Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Mutations in HNF1A result in marked alterations of plasma glycan profile.

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥ 0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

Robustness testing of the high throughput HPLC-based analysis of plasma N-glycans.

BACKGROUND: Plasma glycan analysis using high throughput HPLC-based 96 well platform became a standard procedure for analyzing a large pool of samples for studies comprising thousands of observed individuals. An analytical method which is used to obtain such a huge amount of data should be well characterized and all potentially critical steps should be known. METHODS: Robustness of the high throughput method was tested by Plackett Burman two level, 11-factor, 12 experiment screening design. It provides valuable information about the few most important factors on which further optimization should be focused. According to a long-term laboratory experience, eleven potentially critical factors were chosen for initial screening. Response variable was calculated as coefficient of variance between area % of each peak in each reaction and the area % obtained after performing the procedure according to the laboratory standard operating procedure. RESULTS: Six out of 16, by HPLC separated, glycan groups revealed significant changes according to changes in factor levels. As expected due to their structural and chemical differences, glycan groups did not display uniform response to 11 factors, but effect estimates for six significant glycan groups showed the same direction regarding high and low factor levels. GENERAL SIGNIFICANCE: Screening experiment provided quality data which resolved the questions about optimal conditions and robustness of the high throughput glycan analysis. Fraction factorial design used in this study enabled us to test a great deal of critical steps in time, labor and money saving manner. This article is part of a Special Issue entitled Glycoproteomics.

Polymorphisms in B3GAT1, SLC9A9 and MGAT5 are associated with variation within the human plasma N-glycome of 3533 European adults.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high-throughput high-performance liquid chromatography (HPLC) analysis method, we have reported, in a pilot genome-wide association study of 13 glycan features in 2705 individuals from three European populations, that polymorphisms at three loci (FUT8, FUT6/FUT3 and HNF1A) affect plasma levels of N-glycans. Here, we extended the analysis to 33 directly measured and 13 derived glycosylation traits in 3533 individuals and identified three novel gene association (MGAT5, B3GAT1 and SLC9A9) as well as replicated the previous findings using an additional European cohort. MGAT5 (meta-analysis association P-value = 1.80 × 10(-10) for rs1257220) encodes a glycosyltransferase which is known to synthesize the associated glycans. In contrast, neither B3GAT1 (rs7928758, P = 1.66 × 10(-08)) nor SLC9A9 (rs4839604, P = 3.50 × 10(-13)) had previously been associated functionally with glycosylation of plasma proteins. Given the glucuronyl transferase activity of B3GAT1, we were able to show that glucuronic acid is present on antennae of plasma glycoproteins underlying the corresponding HPLC peak. SLC9A9 encodes a proton pump which affects pH in the endosomal compartment and it was recently reported that changes in Golgi pH can impair protein sialylation, giving a possible mechanism for the observed association.

High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Human plasma glycome in attention-deficit hyperactivity disorder and autism spectrum disorders.

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

Effects of aging, body mass index, plasma lipid profiles, and smoking on human plasma N-glycans.

Protein glycosylation affects nearly all molecular interactions at the cell surface and in the intercellular space. Many of the physiological variations which are part of homeostatic mechanisms influence glycosylation. However, a comprehensive overview of changes in glycosylation caused by aging and common lifestyle parameters is still lacking. After analyzing N-glycans in the plasma of 1914 individuals from the Croatian islands of Vis and Korcula, we performed a comprehensive analysis of the dependence of different glycosylation features (position of fucose, level of galactosylation, sialylation and branching) on aging, smoking, body fat and plasma lipid status. A number of statistically significant associations were observed. Glycosylation changes with aging were especially evident in females, mostly in association with the transition from pre-menopausal to post-menopausal age. Levels of core-fucosylated, non-galactosylated, digalactosylated and disialylated biantennary glycans were shown to be mainly age dependent, but the level of branching and higher levels of galactosylation were found to correlate with lipid status. For the majority of glycans which we analyzed, all examined parameters explained up to 5% of the variance. The only notable exception were non-galactosylated glycans where 20% of the variance was explained mostly by age and blood pressure. In general, only a small fraction of the variability in glycan levels observed in a population was explained by age and other measured parameters, indicating that even in the absence of a genetic template, glycan levels are mostly determined by genetic background and/or specific pathophysiological processes.

Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation.

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

Stability of N-glycan profiles in human plasma.

Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.