New pseudonucleosides containing chiral oxazolidin-2-ones and cyclosulfamides as aglycones: synthesis and antiviral evaluation

A series of chiral cyclosulfamides and oxazolidinon-2-ones have been synthesized starting from aminoacids. Regioselective substitution of these pseudopyrimidic heterocyles was carried out under Mitsunobu conditions. Best substitution results were obtained by preliminary deprotection of cyclosulfamides and their condensation with beta-D-ribofuranose. Chiral oxazolidin-2-ones were coupled directly with D-ribofuranose. All compounds were tested against HSV-2, VV and SV viruses. Two compounds 6b and 6e showed significant activities against HSV-type 1.     

Specificity of enzymatic in vitro glycosylation by PNGase F: a comparison of enzymatic and non-enzymatic glycosylation

Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.     

Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer

Monoclonal antibodies have been used increasingly as therapeutic agents to target various diseases. Although most monoclonal antibodies have only one N-linked glycosylation site in the Fc region, N-linked glycosylation sites in the Fab region have also been observed. Because glycosylation of a monoclonal antibody can have a significant impact on its effector function, efficacy, clearance, and immunogenicity, it is essential to assess the glycosylation profile during cell line and clone selection studies and to assess the impact of cell culture conditions on the glycoform distribution during process optimization studies to ensure that the antibody is being produced with appropriate and consistent glycosylation. This article describes a liquid chromatography-mass spectrometry-based approach, in combination with papain digestion and partial reduction, to obtain site-specific glycosylation profile information for a therapeutic monoclonal antibody with two N-linked glycosylation sites in the heavy chain.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.     

Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others. Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.     

Dynamics of immature mAb glycoform secretion during CHO cell culture: An integrated modelling framework

Ensuring consistent glycosylation‐associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immunogenicity. However, the interaction between bioprocess conditions, cellular metabolism and the intracellular process of glycosylation remains to be fully understood. To gain further insight into these interactions, we present a novel integrated modelling platform that links dynamic variations in mAb glycosylation with cellular secretory capacity. Two alternative mechanistic representations of how mAb specific productivity (q_p) influences glycosylation are compared. In the first, mAb glycosylation is modulated by the linear velocity with which secretory cargo traverses the Golgi apparatus. In the second, glycosylation is influenced by variations in Golgi volume. Within our modelling framework, both mechanisms accurately reproduce experimentally‐observed dynamic changes in mAb glycosylation. In addition, an optimisation‐based strategy has been developed to estimate the concentration of glycosylation enzymes required to minimise mAb glycoform variability. Our results suggest that the availability of glycosylation machinery relative to cellular secretory capacity may play a crucial role in mAb glycosylation. In the future, the modelling framework presented here may aid in selecting and engineering cell lines that ensure consistent mAb glycosylatio.     

Distinctive roles of endoplasmic reticulum and golgi glycosylation in functional surface expression of mammalian E-NTPDase1, CD39

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes. The results demonstrate that endoplasmic reticulum glycosylation is required for protein folding and essential for functional surface expression of CD39, while Golgi glycosylation is less important. The study has also shown that N-linked glycosylation of CD39 is dispensable for the activity after the protein is properly folded and targeted.     

Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large receptor that contains extensive glycosylation sites and disulfide bonds. Here we analyzed how N-linked glycosylation and molecular chaperones function during LRP folding. Treatment of cells with a glycosylation inhibitor tunicamycin significantly impaired LRP folding, although binding to receptor-associated protein (RAP), a specialized chaperone for LRP, was not affected. The effects of tunicamycin on LRP folding were not due to an inhibition of RAP glycosylation since a mutant RAP that harbors a mutation at its sole glycosylation site was still capable of promoting LRP folding. The roles of N-linked glycosylation and the lectin chaperone, calnexin, in LRP folding were further dissected using LRP minireceptors that carry mutations at individual glycosylation sites. Interestingly, we found that RAP interacts with oxidoreductase ERp57 and mediates its interaction with LRP. Since previous studies have shown that N-glycan-bound calnexin/calreticulin are also capable of recruiting ERp57, our results suggest that N-linked glycosylation and RAP can independently and cooperatively recruit oxidoreductases to facilitate protein folding and proper disulfide bond formation.     

Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies

Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.     

Glycosylation is important for binding to human calcitonin receptors

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.     

Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence

To understand better the structural requirements of the protein moiety important for N-glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn-Xaa-Ser/Thr. In the first part of the paper, experiments are described using a cell-free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline-containing peptides are compatible with the notion that a proline residue in an N-terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C-terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively. This statement was further substantiated by in vivo studies using site-directed mutagenesis to introduce a proline residue at the C-terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell-free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes, leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

Altered glycosylation of acetylcholinesterase in Creutzfeldt-Jakob disease

Changes in the glycosylation pattern of brain proteins have been associated with Creutzfeldt-Jakob disease (CJD). We have investigated the glycosylation status of acetylcholinesterase (AChE) by lectin binding assay. Our data show that in lumbar CSF from definite and probable sporadic CJD cases AChE activity is lower compared with that in age-matched controls. We also show, for the first time, that AChE glycosylation is altered in CJD CSF and brain. Unlike Alzheimer's disease, in which an alteration in both the glycosylation and levels of AChE molecular forms is observed, the abnormal glycosylation of AChE in CJD appears to be unrelated to changes in molecular forms of this enzyme. These findings suggest that altered AChE glycosylation in CJD may be a consequence of the general perturbation of the glycosylation machinery that affects prion protein, as well as other proteins. The diagnostic potential of these changes remains to be explored.     

Impact of host cell line choice on glycan profile

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.     

Regulation of TRP channels by N-linked glycosylation

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.     

Role of glycosylation in cell surface expression and stability of HERG potassium channels

The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.     

Protein glycosylation in infectious disease pathobiology and treatment

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics.     

Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.