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BACKGROUND: B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide is the only blood biomarker in established risk calculators for pulmonary arterial hypertension (PAH). Profiling systemic-originated plasma immunoglobulin G (IgG) N-glycans, which reflect different components of the pathophysiology of PAH including immune dysregulation and inflammation, may improve PAH risk assessment. OBJECTIVES: This study sought to identify plasma IgG N-glycan biomarkers that predict survival in PAH to improve risk assessment. METHODS: This cohort study examined 622 PAH patients from 2 national centers (Beijing [discovery] cohort: n = 273; Shanghai [validation] cohort: n = 349). Plasma IgG N-glycomes were profiled by a robust mass spectrometry-based method. Prognostic IgG N-glycan traits were identified and validated in the 2 cohorts using Cox regression and Kaplan-Meier survival analyses. The added value of IgG N-glycan traits to previously established risk models was assessed using Harrell C-indexes and survival analysis. RESULTS: Plasma IgG fucosylation was found to predict survival independent of age and sex in the discovery cohort (HR: 0.377; 95% CI: 0.168-0.845; P = 0.018) with confirmation in the validation cohort (HR: 0.445; 95% CI: 0.264-0.751; P = 0.005). IgG fucosylation remained a robust predictor of mortality in combined cohorts after full adjustment and in subgroup analyses. Integrating IgG fucosylation into previously established risk models improved their predictive capacity, marked by an overall elevation in Harrell C-indexes. IgG fucosylation was useful in further stratifying the intermediate-risk patients classified by a previously established model. CONCLUSIONS: Plasma IgG fucosylation informs PAH prognosis independent of established factors, offering additional value for predicting PAH outcomes.

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Introduction: Glyphosate-based herbicides (GBHs) have been shown to have significant neurotoxic effects, affecting both the structure and function of the brain, and potentially contributing to the development of neurodegenerative disorders. Despite the known importance of glycosylation in disease progression, the glycome profile of systems exposed to GBH has not been thoroughly investigated. Methods: In this study, we conducted a comprehensive glycomic profiling using LC-MS/MS, on the hippocampus and prefrontal cortex (PFC) of juvenile rats exposed to GBH orally, aiming to identify glyco-signature aberrations after herbicide exposure. Results: We observed changes in the glycome profile, particularly in fucosylated, high mannose, and sialofucosylated N-glycans, which may be triggered by GBH exposure. Moreover, we found major significant differences in the N-glycan profiles between the GBH-exposed group and the control group when analyzing each gender independently, in contrast to the analysis that included both genders. Notably, gender differences in the behavioral test of object recognition showed a decreased performance in female animals exposed to GBH compared to controls (p < 0.05), while normal behavior was recorded in GBH-exposed male rats (p > 0.05). Conclusion: These findings suggest that glycans may play a role in the neurotoxic effect caused by GBH. The result suggests that gender variation may influence the response to GBH exposure, with potential implications for disease progression and specifically the neurotoxic effects of GBHs. Understanding these gender-specific responses could enhance knowledge of the mechanisms underlying GBH-induced toxicity and its impact on brain health. Overall, our study represents the first detailed analysis of N-glycome profiles in the hippocampus and PFC of rats chronically exposed to GBH. The observed alterations in the expression of N-glycan structures suggest a potential neurotoxic effect associated with chronic GBH exposure, highlighting the importance of further research in this area.

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Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated. Therefore, cloning these glycosyltransferases from different model systems will provide valuable insights into this process. Little is known about fucosyltransferases in molluscs. For this study, a sequence of the Pacific oyster, Crassostrea gigas, based on amino acid sequence homologies with rabbit and human α-1,2-fucosyltransferases, was chosen. The recombinant enzyme (350 amino acids) was able to transfer fucose from GDP-fucose to the galactose residue of type II disaccharides, terminal galactoses in complex N-glycan structures and several linear and branched galactans which were tested using a glycan microarray. The α-1,2-linkage formed was confirmed by NMR analysis. The enzyme was active in a broad pH-range, it was relatively stable upon storage conditions and its activity was not dependent on the presence of divalent cations. In this study, we were able to clone, express and characterise a novel α-1,2-fucosyltrasferase from Crassostrea gigas (CgFUT2).

