Glycoprotein Quality Control Research Articles

The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered, stabilizing association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

Analysis of enzyme activity and substrate specificity of dolichyl-phosphate β-glucosyltransferase

Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated N-glycan moieties on peptide. It is of great significance to study the N-glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues in vitro. In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate β-glucosyltransferase E (Alg5E) derived from Trichomonas vaginalis. The results demonstrated that the recombinant Alg5E, expressed in Escherichia coli, exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).

Uncloaking the viral glycocalyx: How do viruses exploit glycoimmune checkpoints?

The surfaces of cells and enveloped viruses alike are coated in carbohydrates that play multifarious roles in infection and immunity. Organisms across all kingdoms of life make use of a diverse set of monosaccharide subunits, glycosidic linkages, and branching patterns to encode information within glycans. Accordingly, sugar-patterning enzymes and glycan binding proteins play integral roles in cell and organismal biology, ranging from glycoprotein quality control within the endoplasmic reticulum to lymphocyte migration, coagulation, inflammation, and tissue homeostasis. Unsurprisingly, genes involved in generating and recognizing oligosaccharide patterns are playgrounds for evolutionary conflicts that abound in cross-species interactions, exemplified by the myriad plant lectins that function as toxins. In vertebrates, glycans bearing acidic nine-carbon sugars called sialic acids are key regulators of immune responses. Various bacterial and fungal pathogens adorn their cells in sialic acids that either mimic their hosts' or are stolen from them. Yet, how viruses commandeer host sugar-patterning enzymes to thwart immune responses remains poorly studied. Here, we review examples of viruses that interact with sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immune cell receptors that regulate toll-like receptor signaling and govern glycoimmune checkpoints, while highlighting knowledge gaps that merit investigation. Efforts to illuminate how viruses leverage glycan-dependent checkpoints may translate into new clinical treatments that uncloak viral antigens and infected cell surfaces by removing or masking immunosuppressive sialoglycans, or by inhibiting viral gene products that induce their biosynthesis. Such approaches may hold the potential to unleash the immune system to clear long intractable chronic viral infections.

Multisite Partial Glycosylation Approach for Preparation of Biologically Relevant Oligomannosyl Branches Contribute to Lectin Affinity Analysis.

High-mannose-type glycans play essential biological roles, e.g., immune response and glycoprotein quality control, and preparing a series of oligomannosyl branches of high-mannose-type glycans is critical for biological studies. However, obtaining sufficient amounts of the various oligomannosyl branches is challenging. In this study, we demonstrated a partial glycosylation strategy for the single-step synthesis of various biologically relevant oligomannosyl-branched structures. First, Manα1-6(Manα1-3)Man-type oligomannosyl branch was synthesized via double glycosylation from a 3,6-di-OH mannosyl acceptor and fluorinated mannosyl donor with perfect α-selectivity. Subsequent partial glycosylation by reducing the equivalent of the mannosyl donor enabled to obtain biologically relevant Manα1-2Manα1-6(Manα1-2Manα1-3)Man, Manα1-6(Manα1-2Manα1-3)Man, Manα1-2Manα1-6(Manα1-3)Man, and Manα1-6(Manα1-3)Man in one-pot. Each oligomannosyl branch could be easily purified by liquid chromatography. The resulting structural isomers were identified by 2D-HMBC NMR. A systematic lectin affinity assay using the prepared oligomannosyl branches showed different specificities for the Galanthus nivalis lectin between structural isomers of the oligomannosyl branches with the same number of mannose residues..

Lectin Blotting of Cell Lysates.

Glycans are composed of branched structures consisting of monosaccharides, such as glucose and galactose linked by glycosidic bonds. Glycans are often bound to proteins and lipids and are localized at the cell surface. They are deeply involved in a wide range of multicellular systems inside and outside the cells, such as the quality control of glycoproteins, cell-cell communication, and various diseases. While western blotting uses antibodies to detect proteins, lectin blotting uses lectins, which are glycan-binding proteins, to detect glycans on glycoconjugates, such as glycoproteins. Lectin blotting was first reported in the early 1980s and has been widely used in life science for several decades. However, it is not straightforward to obtain consistent data using lectin blotting, which tends to show high backgrounds and lab-to-lab variation. Here, we describe the protocol used in our laboratory for lectin blotting following protein separation by SDS-PAGE to detect glycoproteins extracted from cell membrane fractions. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Extraction and quantification of proteins from cell lysate Basic Protocol 2: Lectin peroxidase labeling and lectin blotting.

