Salinomycin disturbs Golgi function and specifically affects cells in epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) gives rise to cells with properties similar to cancer stem cells (CSCs). Targeting the EMT program to selectively eliminate CSCs is a promising way to improve cancer therapy. Salinomycin (Sal), a K+/H+ ionophore, was identified as highly selective towards CSC-like cells, but its mechanism of action and selectivity remains elusive. Here, we show that Sal, similar to monensin and nigericin, disturbs the function of the Golgi. Sal alters the expression of Golgi-related genes and leads to marked changes in Golgi morphology, particularly in cells that have undergone EMT. Moreover, Golgi-disturbing agents severely affect post-translational modifications of proteins, including protein processing, glycosylation and secretion. We discover that the alterations induced by Golgi-disturbing agents specifically affect the viability of EMT cells. Collectively, our work reveals a novel vulnerability related to the EMT, suggesting an important role for the Golgi in the EMT and that targeting the Golgi could represent a novel therapeutic approach against CSCs.

Variability of human Alpha-1-acid glycoprotein N-glycome in a Caucasian population.

AIM: Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein in human plasma and one of the most abundant acute phase proteins in humans. Glycosylation plays a crucial role in its biological functions, and alterations in AGP N-glycome have been associated with various diseases and inflammatory conditions. However, large-scale studies of AGP N-glycosylation in the general population are lacking. METHODS: Using recently developed high-throughput glycoproteomic workflow for site-specific AGP N-glycosylation analysis, 803 individuals from the Croatian island of Korcula were analyzed and their AGP N-glycome data associated with biochemical and physiological traits, as well as different environmental factors. RESULTS: After regression analysis, we found that AGP N-glycosylation is strongly associated with sex, somewhat less with age, along with multiple biochemical and physiological traits (e.g. BMI, triglycerides, uric acid, glucose, smoking status, fibrinogen). CONCLUSION: For the first time we have extensively explored the inter-individual variability of AGP N-glycome in a general human population, demonstrating its changes with sex, age, biochemical, and physiological status of individuals, providing the baseline for future population and clinical studies.

Probing Blood Plasma Protein Glycosylation with Infrared Spectroscopy.

The health state of an individual is closely linked to the glycosylation patterns of his or her blood plasma proteins. However, obtaining this information requires cost- and time-efficient analytical methods. We put forward infrared spectroscopy, which allows label-free analysis of protein glycosylation but so far has only been applied to analysis of individual proteins. Although spectral information does not directly provide the molecular structure of the glycans, it is sensitive to changes therein and covers all types of glycosidic linkages. Combining single-step ion exchange chromatography with infrared spectroscopy, we developed a workflow that enables the separation and analysis of major protein classes in blood plasma. Our results demonstrate that infrared spectroscopy can identify different patterns and global levels of glycosylation of intact plasma proteins. To showcase the strengths and limitations of the proposed approach, we compare the glycoforms of human and bovine alpha-1-acid glycoproteins, which exhibit highly variable global levels of glycosylation. To independently evaluate our conclusions, the glycan moieties of human alpha-1-acid glycoprotein were further analyzed using an established glycomics workflow. Importantly, the chromatographic separation of blood plasma improves the detection of aberrant glycoforms of a given protein as compared to infrared spectroscopy of bulk plasma. The presented approach allows a time-efficient comparison of glycosylation patterns of multiple plasma proteins, opening new avenues for biomedical probing.

High-Throughput Human Complement C3 N-Glycoprofiling Identifies Markers of Early Onset Type 1 Diabetes Mellitus in Children.

Recently, it was shown that children at the onset of type 1 diabetes (T1D) have a higher proportion of oligomannose glycans in their total plasma protein N-glycome compared to their healthy siblings. The most abundant complement component, glycoprotein C3, contains two N-glycosylation sites occupied exclusively by this type of glycans. Furthermore, complement system, as well as C3, was previously associated with T1D. It is also known that changes in glycosylation can modulate inflammatory responses, so our aim was to characterize the glycosylation profile of C3 in T1D. For this purpose, we developed a novel high-throughput workflow for human C3 concanavalin A lectin affinity enrichment and subsequent LC-MS glycopeptide analysis which enables protein-specific N-glycosylation profiling. From the Danish Childhood Diabetes Register, plasma samples of 61 children/adolescents newly diagnosed with T1D and 84 of their unaffected siblings were C3 N-glycoprofiled. Significant changes of C3 N-glycan profiles were found. T1D was associated with an increase in the proportion of unprocessed glycan structures with more mannose units. A regression model including C3 N-glycans showed notable discriminative power between children with early onset T1D and their healthy siblings with area under curve of 0.879. This study confirmed our previous findings of plasma high-mannose glycan changes in a cohort of recent onset T1D cases, suggesting the involvement of C3 N-glycome in T1D development. Our C3 glycan-based discriminative model could be valuable in assessment of T1D risk in children.

