N‐glycosylation is a ubiquitous protein modification, and N‐glycosylation profiles are emerging as both biomarkers and functional effectors in various types of diabetes. Genome‐wide association ...studies identified glycosyltransferase genes as candidate causal genes for type 1 and type 2 diabetes. Studies focused on N‐glycosylation changes in type 2 diabetes demonstrated that patients can be distinguished from healthy controls based on N‐glycome composition. In addition, individuals at an increased risk of future disease development could be identified based on N‐glycome profiles. Moreover, accumulating evidence indicates that N‐glycans have a major role in preventing the impairment of glucose‐stimulated insulin secretion by maintaining the glucose transporter in proper orientation, indicating that interindividual variation in protein N‐glycosylation might be a novel risk factor contributing to diabetes development. Defective N‐glycosylation of T cells has been implicated in type 1 diabetes pathogenesis. Furthermore, studies of N‐glycan alterations have successfully been used to identify individuals with rare types of diabetes (such as the HNF1A‐MODY), and also to evaluate functional significance of novel diabetes‐associated mutations. In conclusion, both N‐glycans and glycosyltransferases emerge as potential therapeutic targets in diabetes.
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.
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•Cost-effective method for high-throughput detailed AGP glycoprofiling is presented.•Site-specific AGP N-glycan profile can be obtained from 5 μl of blood plasma.•AGP N-glycan profile is stable in a healthy individual.•AGP glycoprofile could help identify individuals who are at risk of type 2 diabetes.
For the first time, a cost-effective method for a high-throughput detailed AGP N-glycosylation profiling was developed, which includes site-specific glycosylation information. Using the method, it was demonstrated that AGP N-glycan profile is stable in a healthy individual. Furthermore, using the method on a pilot cohort, it was found that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes. The method presents a new valuable tool for investigation of AGP’s large biomarker potential.
Nearly all membrane and secreted proteins, as well as numerous intracellular proteins are glycosylated. However, contrary to proteins which are defined by their individual genetic templates, glycans ...are encoded in a complex dynamic network of hundreds of genes which participate in the complex biosynthetic pathway of protein glycosylation.
This review summarizes present knowledge about the importance of alternative glycosylation of IgG and other proteins.
Numerous proteins depend on correct glycosylation for proper function. Very good example for this is the alternative glycosylation of IgG whose effector functions can be completely changed by the addition or removal of a single monosaccharide residue from its glycans.
The change in the structure of a protein requires mutations in DNA and subsequent selection in the next generation, while even slight alterations in activity or intracellular localization of one or more biosynthetic enzymes are sufficient for the creation of novel glycan structures, which can then perform new functions. Glycome composition varies significantly between individuals, which makes them slightly or even significantly different in their ability to execute specific molecular pathways with numerous implications for development and progression of various diseases. This article is part of a Special Issue entitled Glycoproteomics.
► Glycans are encoded in a complex dynamic network of hundreds of genes. ► Glycome composition varies significantly between individuals. ► Alternative glycosylation modulates function of numerous proteins.
Inflammatory diseases are accompanied by numerous changes at the site of inflammation as well as many systemic physiological and biochemical changes. In the past two decades more and more attention ...is being paid to changes in glycosylation and in this review we describe some of the changes found on main serum proteins (alpha1-acid glycoprotein, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha2-macroglobulin, C-reactive protein, and others). Molecular background and physiological importance of most of these changes are yet to be discovered, but it is evident that glycosylation plays an important role in the inflammatory response. Maybe the greatest value of these changes currently lays in their potential diagnostic and prognostic usage, either in combination with current diagnostic markers or on their own. However, determining glycan structures is still technically too complex for most clinical laboratories and further efforts have to be made to develop simple analytical tools to study changes in glycosylation.
Rising awareness of the universal importance of protein N-glycosylation governs the development of further advances in N-glycan analysis. Nowadays it is well known that correct glycosylation is ...essential for proper protein function, which emanates from its important role in many physiological processes. Furthermore, glycosylation is involved in pathophysiology of multiple common complex diseases. In the vast majority of cases, N-glycosylation profiles are analyzed from enzymatically released glycans, which can be further derivatized in order to enhance the sensitivity of the analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds −2-aminiobenzamide (2-AB) and procainamide (ProA), and the recently introduced
Rapi
Fluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derivatized, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification (LOQ), repeatability and labeling efficiency for all three labels. For this purpose, we employed in-house prepared IgG and a commercially available IgG as a model glycoprotein. All samples were analyzed in triplicates using different amounts of starting material. We also tested the performance of all three labels in a high-throughput setting on 68 different IgG samples, all in duplicates and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limits of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. Furthermore, the results indicated that labeling efficiency was very similar for all three labels. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.
Plasma protein
-glycan profiling integrates information on enzymatic protein glycosylation, which is a highly controlled ubiquitous posttranslational modification. Here we investigate the ability of ...the plasma
-glycome to predict incidence of type 2 diabetes and cardiovascular diseases (CVDs; i.e., myocardial infarction and stroke).
Based on the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort (
= 27,548), we constructed case-cohorts including a random subsample of 2,500 participants and all physician-verified incident cases of type 2 diabetes (
= 820; median follow-up time 6.5 years) and CVD (
= 508; median follow-up time 8.2 years). Information on the relative abundance of 39
-glycan groups in baseline plasma samples was generated by chromatographic profiling. We selected predictive
-glycans for type 2 diabetes and CVD separately, based on cross-validated machine learning, nonlinear model building, and construction of weighted prediction scores. This workflow for CVD was applied separately in men and women.
The
-glycan-based type 2 diabetes score was strongly predictive for diabetes risk in an internal validation cohort (weighted C-index 0.83, 95% CI 0.78-0.88), and this finding was externally validated in the Finland Cardiovascular Risk Study (FINRISK) cohort.