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The attachment of sugar to proteins and lipids is a basic modification needed for organismal survival, and perturbations in glycosylation cause severe developmental and neurological difficulties. Here, we investigated the neurological consequences of N-glycan populations in the spinal cord of Wt AB and mgat1b mutant zebrafish. Mutant fish have reduced N-acetylglucosaminyltransferase-I (GnT-I) activity as mgat1a remains intact. GnT-I converts oligomannose N-glycans to hybrid N-glycans, which is needed for complex N-glycan production. MALDI-TOF MS profiles identified N-glycans in the spinal cord for the first time and revealed reduced amounts of complex N-glycans in mutant fish, supporting a lesion in mgat1b. Further lectin blotting showed that oligomannose N-glycans were more prevalent in the spinal cord, skeletal muscle, heart, swim bladder, skin, and testis in mutant fish relative to WT AB, supporting lowered GnT- I activity in a global manner. Developmental delays were noted in hatching and in the swim bladder. Microscopic images of caudal primary (CaP) motor neurons of the spinal cord transiently expressing EGFP in mutant fish were abnormal with significant reductions in collateral branches. Further motor coordination skills were impaired in mutant fish. We conclude that identifying the neurological consequences of aberrant N-glycan processing will enhance our understanding of the role of complex N-glycans in development and nervous system health.

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Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by ß-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

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Gilthead seabream (Sparus aurata) is a marine finfish of economic importance in aquaculture. Despite its adaptability to varying culture conditions, gilthead seabream culture can be affected by viral, bacterial or parasitic diseases. The main route of entry of pathogens is through mucosal surfaces. Teleost external and internal surfaces are covered by mucus, mainly comprised of highly glycosylated proteins called mucins. The mucin glycans regulate pathogen growth, adhesion, virulence and inter and intra species communication. Here, we characterized the gilthead seabream mucus glycosylation, compared it to previously described species and investigated associations with microbiota. 214 glycans were identified. The majority of the glycans were found at more than one epithelial surface, but 27, 22 and 89 O-glycan structures were unique to skin, gill and intestinal sample groups, respectively. Six O-glycan core types were observed. The majority of the seabream skin and gill O-glycans were neutral with unusual poly HexNAc motifs. In contrast, seabream intestinal O-glycans were highly acidic and not of the 'poly HexNAc' type observed in skin and gill. Furthermore, gilthead seabream gill mucosa had less oligomannose and more complex N-glycans compared to skin and intestine. The concentration and diversity of bacteria was similar in skin, gill and intestine, but the bacterial species differed between epithelia and co-varied with glycan epitopes. The presence of a complex mucus glycosylation with plenty of glycan epitopes for bacterial foraging, suggest that the skin mucosal defense in seabream includes an abundant resident microbiota. This large library of structures provides a platform for further studies, for example aiming to identifying glycans to use for diagnostic purposes, to study host-microbe interactions or disease intervention therapies.

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Glycosylation is the most abundant and diverse post-translational modification occurring on proteins. Glycans play important roles in modulating cell adhesion, growth, development, and differentiation. Changes in glycosylation affect protein structure and function and contribute to disease processes. Therefore, understanding glycosylation patterns is key for the identification of targets for the diagnosis of diseases, cellular states, and therapy. Glycosylation is a non template-driven process governed by the action of numerous enzymes and substrate availability that varies among cell types and species. Therefore, qualitative and quantitative assessment of global glycosylation and individual glycans remains challenging because it requires integration of multiple complex data types. Glycan structure and quantity data are often integrated with assessments of gene expression to aid contextualization of observed glycosylation changes within biological processes. However, correlating glycogene expression to the glycan structure is challenging because transcriptional changes may not always concur with the final gene product; there is often a lack of information on nucleotide sugar pools, and the final glycan structure is the result of many different glycogenes acting in concert. To overcome these challenges, interactive online tools are emerging as key resources for facilitating the analysis and integration of glycomics and glycogene expression data. Importantly, these tools work in concurrence with glycan biosynthetic schemes and therefore provide a clear indication of the molecular pathways where the glycan and glycogene are involved. In this chapter, we describe the applications of four freely available online tools that can be used for integrated visualization, interpretation, and presentation of RNAseq and glycomics results.