Correction: Site-specific analysis of N-glycans from different sheep prion strains.

[This corrects the article DOI: 10.1371/journal.ppat.1009232.].

Site-specific analysis of N-glycans from different sheep prion strains.

Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian species, including humans. During the course of the disease, the abnormally folded scrapie prion protein (PrPSc) accumulates in the central nervous system where it causes neurodegeneration. In prion disorders, the diverse spectrum of illnesses exists because of the presence of different isoforms of PrPSc where they occupy distinct conformational states called strains. Strains are biochemically distinguished by a characteristic three-band immunoblot pattern, defined by differences in the occupancy of two glycosylation sites on the prion protein (PrP). Characterization of the exact N-glycan structures attached on either PrPC or PrPSc is lacking. Here we report the characterization and comparison of N-glycans from two different sheep prion strains. PrPSc from both strains was isolated from brain tissue and enzymatically digested with trypsin. By using liquid chromatography coupled to electrospray mass spectrometry, a site-specific analysis was performed. A total of 100 structures were detected on both glycosylation sites. The N-glycan profile was shown to be similar to the one on mouse PrP, however, with additional 40 structures reported. The results presented here show no major differences in glycan composition, suggesting that glycans may not be responsible for the differences in the two analyzed prion strains.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Mouse milk immunoglobulin G Fc-linked N-glycosylation nano-LC-MS analysis in a model of vancomycin exposure during pregnancy.

Breast milk immunoglobulin G (IgG) plays an important role in the transfer of passive immunity in early life and in shaping the neonatal immune system through N-glycan-mediated effector functions. Currently, there are no protocols available to analyze breast milk IgG-Fc glycosylation in mouse models. Therefore, we developed and validated a glycoproteomic workflow for the medium-throughput subclass-specific nano-LC-MS analysis of IgG enriched from small milk volumes of lactating mice. With the established methods, the IgG glycopatterns in a mouse model of antibiotic use during pregnancy and increased asthma susceptibility in the offspring were analyzed. Pregnant BALB/c mice were treated with vancomycin during gestation days 8-17 and IgG1F, IgG2, and IgG3-Fc glycosylation was subsequently analyzed in maternal serum, maternal breast milk, and offspring serum on postnatal day 15. The IgG glycosylation profiles of mouse maternal milk and serum revealed no significant differences within the glycoforms quantified across subclasses. However, vancomycin use during pregnancy was associated with changes in IgG-Fc glycosylation in offspring serum, shown by the decreased relative abundance of the IgG1F-G1 and IgG3-G0 glycoforms, together with the increased relative abundance of the IgG3-G2 and S1 glycoforms. The workflow presented will aid in the emerging integrative multi-omics- and glycomics-oriented milk analyses both in rodent models and human cohorts for a better understanding of mother-infant immunological interactions.

High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery.

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP's role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 µl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP's second glycosylation site and lower sialylation of N-glycans on AGP's third and AGP1's fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

Developments and perspectives in high-throughput protein glycomics: enabling the analysis of thousands of samples.

Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins, with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since interindividual differences in glycosylation are extensive, large studies are needed to map the structures and to understand the role of glycosylation in human (patho)physiology. Driven by these challenges, methods have emerged, which can tackle the complexity of glycosylation in thousands of samples, also known as high-throughput (HT) glycomics. For facile dissemination and implementation of HT glycomics technology, the sample preparation, analysis, as well as data mining, need to be stable over a long period of time (months/years), amenable to automation, and available to non-specialized laboratories. Current HT glycomics methods mainly focus on protein N-glycosylation and allow to extensively characterize this subset of the human glycome in large numbers of various biological samples. The ultimate goal in HT glycomics is to gain better knowledge and understanding of the complete human glycome using methods that are easy to adapt and implement in (basic) biomedical research. Aiming to promote wider use and development of HT glycomics, here, we present currently available, emerging, and prospective methods and some of their applications, revealing a largely unexplored molecular layer of the complexity of life.

Distinct Longitudinal Changes in Immunoglobulin G N-Glycosylation Associate with Therapy Response in Chronic Inflammatory Diseases.

