N-linked Protein Glycosylation Research Articles

GlyCompute: towards the automated analysis of protein N-linked glycosylation kinetics via an open-source computational framework.

Understanding the complex biosynthetic pathways of glycosylation is crucial for the expanding field of glycosciences. Computer-aided glycosylation analysis has greatly benefited in recent years from the development of tools found in web-based portals and open-source libraries. However, the in silico analysis of cellular glycosylation kinetics is underrepresented in current glycoscience-related tools and databases. This could be partly attributed to the limited accessibility of kinetic models developed using proprietary software and the difficulty in reliably parameterising such models. This work aims to address these challenges by proposing GlyCompute, an open-source framework demonstrating a novel, streamlined approach for the assembly, simulation, and parameterisation of kinetic models of protein N-linked glycosylation. Specifically, given one or more sets of experimentally observed N-glycan structures and their relative abundances, minimum representations of a glycosylation reaction network are generated. The topology of the resulting networks is then used to automatically assemble the material balances and kinetic mechanisms underpinning the mathematical model. To match the experimentally observed relative abundances, a sequential parameter estimation strategy using Bayesian inference is proposed, with stages determined automatically based on the underlying network topology. The proposed framework was tested on a case study involving the simultaneous fitting of the kinetic model to two protein N-linked glycoprofiles produced by the same CHO cell culture, showing good agreement with experimental observations. We envision that GlyCompute could help glycoscientists gain quantitative insights into the effect of enzyme kinetics and their perturbations on experimentally observed glycoprofiles in biomanufacturing and clinical settings.

N-linked protein glycosylation in Nanobdellati (formerly DPANN) archaea and their hosts.

Members of the kingdom Nanobdellati, previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati. Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N-acetylhexosamine, and sulfonated deoxyhexose. However, the N-glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N-glycan in a host archaeon when co-cultured with Nanobdella. These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCENanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments.

Sensitivity of transferrin isoform analysis for PMM2-CDG

Transferrin isoform analysis is an established laboratory test for congenital disorders of glycosylation (CDG). Despite its long history of clinical use, little has been published about its empirical sensitivity for specific conditions. We conducted a retrospective analysis of ten years of testing data and report our experience with transferrin testing for type I profiles and its sensitivity for the most common congenital disorder of glycosylation, PMM2-CDG. The data demonstrate 94% overall test sensitivity for PMM2-CDG and importantly demonstrate two known, recurrent variants enriched in false positive cases highlighting an important limitation of the test. The data confirm the clinical validity of transferrin isotype analysis as a screening test for disorders of protein N-linked glycosylation and as functional test for PMM2 genotypes of uncertain significance.

N-Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg2+ influx in conditions of high glucose or impaired respiratory chain function.

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.

Frontiers in congenital disorders of glycosylation consortium, a cross-sectional study report at year 5 of 280 individuals in the natural history cohort