Convergent synthesis of oligomannose-type glycans via step-economical construction of branch structures

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection–deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS

Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

Isolation and characterization of high-mannose type glycans containing five or six mannose residues from hen egg yolk

High-mannose type glycans play important roles in biosynthesis of glycoproteins including glycoprotein quality control system. In the endoplasmic reticulum (ER), α1,2-mannosidases cleave several mannose (Man) residues to give small high-mannose type glycans, such as glycans containing five or six mannose residues (M5-glycan or M6-glycan). These glycans are reported to act as a signal for degradation processes of glycoproteins in the ER. In this work, we isolated the M5-glycan and the M6-glycan from delipidated egg yolk and confirmed that their structures were identical to human type glycans based on rigorous NMR experiments, suggesting the potential use for semisynthesis of glycoconjugates and glycan analysis.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and β-human chorionic gonadotropin (β-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a β-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of β-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

The Rice Malectin Regulates Plant Cell Death and Disease Resistance by Participating in Glycoprotein Quality Control.

In animals, malectin is well known to play an essential role in endoplasmic reticulum quality control (ERQC) by interacting with ribophorin I, one unit of the oligosaccharyltransferase (OST) complex. However, the functions of malectin in plants remain largely unknown. Here, we demonstrate the rice OsMLD1 is an ER- and Golgi-associated malectin protein and physically interacts with rice homolog of ribophorin I (OsRpn1), and its disruption leads to spontaneous lesion mimic lesions, enhanced disease resistance, and prolonged ER stress. In addition, there are many more N-glycosites and N-glycoproteins identified from the mld1 mutant than wildtype. Furthermore, OsSERK1 and OsSERK2, which have more N-glycosites in mld1, were demonstrated to interact with OsMLD1. OsMLD1 can suppress OsSERK1- or OsSERK2-induced cell death. Thus, OsMLD1 may play a similar role to its mammalian homologs in glycoprotein quality control, thereby regulating cell death and immunity of rice, which uncovers the function of malectin in plants.

Phenotypic analysis of α1,2-mannosidase-like protein deletion mutants in Saccharomyces cerevisiae.

α1,2‑mannosidase-like proteins mediate quality control of glycoproteins in the endoplasmic reticulum. This study explored α1,2‑mannosidase-like protein functions in Saccharomyces cerevisiae. Single disruptants in targeted protein-coding genes were found to be viable; however, deletion of MNL2 resulted in declined yeast growth at 37 °C. The normal growth rate was recovered in double-deletion strains where one of the deletions was in MNS1 . We also measured the mannosidase activity of microsomal fractions of deficient strains using artificial glycan. Increased mannose trimming activities were demonstrated by the microsomes of MNL2 -deletion strains compared to levels of activity exhibited by the microsomes of the control strain.

Possible Roles of Calreticulin in Uterine Decidualization and Receptivity in Rats and Humans.

Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17β in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.

The glycosylation status of MHC class I molecules impacts their interactions with TAPBPR

Glycosylation plays a crucial role in the folding, structure, quality control and trafficking of glycoproteins. Here, we explored whether the glycosylation status of MHC class I (MHC-I) molecules impacts their affinity for the peptide editor, TAPBPR. We demonstrate that the interaction between TAPBPR and MHC-I is stronger when MHC-I lacks a glycan. Subsequently, TAPBPR can dissociate peptides, even those of high affinity, more easily from non-glycosylated MHC-I compared to their glycosylated counterparts. In addition, TAPBPR is more resistant to peptide-mediated allosteric release from non-glycosylated MHC-I compared to species with a glycan attached. Consequently, we find the glycosylation status of HLA-A*68:02, -A*02:01 and –B*27:05 influences their ability to undergo TAPBPR-mediated peptide exchange. The discovery that the glycan attached to MHC-I significantly influences the affinity of their interactions with TAPBPR has important implications, on both an experimental level and in a biological context.

WLS/wntless is essential in controlling dendritic cell homeostasis via a WNT signaling-independent mechanism