Immunosuppressants and biologicals are widely used therapeutics for various chronic inflammatory diseases (CID). To gain more detailed insight into their downstream effects, we examined their impact on serum immunoglobulin G (IgG) glycosylation. We analyzed IgG subclass-specific fragment crystallizable (Fc) N-glycosylation in patients suffering from various CID using the LC-MS approach. Firstly, we compared IgG Fc N-glycosylation between 128 CID patients and 204 healthy controls. Our results replicated previously observed CID-related decrease in IgG Fc galactosylation (adjusted p-value range 1.70 × 10-2-5.95 × 10-22) and sialylation (adjusted p-value range 1.85 × 10-2-1.71 × 10-18). Secondly, to assess changes in IgG Fc N-glycosylation associated with therapy and remission status, we compared 139 CID patients receiving either azathioprine, infliximab, or vedolizumab therapy. We observed an increase in IgG Fc galactosylation (adjusted p-value range 1.98 × 10-2-1.30 × 10-15) and sialylation (adjusted p-value range 3.28 × 10-6-4.34 × 10-18) during the treatment. Furthermore, patients who reached remission displayed increased Fc galactosylation levels (p-value range 2.25 × 10-2-5.44 × 10-3) in comparison to patients with active disease. In conclusion, the alterations in IgG Fc glycosylation and the fact these changes are even more pronounced in patients who achieved remission, suggest modulation of IgG inflammatory potential associated with CID therapy.

Heritability of Human Plasma N-Glycome.

The N-glycosylation profile of total human plasma proteins could be a useful biomarker for various pathological states. Reliable high-throughput methods for such profiling have been developed. However, studies of relative importance of genetic and environmental factors in regulating plasma N-glycome are scarce. The aim of our study was to determine the role of genetic factors in phenotypic variation of plasma N-glycan profile through the estimates of its heritability. Thirty-nine total plasma N-glycome traits were analyzed in 2816 individuals from the TwinsUK data set. For the majority of the traits, high heritability estimates (>50%) were obtained pointing at a significant contribution of genetic factors in plasma N-glycome variation, especially for glycans mostly attached to immunoglobulins. We have also found several structures with higher environmental contribution to their variation.

Profiling and genetic control of the murine immunoglobulin G glycome.

Immunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter-individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences; (ii) five genetic loci were found to be associated with murine IgG glycosylation; (iii) variants outside traditional glycosylation site motifs affected glycome variation; (iv) bisecting N-acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.

Plasma N-Glycan Signatures Are Associated With Features of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Biomarkers are needed for early detection of Crohn's disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls). METHODS: We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. RESULTS: Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. CONCLUSIONS: We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients' responses to treatment.

Plasma N-glycome composition associates with chronic low back pain.

BACKGROUND: Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease. METHODS: Plasma samples of 1128 CLBP patients and 760 healthy controls were collected in clinical centers in Italy, Belgium and Croatia and used for N-glycosylation profiling by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) after N-glycans release, fluorescent labeling and clean-up. Observed N-glycosylation profiles have been compared with a cohort of 126 patients with acute inflammation that underwent abdominal surgery. RESULTS: We have found a statistically significant increase in the relative amount of high-branched (tri-antennary and tetra-antennary) N-glycan structures on CLBP patients' plasma glycoproteins compared to healthy controls. Furthermore, relative amounts of disialylated and trisialylated glycan structures were increased, while high-mannose and glycans containing bisecting N-acetylglucosamine decreased in CLBP. CONCLUSIONS: Observed changes in CLBP on the plasma N-glycome level are consistent with N-glycosylation changes usually seen in chronic inflammation. GENERAL SIGNIFICANCE: To our knowledge, this is a first large clinical study on CLBP patients and plasma N-glycome providing a new glycomics perspective on potential disease pathology.

The N-glycosylation of immunoglobulin G as a novel biomarker of Parkinson's disease.

The use of the emerging "omics" technologies for large scale population screening is promising in terms of predictive, preventive and personalized medicine. For Parkinson's disease, it is essential that an accurate diagnosis is obtained and disease progression can be monitored. Immunoglobulin G (IgG) has the ability to exert both anti-inflammatory and pro-inflammatory effects, and the N-glycosylation of the fragment crystallizable portion of IgG is involved in this process. This study aimed to determine whether the IgG glycome could be a candidate biomarker for Parkinson's disease. Ninety-four community-based individuals with Parkinson's disease and a sex-, age- and ethnically-matched cohort of 102 individuals with mixed phenotypes, representative of a "normally" aged Caucasian controls, were investigated. Plasma IgG glycans were analyzed by ultra-performance liquid chromatography. Overall, seven glycan peaks and 11 derived traits had statistically significant differences (P < 8.06 × 10-4) between Parkinson's disease cases and healthy controls. Out of the seven significantly different glycan peaks, four were selected by Akaike's Information Criterion to be included in the logistic regression model, with a sensitivity of 87.2% and a specificity of 92.2%. The study suggested that there may be a reduced capacity for the IgG to inhibit Fcγ-RIIIa binding, which would allow an increased ability for the IgG to cause antibody-dependent cell cytotoxicity and a possible state of low-grade inflammation in individuals with Parkinson's disease.

Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.

Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. Materials and methods: The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. Results: The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. Conclusions: The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.