ObjectiveOur report describes clinical, genetic, and biochemical features of participants with a molecularly confirmed congenital disorder of glycosylation (CDG) enrolled in the Frontiers in Congenital Disorders of Glycosylation (FCDGC) Natural History cohort at year 5 of the study. MethodsWe enrolled individuals with a known or suspected CDG into the FCDGC Natural History Study, a multicenter prospective and retrospective natural history study of all genetic causes of CDG. We conducted a cross-sectional analysis of baseline study visit data from participants with confirmed CDG who were consented into the FCDGC Natural History Study (5U54NS115198) from October 2019 to November 2023. ResultsThree hundred thirty-three subjects consented to the FCDGC Natural History Study. Of these, 280 unique individuals had genetic data available that was consistent with a diagnosis of CDG. These 280 individuals were enrolled into the study between October 8, 2019 and November 29, 2023. One hundred forty-one (50.4%) were female, and 139 (49.6%) were male. Mean and median age at enrollment was 10.1 and 6.5 years, respectively, with a range of 0.22 to 71.4 years. The cohort encompassed individuals with disorders of N-linked protein glycosylation (57%), glycosylphosphatidylinositol anchor disorder (GPI anchor) (15%), disorders of Golgi homeostasis, trafficking and transport (12%), dolichol metabolism disorders (5%), disorders of multiple pathways (6%), and other (5%). The most frequent presenting symptom(s) leading to diagnosis were developmental delay/disability (77%), followed by hypotonia (56%) and feeding difficulties (42%). Mean and median time between first related symptom and diagnosis was 2.7 and 0.8 years, respectively. One hundred percent of individuals in our cohort had developmental differences/disabilities at the time of their baseline visit, followed by 97% with neurologic involvement, 91% with gastrointestinal (GI)/liver involvement, and 88% with musculoskeletal involvement. Severity of disease in individuals was scored on the Nijmegen Progression CDG Rating Scale (NPCRS) with 27% of scores categorized as mild, 44% moderate, and 29% severe. Of the individuals with N-linked protein glycosylation defects, 83% of those with data showed a type 1 pattern on carbohydrate deficient transferrin (CDT) analysis including 82/84 individuals with PMM2-CDG, 6% a type 2 pattern, 1% both type 1 and type 2 pattern and 10% a normal or nonspecific pattern. One hundred percent of individuals with Golgi homeostasis and trafficking defects with data showed a type 2 pattern on CDT analysis, while Golgi transport defect showed a type II pattern 73% of the time, a type 1 pattern for 7%, and 20% had a normal or nonspecific pattern. Most of the variants documented were classified as pathogenic or likely pathogenic using ACMG criteria. For the majority of the variants, the predicted molecular consequence was missense followed by nonsense and splice site, and the majority of the diagnoses are inherited in an autosomal recessive pattern but with disorders of all major nuclear inheritance included. DiscussionThe FCDGC Natural History Study serves as an important resource to build future research studies, improve clinical care, and prepare for clinical trial readiness. Herein is the first overview of CDG participants of the FCDGC Natural History Study.

Altered N-linked glycosylation in depression: A pre-clinical study

BackgroundNeuroimmune plays an important role in major depressive disorders (MDD). N-linked protein glycosylation (NLG) might contribute to depression by regulating the neuroinflammatory response. As microglia is the main executor of neuroimmune function in the central neural system (CNS), targeting the process of N-linked protein glycosylation of microglia in the mice used for studying depression might potentially offer new avenues for the strategy for MDD. MethodsThe chronic unpredictable mild stress (CUMS) mouse model was established for the whole brain microglia isolating. Then, RNA samples of microglia were extracted for transcriptome sequencing and mRNA analysis. Immunofluorescence (IF) was used to identify the expression level of NLG-related enzyme, B4galt1, in microglia. ResultsThe data showed that NLG was positively related to depression. Moreover, the NLG-related gene, B4galt1 increased expression in the microglia of CUMS mice. Then, the inhibition of NLG reversed the depressive behavior in CUMS mice. The expression level of B4galt1 in CUMS mice was upregulating following the NLG-inhibitor treatment. Similar results haven't been observed in neurons. Information obtained from these experiments showed increasing expression of B4galt1 in microglia following depressive-like behaviors. ConclusionsThese findings indicate that NLG in microglia is associated with MDD, and suggest that therapeutically targeting NLG might be an effective strategy for depression. LimitationsHow to modulate the B4galt1 or NLG pathways in microglia efficiently and economically request new technologies.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17–34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8–12%), with hair and dental abnormalities present in only 4–7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Brucella-driven host N-glycome remodeling controls infection

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

Protein glycosylation in cardiovascular health and disease.

Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.

Dolichol kinases from yeast, nematode and human can replace each other and exchange their domains creating active chimeric enzymes in yeast.