-glycans were moderately predictive for CVD incidence (weighted C-indices 0.66, 95% CI 0.60-0.72, for men; 0.64, 95% CI 0.55-0.73, for women). Information on the selected
-glycans improved the accuracy of established and clinically applied risk prediction scores for type 2 diabetes and CVD.
Selected
-glycans improve type 2 diabetes and CVD prediction beyond established risk markers. Plasma protein
-glycan profiling may thus be useful for risk stratification in the context of precisely targeted primary prevention of cardiometabolic diseases.
Monitoring human circulating N-glycome could provide valuable insight into an individual's metabolic status. Therefore, we examined if aberrant carbohydrate metabolism in gestational diabetes ...mellitus (GDM) associates with alterations in plasma protein, immunoglobulin G (IgG) and immunoglobulin A (IgA) N-glycosylation.
Plasma protein, IgG and IgA N-glycans were enzymatically released, purified and chromatographically profiled in 48 pregnant women with normal glucose tolerance and 41 pregnant women with GDM, all sampled at 24-28 weeks of gestation. Linear mixed models adjusting for age and multiple testing (FDR<0.05) were used to investigate the associations between glycosylation features, metabolic markers and GDM status.
Fasting insulin exhibited significant associations to numerous glycan traits, including plasma protein galactosylation, sialylation, branching, core fucosylation and bisection, to IgG core fucosylated, bisected (FA2B) and afucosylated disialylated (A2G2S2) glycan and to IgA trisialylated triantennary (A3G3S3) glycan (padj range: 4.37x10-05-4.94x10-02). Insulin resistance markers HOMA2-IR and HOMA2-%B were mostly associated to the same glycan structures as fasting insulin. Both markers showed positive association with high-branched plasma glycans (padj = 1.12x10-02 and 2.03x10-03) and negative association with low-branched plasma glycans (padj = 1.21x10-02 and 2.05x10-03). Additionally, HOMA2-%B index was significantly correlated with glycosylation features describing IgG sialylation. Multiple plasma protein IgG and IgA glycans showed significant associations with total cholesterol and triglyceride levels. None of the tested glycan traits showed a significant difference between GDM and normoglycemic pregnancies.
Markers of glucose homeostasis and lipid metabolism in pregnancy show extensive associations to various N-glycosylation features. However, plasma protein, IgG and IgA N-glycans were not able to differentiate pregnant women with and without GDM, possibly due to numerous physiological changes accompanying pregnancy, which confound the impact of GDM on protein glycosylation.
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.
Poorer glycemic control in type 1 diabetes may alter N-glycosylation patterns on circulating glycoproteins, and these alterations may be linked with diabetic kidney disease (DKD). We investigated ...associations between N-glycans and glycemic control and renal function in type 1 diabetes.
Using serum samples from 818 adults who were considered to have extreme annual loss in estimated glomerular filtration rate (eGFR; i.e., slope) based on retrospective clinical records, from among 6,127 adults in the Scottish Diabetes Research Network Type 1 Bioresource Study, we measured total and IgG-specific N-glycan profiles. This yielded a relative abundance of 39 total (GP) and 24 IgG (IGP) N-glycans. Linear regression models were used to investigate associations between N-glycan structures and HbA
, albumin-to-creatinine ratio (ACR), and eGFR slope. Models were adjusted for age, sex, duration of type 1 diabetes, and total serum IgG.
Higher HbA
was associated with a lower relative abundance of simple biantennary N-glycans and a higher relative abundance of more complex structures with more branching, galactosylation, and sialylation (GP12, 26, 31, 32, and 34, and IGP19 and 23; all
< 3.79 × 10
). Similar patterns were seen for ACR and greater mean annual loss of eGFR, which were also associated with fewer of the simpler N-glycans (all
< 3.79 × 10
).
Higher HbA
in type 1 diabetes is associated with changes in the serum N-glycome that have elsewhere been shown to regulate the epidermal growth factor receptor and transforming growth factor-β pathways that are implicated in DKD. Furthermore, N-glycans are associated with ACR and eGFR slope. These data suggest that the role of altered N-glycans in DKD warrants further investigation.
Chronic obstructive pulmonary disease (COPD) is a complex condition, whose diagnosis requires spirometric assessment. However, considering its heterogeneity, subjects with similar spirometric ...parameters do not necessarily have the same functional status. To overcome this limitation novel biomarkers for COPD have been investigated. Therefore, we aimed to explore the potential value of N-glycans as COPD biomarkers and to examine the individual variation of plasma protein and immunoglobulin G (IgG) glycosylation profiles in subjects with COPD and healthy controls.
Both the total plasma protein and IgG N-glycome have been profiled in the total of 137 patients with COPD and 95 matching controls from Croatia. Replication cohort consisted of 61 subjects with COPD and 148 controls recruited at another Croatian medical centre.
Plasma protein N-glycome in COPD subjects exhibited significant decrease in low branched and conversely, an increase in more complex glycan structures (tetragalactosylated, trisialylated, tetrasialylated and antennary fucosylated glycoforms). We also observed a significant decline in plasma monogalactosylated species, and the same change replicated in IgG glycome. N-glycans also showed value in distinguishing subjects in different COPD GOLD stages, where the relative abundance of more complex glycan structures increased as the disease progressed. Glycans also showed statistically significant associations with the frequency of exacerbations and demonstrated to be affected by smoking, which is the major risk factor for COPD development.
This study showed that complexity of glycans associates with COPD, mirroring also the disease severity. Moreover, changes in N-glycome associate with exacerbation frequency and are affected by smoking. In general, this study provided new insights into plasma protein and IgG N-glycome changes occurring in COPD and pointed out potential novel markers of the disease progression and severity.
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