycosylation sites has relevance for studies involving expression of the proteins. Six proteins associated with gliding motility or the T9SS were demonstrated to be -glycosylated, which may impact the structure and function of these components.
Topics: Bacterial Proteins; Flavobacterium; Polysaccharides; Glycosylation; Proteome
PubMed: 37162352
DOI: 10.1128/jb.00093-23 -
Redox Biology Oct 2022N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each...
N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.
Topics: Disulfides; Glycosylation; Humans; Lectins; Membrane Glycoproteins; Neovascularization, Pathologic; Oxidation-Reduction; Oxidoreductases; Protein Folding; Thioredoxins; Vascular Endothelial Growth Factor A
PubMed: 36063727
DOI: 10.1016/j.redox.2022.102455