Protein glycosylation is a fundamental modification crucial for numerous intra- and extracellular functions in all eukaryotes. The phosphorylated dolichol (Dol-P) is utilized in N-linked protein glycosylation and other glycosylation pathways. Dolichol kinase (DK) plays a key role in catalyzing the phosphorylation of dolichol. The glycosylation patterns in the Kluyveromyces lactis DK mutant revealed that the yeast well tolerated a minor deficiency in Dol-P by adjusting protein glycosylation. Comparative analysis of sequences of DK homologs from different species of eukaryotes, archaea and bacteria and AlphaFold3 structural model studies, allowed us to predict that DK is most likely composed of two structural/functional domains. The activity of predicted K. lactis DK C-terminal domain expressed from the single copy in the chromosome was not sufficient to keep protein glycosylation level necessary for survival of K. lactis. However, the glycosylation level was partially restored by additionally provided and overexpressed N- or C-terminal domain. Moreover, co-expression of the individual N-and C-terminal domains restored the glycosylation of vacuolar carboxypeptidase Y in both K. lactis and Saccharomyces cerevisiae. Despite the differences in length and non-homologous sequences of the N-terminal domains the human and nematode Caenorhabditis elegans DKs successfully complemented DK functions in both yeast species. Additionally, the N-terminal domains of K. lactis and C. elegans DK could functionally substitute for one another, creating active chimeric enzymes. Our results suggest that while the C-terminal domain remains crucial for DK activity, the N-terminal domain may serve not only as a structural domain but also as a possible regulator of DK activity.

CSF N-Glycomics Using MALDI MS Techniques.

In this chapter, we will present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease (AD).

Characterization of PglJ, a Glycosyltransferase in the Campylobacter concisus N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The Campylobacter genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As Campylobacter concisus is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of C. concisus strains have been reported to diverge from other Campylobacter in steps after the biosynthesis of N-acetylgalactosamine-α1,3-N,N'-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of C. concisus. Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the Pseudomonas aeruginosa WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the Campylobacter genus. These studies now allow detailed characterization of the later steps in the pgl pathway in C. concisus strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.

N-glycans show distinct spatial distribution in mouse brain.

The development and function of the brain requires N-linked glycosylation of proteins, which is a ubiquitous modification in the secretory pathway. N-glycans have a distinct composition and undergo tight regulation in the brain, but the spatial distribution of these structures remains relatively unexplored. Here, we systematically employed carbohydrate binding lectins with differing specificities to various classes of N-glycans and appropriate controls to identify glycan expression in multiple regions of the mouse brain. Lectins binding high-mannose-type N-glycans, the most abundant class of brain N-glycans, showed diffuse staining with some punctate structures observed on high magnification. Lectins binding specific motifs of complex N-glycans, including fucose and bisecting GlcNAc, showed more partitioned labeling, including to the synapse-rich molecular layer of the cerebellum. Understanding the spatial distribution of N-glycans across the brain will aid future studies of these critical protein modifications in development and disease of the brain.

Probing enzymatic properties of N-glycosyltransferase isoforms from Mannheimia haemolytica

N-Glycosyltransferase (NGT) is an inverting glycosyltransferase for an unusual pathway of N-linked protein glycosylation and glycosylates polypeptides in the consensus sequon (N-(X≠P)-T/S) with hexose monosaccharides. Here, we expressed and characterized a novel N-glycosyltransferase from Mannheimia haemolytica (named MhNGT). RP-HPLC and Mass Spectrometry were used to assay and quantify glycopeptide formation by MhNGT and determine its substrate specificities. MhNGT could utilize a variety of nucleotide-activated sugar donors, including UDP-Glc, UDP-Gal and UDP-Xyl, to glycosylate the tested peptides DANYTK, GGNWTT and PAVGNCSSALR with higher efficiency than ApNGT which was comprehensive studied. The optimum temperature of MhNGT was about 30 °C and the optimum pH was 7.5–8.0 in PBS-NaOH buffer. MhNGT exhibited a different position-specific residue preference of substrate peptides from the NGT of Actinobacillus pleuropneumoniae (ApNGT). The effective glycosylation of common short peptides by MhNGT demonstrated the enzyme's potential to alter therapeutically significant mammalian N-glycoproteins.