ABSTRACT We propose that beyond its role in WNT secretion, WLS/GPR177 (wntless, WNT ligand secretion mediator) acts as an essential regulator controlling protein glycosylation, endoplasmic reticulum (ER) homeostasis, and dendritic cell (DC)-mediated immunity. WLS deficiency in bone marrow-derived DCs (BMDCs) resulted in poor growth and an inability to mount cytokine and T-cell responses in vitro, phenotypes that were irreversible by the addition of exogenous WNTs. In fact, WLS was discovered to integrate a protein complex in N-glycan-dependent and WLS domain-selective manners, comprising ER stress sensors and lectin chaperones. WLS deficiency in BMDCs led to increased ER stress response and macroautophagy/autophagy, decreased calcium efflux from the ER, and the loss of CALR (calreticulin)-CANX (calnexin) cycle, and hence protein hypo-glycosylation. Consequently, DC-specific wls-null mice were unable to develop both Th1-, Th2- and Th17-associated responses in the respective autoimmune and allergic disease models. These results suggest that WLS is a critical chaperone in maintaining ER homeostasis, glycoprotein quality control and calcium dynamics in DCs. Abbreviations: ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; ATP2A1/SERCA1: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1; BALF: bronchoalveolar lavage fluid; BFA: brefeldin A; BMDC: bone marrow-derived dendritic cell; CALR: calreticulin; CANX: calnexin; CCL2/MCP-1: C-C motif chemokine ligand 2; CNS: central nervous system; CT: C-terminal domain; DTT: dithiothreitol; DNAJB9/ERDJ4: DnaJ heat shock protein family (Hsp40) member B9; EAE: experimental autoimmune encephalomyelitis; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein A5; IFNA: interferon alpha; IFNAR1: interferon alpha and beta receptor subunit 1; IFNB: interferon beta; IFNG/INFγ: interferon gamma; IFNGR2: interferon gamma receptor 2; IL6: interleukin 6; IL10: interleukin 10; IL12A: interleukin 12A; IL23A: interleukin 23 subunit alpha; ITGAX/CD11c: integrin subunit alpha X; ITPR1/InsP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OVA: ovalbumin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLF: predicted lipocalin fold; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; RYR1/RyanR1: ryanodine receptor 1, skeletal muscle; SD: signal domain; TGFB/TGF-β: transforming growth factor beta family; Th1: T helper cell type 1; Th17: T helper cell type 17; TM: tunicamycin; TNF/TNF-α: tumor necrosis factor; UPR: unfolded protein response; WLS/wntless: WNT ligand secretion mediator.

Design principles for the glycoprotein quality control pathway.

Newly-translated glycoproteins in the endoplasmic reticulum (ER) often undergo cycles of chaperone binding and release in order to assist in folding. Quality control is required to distinguish between proteins that have completed native folding, those that have yet to fold, and those that have misfolded. Using quantitative modeling, we explore how the design of the quality-control pathway modulates its efficiency. Our results show that an energy-consuming cyclic quality-control process, similar to the observed physiological system, outperforms alternative designs. The kinetic parameters that optimize the performance of this system drastically change with protein production levels, while remaining relatively insensitive to the protein folding rate. Adjusting only the degradation rate, while fixing other parameters, allows the pathway to adapt across a range of protein production levels, aligning with in vivo measurements that implicate the release of degradation-associated enzymes as a rapid-response system for perturbations in protein homeostasis. The quantitative models developed here elucidate design principles for effective glycoprotein quality control in the ER, improving our mechanistic understanding of a system crucial to maintaining cellular health.

The translocon-associated protein (TRAP) complex regulates quality control of N-linked glycosylation during ER stress.

Asparagine (N)-linked glycosylation is required for endoplasmic reticulum (ER) homeostasis, but how this co- and posttranslational modification is maintained during ER stress is unknown. Here, we introduce a fluorescence-based strategy to detect aberrant N-glycosylation in individual cells and identify a regulatory role for the heterotetrameric translocon-associated protein (TRAP) complex. Unexpectedly, cells with knockout of SSR3 or SSR4 subunits restore N-glycosylation over time concurrent with a diminished ER stress transcriptional signature. Activation of ER stress or silencing of the ER chaperone BiP exacerbates or rescues the glycosylation defects, respectively, indicating that SSR3 and SSR4 enable N-glycosylation during ER stress. Protein levels of the SSR3 subunit are ER stress and UBE2J1 dependent, revealing a mechanism that coordinates upstream N-glycosylation proficiency with downstream ER-associated degradation and proteostasis. The fidelity of N-glycosylation is not static in both nontransformed and tumor cells, and the TRAP complex regulates ER glycoprotein quality control under conditions of stress.

Dimerization of ER-resident molecular chaperones mediated by ERp29

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX–CRT were formed via ERp29.

Chemical-Synthesis-Based Approach to Glycoprotein Functions in the Endoplasmic Reticulum.

The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins' entry and exit from the cycle, and UGGT serves as the "folding sensor". This account summarizes our effort to analyze the properties of enzymes and lectins that play important roles in GPQC, especially those involved in the CNX/CRT cycle. To commence our study, general methods for the synthesis of high-mannose-type glycans and glycoproteins were established. Based on these, various substrates to analyze components of the GPQC were created, and properties of CRT, G-II, and UGGT have been clarified.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.