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INTRODUCTION: Congenital disorders of glycosylation (CDG) are a continuously expanding group of monogenic disorders that disrupt glycoprotein and glycolipid biosynthesis, leading to multi-systemic manifestations. These disorders are categorized into various groups depending on which part of the glycosylation process is impaired. The cardiac manifestations in CDG can significantly differ, not only across different types but also among individuals with the same genetic cause of CDG. Cardiomyopathy is an important phenotype in CDG. The clinical manifestations and progression of cardiomyopathy in CDG patients have not been well characterized. This study aims to delineate common patterns of cardiomyopathy across a range of genetic causes of CDG and to propose baseline screening and follow-up evaluation for this patient population. METHODS: Patients with molecular confirmation of CDG who were enrolled in the prospective or memorial arms of the Frontiers in Congenital Disorders of Glycosylation Consortium (FCDGC) natural history study were ascertained for the presence of cardiomyopathy based on a retrospective review of their medical records. All patients were evaluated by clinical geneticists who are members of FCDGC at their respective academic centers. Patients were screened for cardiomyopathy, and detailed data were retrospectively collected. We analyzed their clinical and molecular history, imaging characteristics of cardiac involvement, type of cardiomyopathy, age at initial presentation of cardiomyopathy, additional cardiac features, the treatments administered, and their clinical outcomes. RESULTS: Of the 305 patients with molecularly confirmed CDG participating in the FCDGC natural history study as of June 2023, 17 individuals, nine females and eight males, were identified with concurrent diagnoses of cardiomyopathy. Most of these patients were diagnosed with PMM2-CDG (n = 10). However, cardiomyopathy was also observed in other diagnoses, including PGM1-CDG (n = 3), ALG3-CDG (n = 1), DPM1-CDG (n = 1), DPAGT1-CDG (n = 1), and SSR4-CDG (n = 1). All PMM2-CDG patients were reported to have hypertrophic cardiomyopathy. Dilated cardiomyopathy was observed in three patients, two with PGM1-CDG and one with ALG3-CDG; left ventricular non-compaction cardiomyopathy was diagnosed in two patients, one with PGM1-CDG and one with DPAGT1-CDG; two patients, one with DPM1-CDG and one with SSR4-CDG, were diagnosed with non-ischemic cardiomyopathy. The estimated median age of diagnosis for cardiomyopathy was 5 months (range: prenatal-27 years). Cardiac improvement was observed in three patients with PMM2-CDG. Five patients showed a progressive course of cardiomyopathy, while the condition remained unchanged in eight individuals. Six patients demonstrated pericardial effusion, with three patients exhibiting cardiac tamponade. One patient with SSR4-CDG has been recently diagnosed with cardiomyopathy; thus, the progression of the disease is yet to be determined. One patient with PGM1-CDG underwent cardiac transplantation. Seven patients were deceased, including five with PMM2-CDG, one with DPAGT1-CDG, and one with ALG3-CDG. Two patients died of cardiac tamponade from pericardial effusion; for the remaining patients, cardiomyopathy was not necessarily the primary cause of death. CONCLUSIONS: In this retrospective study, cardiomyopathy was identified in ∼6% of patients with CDG. Notably, the majority, including all those with PMM2-CDG, exhibited hypertrophic cardiomyopathy. Some cases did not show progression, yet pericardial effusions were commonly observed, especially in PMM2-CDG patients, occasionally escalating to life-threatening cardiac tamponade. It is recommended that clinicians managing CDG patients, particularly those with PMM2-CDG and PGM1-CDG, be vigilant of the cardiomyopathy risk and risk for potentially life-threatening pericardial effusions. Cardiac surveillance, including an echocardiogram and EKG, should be conducted at the time of diagnosis, annually throughout the first 5 years, followed by check-ups every 2-3 years if no concerns arise until adulthood. Subsequently, routine cardiac examinations every five years are advisable. Additionally, patients with diagnosed cardiomyopathy should receive ongoing cardiac care to ensure the effective management and monitoring of their condition. A prospective study will be required to determine the true prevalence of cardiomyopathy in CDG.

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Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting a growing number of elderly people. In order to improve the early and differential diagnosis of AD, better biomarkers are needed. Glycosylation is a protein post-translational modification that is modulated in the course of many diseases, including neurodegeneration. Aiming to improve AD diagnosis and differential diagnosis through glycan analytics methods, we report the glycoprotein glycome of cerebrospinal fluid (CSF) isolated from a total study cohort of 262 subjects. The study cohort consisted of patients with AD, healthy controls and patients suffering from other types of dementia. CSF free-glycans were also isolated and analyzed in this study, and the results reported for the first time the presence of 19 free glycans in this body fluid. The free-glycans consisted of complete or truncated N-/O-glycans as well as free monosaccharides. The free-glycans Hex1 and HexNAc1Hex1Neu5Ac1 were able to discriminate AD from controls and from patients suffering from other types of dementia. Regarding CSF N-glycosylation, high proportions of high-mannose, biantennary bisecting core-fucosylated N-glycans were found, whereby only about 20% of the N-glycans were sialylated. O-Glycans and free-glycan fragments were less sialylated in AD patients than in controls. To conclude, this comprehensive study revealed for the first time the biomarker potential of free glycans for the differential diagnosis of AD.

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The remediation of the carbohydrate data of the Protein Data Bank (PDB) has brought numerous enhancements to the findability and interpretability of deposited glycan structures, yet crucial quality indicators are either missing or hard to find on the PDB pages. Without a way to access wider glycochemical context, problematic structures may be taken as fact by keen but inexperienced scientists. The Privateer software is a validation and analysis tool that provides access to a number of metrics and links to external experimental resources, allowing users to evaluate structures using carbohydrate-specific methods. Here, we present the Privateer database, a free resource that aims to complement the growing glycan content of the PDB.