Effector secretion and stability in the maize anthracnose pathogen Colletotrichum graminicola requires N-linked protein glycosylation and the ER chaperone pathway.

N-linked protein glycosylation is a conserved and essential modification mediating protein processing and quality control in the endoplasmic reticulum (ER), but how this contributes to the infection cycle of phytopathogenic fungi is largely unknown. In this study, we discovered that inhibition of protein N-glycosylation severely affected vegetative growth, hyphal tip development, conidial germination, appressorium formation, and, ultimately, the ability of the maize (Zea mays) anthracnose pathogen Colletotrichum graminicola to infect its host. Quantitative proteomics analysis showed that N-glycosylation can coordinate protein O-glycosylation, glycosylphosphatidylinositol anchor modification, and endoplasmic reticulum quality control (ERQC) by directly targeting the proteins from the corresponding pathway in the ER. We performed a functional study of the N-glycosylation pathway-related protein CgALG3 and of the ERQC pathway-related protein CgCNX1, which demonstrated that N-glycosylation of ER chaperone proteins is essential for effector stability, secretion, and pathogenicity of C. graminicola. Our study provides concrete evidence for the regulation of effector protein stability and secretion by N-glycosylation.

B4GALT1 promotes immune escape by regulating the expression of PD-L1 at multiple levels in lung adenocarcinoma

BackgroundInvasive adenocarcinoma (IAC), which is typically preceded by minimally invasive adenocarcinoma (MIA), is the dominant pathological subtype of early-stage lung adenocarcinoma (LUAD). Identifying the molecular events underlying the progression from MIA to IAC may provide a crucial perspective and boost the exploration of novel strategies for early-stage LUAD diagnosis and treatment.MethodsTranscriptome sequencing of four pairs of MIA and IAC tumours obtained from four multiple primary lung cancer patients was performed to screen out beta-1,4-galactosyltransferase1 (B4GALT1). Function and mechanism experiments in vitro and in vivo were performed to explore the regulatory mechanism of B4GALT1-mediated immune evasion by regulating programmed cell death ligand 1 (PD-L1).ResultsB4GALT1, a key gene involved in N-glycan biosynthesis, was highly expressed in IAC samples. Further experiments revealed that B4GALT1 regulated LUAD cell proliferation and invasion both in vitro and in vivo and was related to the impaired antitumour capacity of CD8 + T cells. Mechanistically, B4GALT1 directly mediates the N-linked glycosylation of PD-L1 protein, thus preventing PD-L1 degradation at the posttranscriptional level. In addition, B4GALT1 stabilized the TAZ protein via glycosylation, which activated CD274 at the transcriptional level. These factors lead to lung cancer immune escape. Importantly, inhibition of B4GALT1 increased CD8 + T-cell abundance and activity and enhanced the antitumour immunity of anti-PD-1 therapy in vivo.ConclusionB4GALT1 is a critical molecule in the development of early-stage LUAD and may be a novel target for LUAD intervention and immunotherapy.

Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research.

Antibody-based therapeutics and recombinant protein reagents are often produced in mammalian expression systems, which provide human-like post-translational modifications. Among the available mammalian cell lines used for recombinant protein expression, Chinese hamster ovary (CHO)-derived suspension cells are generally utilized because they are easy to culture and tend to produce proteins in high yield. However, some proteins purified from CHO cell overexpression suffer from clipping and display undesired non-human post translational modifications (PTMs). In addition, CHO cell lines are often not suitable for producing proteins with many glycosylation motifs for structural biology studies, as N-linked glycosylation of proteins poses challenges for structure determination by X-ray crystallography. Hence, alternative and complementary cell lines are required to address these issues. Here, we present a robust method for expressing proteins in human embryonic kidney 293 (HEK293)-derived stable pools, leading to recombinant protein products with much less clipped species compared to those expressed in CHO cells and with higher yield compared to those expressed in transiently-transfected HEK293 cells. Importantly, the stable pool generation protocol is also applicable to HEK293S GnTI- (N-acetylglucosaminyltransferase I-negative) and Expi293F GnTI- suspension cells, facilitating production of high yields of proteins with less complex glycans for use in structural biology projects. Compared to HEK293S GnTI- stable pools, Expi293F GnTI- stable pools consistently produce proteins with similar or higher expression levels. HEK293-derived stable pools can lead to a significant cost reduction and greatly promote the production of high-quality proteins for diverse research projects.

