Phylum‐wide general protein O‐glycosylation system of the Bacteroidetes

The human gut symbiont Bacteroides fragilis has a general protein O‐glycosylation system in which numerous extracytoplasmic proteins are glycosylated at a three amino acid motif. In B. fragilis, protein glycosylation is a fundamental and essential property as mutants with protein glycosylation defects have impaired growth and are unable to competitively colonize the mammalian intestine. In this study, we analysed the phenotype of B. fragilis mutants with defective protein glycosylation and found that the glycan added to proteins is comprised of a core glycan and an outer glycan. The genetic region encoding proteins for the synthesis of the outer glycan is conserved within a Bacteroides species but divergent between species. Unlike the outer glycan, an antiserum raised to the core glycan reacted with all Bacteroidetes species tested, from all four classes of the phylum. We found that diverse Bacteroidetes species synthesize numerous glycoproteins and glycosylate proteins at the same three amino acid motif. The wide‐spread conservation of this protein glycosylation system within the phylum suggests that this system of post‐translational protein modification evolved early, before the divergence of the four classes of Bacteroidetes, and has been maintained due to its physiological importance to the diverse species of this phylum.     

The expanding horizons of asparagine-linked glycosylation

Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.     

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Glycan analysis of therapeutic glycoproteins

Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content.     

Diversity and functions of protein glycosylation in insects

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility.Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system.At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.     

Sweet new world: glycoproteins in bacterial pathogens

In eukaryotes, the combinatorial potential of carbohydrates is used for the modulation of protein function. However, despite the wealth of cell wall and surface-associated carbohydrates and glycoconjugates, the accepted dogma has been that prokaryotes are not able to glycosylate proteins. This has now changed and protein glycosylation in prokaryotes is an accepted fact. Intriguingly, in Gram-negative bacteria most glycoproteins are associated with virulence factors of medically significant pathogens. Also, important steps in pathogenesis have been linked to the glycan substitution of surface proteins, indicating that the glycosylation of bacterial proteins might serve specific functions in infection and pathogenesis and interfere with inflammatory immune responses. Therefore, the carbohydrate modifications and glycosylation pathways of bacterial proteins will become new targets for therapeutic and prophylactic measures. Here we discuss recent findings on the structure, genetics and function of glycoproteins of medically important bacteria and potential applications of bacterial glycosylation systems for the generation of novel glycoconjugates.     

Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.     

Protein glycosylation pathways in filamentous fungi

Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.     

Conformational dynamics of α-1 acid glycoprotein (AGP) in cancer: A comparative study of glycosylated and unglycosylated AGP

α-1 acid glycoprotein (AGP) is one of the most abundant plasma proteins. It fulfills two important functions: immunomodulation, and binding to various drugs and receptors. These different functions are closely associated and modulated via changes in glycosylation and cancer missense mutations. From a structural point of view, glycans alter the local biophysical properties of the protein leading to a diverse ligand-binding spectrum. However, glycans can typically not be observed in the resolved X-ray crystallography structure of AGP due to their high flexibility and microheterogeneity, so limiting our understanding of AGP's conformational dynamics 70 years after its discovery. We here investigate how mutations and glycosylation interfere with AGP's conformational dynamics changing its biophysical behavior, by using molecular dynamics (MD) simulations and sequence-based dynamics predictions. The MD trajectories show that glycosylation decreases the local backbone flexibility of AGP and increases the flexibility of distant regions through allosteric effects. We observe that mutations near the glycosylation site affect glycan's conformational preferences. Thus, we conclude that mutations control glycan dynamics which modulates the protein's backbone flexibility directly affecting its accessibility. These findings may assist in the drug design targeting AGP's glycosylation and mutations in cancer.     

Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.     

Genetic defects in the human glycome

The spectrum of all glycan structures--the glycome--is immense. In humans, its size is orders of magnitude greater than the number of proteins that are encoded by the genome, one percent of which encodes proteins that make, modify, localize or bind sugar chains, which are known as glycans. In the past decade, over 30 genetic diseases have been identified that alter glycan synthesis and structure, and ultimately the function of nearly all organ systems. Many of the causal mutations affect key biosynthetic enzymes, but more recent discoveries point to defects in chaperones and Golgi-trafficking complexes that impair several glycosylation pathways. As more glycosylation disorders and patients with these disorders are identified, the functions of the glycome are starting to be revealed.     

Understanding glycoprotein structural heterogeneity and interactions: Insights from native mass spectrometry

Protein glycosylation is critical since it connects complex metabolic pathways to diverse proteoforms, fine-tunes protein structures and exerts biological functions. Aberrant glycosylation on the other hand is associated with many diseases, including cancers, inflammation and metabolic disorders. By resolving monosaccharide residues on intact glycoprotein complexes, native mass spectrometry can shed light on glycan heterogeneity, glycoprotein structure and molecular recognition. Here, we focus on the two most prevalent forms of glycosylation, namely N- and O- linked, and discuss recent progress in native mass spectrometry for elucidating glycoprotein structural heterogeneity and relating specific glycan repertoires to glycoprotein interactions.     

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.     

S-layer nanoglycobiology of bacteria

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

N-Linked glycosylation of antibody fragments in Escherichia coli

Glycosylation is the predominant protein modification to diversify the functionality of proteins. In particular, N-linked protein glycosylation can increase the biophysical and pharmacokinetic properties of therapeutic proteins. However, the major challenges in studying the consequences of protein glycosylation on a molecular level are caused by glycan heterogeneities of currently used eukaryotic expression systems, but the discovery of the N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni and its functional transfer to Escherichia coli opened up the possibility to produce glycoproteins in bacteria. Toward this goal, we elucidated whether antibody fragments, a potential class of therapeutic proteins, are amenable to bacterial N-linked glycosylation, thereby improving their biophysical properties. We describe a new strategy for glycoengineering and production of quantitative amounts of glycosylated scFv 3D5 at high purity. The analysis revealed the presence of a homogeneous N-glycan that significantly increased the stability and the solubility of the 3D5 antibody fragment. The process of bacterial N-linked glycosylation offers the possibility to specifically address and alter the biophysical properties of proteins.