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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival rate of less than 10%, owing to its late-stage diagnosis. Early detection of pancreatic cancer (PC) can significantly increase survival rates. AIM: To identify the serum biomarker signatures associated with early-stage PDAC by serum N-glycan analysis. METHODS: An extensive patient cohort was used to determine a biomarker signature, including patients with PDAC that was well-defined at an early stage (stages I and II). The biomarker signature was derived from a case-control study using a case-cohort design consisting of 29 patients with stage I, 22 with stage II, 4 with stage III, 16 with stage IV PDAC, and 88 controls. We used multiparametric analysis to identify early-stage PDAC N-glycan signatures and developed an N-glycan signature-based diagnosis model called the "Glyco-model". RESULTS: The biomarker signature was created to discriminate samples derived from patients with PC from those of controls, with a receiver operating characteristic area under the curve of 0.86. In addition, the biomarker signature combined with cancer antigen 19-9 could discriminate patients with PDAC from controls, with a receiver operating characteristic area under the curve of 0.919. Glyco-model demonstrated favorable diagnostic performance in all stages of PC. The diagnostic sensitivity for stage I PDAC was 89.66%. CONCLUSION: In a prospective validation study, this serum biomarker signature may offer a viable method for detecting early-stage PDAC.

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Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

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ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.

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In the beginning was the word. But there were no words for N-glycans, at least, no simple words. Next to chemical formulas, the IUPAC code can be regarded as the best, most reliable and yet immediately comprehensible annotation of oligosaccharide structures of any type from any source. When it comes to N-glycans, the venerable IUPAC code has, however, been widely supplanted by highly simplified terms for N-glycans that count the number of antennae or certain components such as galactoses, sialic acids and fucoses and give only limited room for exact structure description. The highly illustrative - and fortunately now standardized - cartoon depictions gained much ground during the last years. By their very nature, cartoons can neither be written nor spoken. The underlying machine codes (e.g., GlycoCT, WURCS) are definitely not intended for direct use in human communication. So, one might feel the need for a simple, yet intelligible and precise system for alphanumeric descriptions of the hundreds and thousands of N-glycan structures. Here, we present a system that describes N-glycans by defining their terminal elements. To minimize redundancy and length of terms, the common elements of N-glycans are taken as granted. The preset reading order facilitates definition of positional isomers. The combination with elements of the condensed IUPAC code allows to describe even rather complex structural elements. Thus, this "proglycan" coding could be the missing link between drawn structures and software-oriented representations of N-glycan structures. On top, it may greatly facilitate keyboard-based mining for glycan substructures in glycan repositories.

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We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

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Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry.

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In this chapter, we will present a high-throughput method applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. This methodology is based on a commercial equipment developed by WATERS™ to speed up N-deglycosylation and N-glycan labeling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies. This analytical kit is sold under the trade name of RapiFluor-MS (RFMS). We have slightly modified the methodology, increasing the glycosylation time and using a high-resolution mass analyzer for the analysis of CSF N-glycans, thus obtaining a high-throughput method (up to 96 samples simultaneously), mass accuracy better than 5 ppm, and the ability to separate and identify isomers.

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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).

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The Hepatitis C Virus (HCV), responsible for causing hepatitis and a significant contributor to liver disorders, presents a challenge for treatment due to its high genetic variability. Despite efforts, there is still no effective medication available for this virus. One of the promising targets for drug development involves targeting glycoprotein E2. However, our understanding of the dynamic behavior of E2 and its associated glycans remains limited. In this study, we investigated the dynamic characteristics of E2 with varying degrees of glycosylation using all-atom molecular dynamics simulations. We also explored glycan's interactions with the protein and among themselves. An overall increase in correlation between the vital protein regions was observed with an increase in glycan number. The protein dynamics is followed by the analysis of glycan dynamics, where the flexibility of the individual glycans was analyzed in their free and bound state, which revealed a decrease in their fluctuation in some cases. Furthermore, we generated the free energy landscape of individual N-glycan linkages in both free and bound states and observed both increases and decreases in flexibility, which can be attributed to the formation and breakage of hydrogen bonds with amino acids. Finally, we found that for a high glycosylation system, glycans interact with glycoprotein and form hydrogen bonds among themselves. Moreover, the hydrogen bond profiles of a given glycan can vary when influenced by other glycans. In summary, our study provides valuable insights into the dynamics of the core region of HCV E2 glycoprotein and its associated glycans.Communicated by Ramaswamy H. Sarma.