2-Deoxy-D-glucose inhibits lymphocytic choriomeningitis virus propagation by targeting glycoprotein N-glycosylation

BackgroundIncreased glucose uptake and utilization via aerobic glycolysis are among the most prominent hallmarks of tumor cell metabolism. Accumulating evidence suggests that similar metabolic changes are also triggered in many virus-infected cells. Viral propagation, like highly proliferative tumor cells, increases the demand for energy and macromolecular synthesis, leading to high bioenergetic and biosynthetic requirements. Although significant progress has been made in understanding the metabolic changes induced by viruses, the interaction between host cell metabolism and arenavirus infection remains unclear. Our study sheds light on these processes during lymphocytic choriomeningitis virus (LCMV) infection, a model representative of the Arenaviridae family.MethodsThe impact of LCMV on glucose metabolism in MRC-5 cells was studied using reverse transcription-quantitative PCR and biochemical assays. A focus-forming assay and western blot analysis were used to determine the effects of glucose deficiency and glycolysis inhibition on the production of infectious LCMV particles.ResultsDespite changes in the expression of glucose transporters and glycolytic enzymes, LCMV infection did not result in increased glucose uptake or lactate excretion. Accordingly, depriving LCMV-infected cells of extracellular glucose or inhibiting lactate production had no impact on viral propagation. However, treatment with the commonly used glycolytic inhibitor 2-deoxy-D-glucose (2-DG) profoundly reduced the production of infectious LCMV particles. This effect of 2-DG was further shown to be the result of suppressed N-linked glycosylation of the viral glycoprotein.ConclusionsAlthough our results showed that the LCMV life cycle is not dependent on glucose supply or utilization, they did confirm the importance of N-glycosylation of LCMV GP-C. 2-DG potently reduces LCMV propagation not by disrupting glycolytic flux but by inhibiting N-linked protein glycosylation. These findings highlight the potential for developing new, targeted antiviral therapies that could be relevant to a wider range of arenaviruses.

Targeting N-linked Glycosylation for the Therapy of Aggressive Lymphomas.

DLBCL depends on constitutive BCR activation and signaling. There are currently no therapeutics that target the BCR directly and attenuate its pathologic signaling. Here, we unraveled a therapeutically exploitable, OST-B-dependent glycosylation pathway that drives BCR organization and proximal BCR signaling. This article is highlighted in the In This Issue feature, p. 1749.

LMNglyPred: prediction of human N-linked glycosylation sites using embeddings from a pre-trained protein language model.

Protein N-linked glycosylation is an important post-translational mechanism in Homo sapiens, playing essential roles in many vital biological processes. It occurs at the N-X-[S/T] sequon in amino acid sequences, where X can be any amino acid except proline. However, not all N-X-[S/T] sequons are glycosylated; thus, the N-X-[S/T] sequon is a necessary but not sufficient determinant for protein glycosylation. In this regard, computational prediction of N-linked glycosylation sites confined to N-X-[S/T] sequons is an important problem that has not been extensively addressed by the existing methods, especially in regard to the creation of negative sets and leveraging the distilled information from protein language models (pLMs). Here, we developed LMNglyPred, a deep learning-based approach, to predict N-linked glycosylated sites in human proteins using embeddings from a pre-trained pLM. LMNglyPred produces sensitivity, specificity, Matthews Correlation Coefficient, precision, and accuracy of 76.50, 75.36, 0.49, 60.99, and 75.74 percent, respectively, on a benchmark-independent test set. These results demonstrate that LMNglyPred is a robust computational tool to predict N-linked glycosylation sites confined to the N-X-[S/T] sequon.