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Aims/hypothesis Individual variation in plasma N-glycosylation has mainly been studied in the context of diabetes complications, and its role in type 1 diabetes onset is largely unknown. Our aims were to undertake a detailed characterisation of the plasma and IgG N-glycomes in patients with recent onset type 1 diabetes, and to evaluate their discriminative potential in risk assessment. Methods In the first part of the study, plasma and IgG N-glycans were chromatographically analysed in a study population from the DanDiabKids registry, comprising 1917 children and adolescents (0.6–19.1 years) who were newly diagnosed with type 1 diabetes. A follow-up study compared the results for 188 of these participants with those for their 244 unaffected siblings. Correlation of N-glycan abundance with the levels and number of various autoantibodies (against IA-2, GAD, ZnT8R, ZnT8W), as well as with sex and age at diagnosis, were estimated by using general linear modelling. A disease predictive model was built using logistic mixed-model elastic net regression, and evaluated using a 10-fold cross-validation. Results Our study showed that onset of type 1 diabetes was associated with an increase in the proportion of plasma and IgG high-mannose and bisecting GlcNAc structures, a decrease in monogalactosylation, and an increase in IgG disialylation. ZnT8R autoantibody levels were associated with higher IgG digalactosylated glycan with bisecting GlcNAc. Finally, an increase in the number of autoantibodies (which is a better predictor of progression to overt diabetes than the level of any individual antibody) was accompanied by a decrease in the proportions of some of the highly branched plasma N-glycans. Models including age, sex and N-glycans yielded notable discriminative power between children with type 1 diabetes and their healthy siblings, with AUCs of 0.915 and 0.869 for addition of plasma and IgG N-glycans, respectively. Conclusions/interpretation We defined N-glycan changes accompanying onset of type 1 diabetes, and developed a predictive model based on N-glycan profiles that could have valuable potential in risk assessment. Increasing the power of tests to identify individuals at risk of disease development would be a considerable asset for type 1 diabetes prevention trials. Graphical abstract

N-glycosylation is a strictly regulated enzymatic reaction during which complex oligosaccharides are covalently attached to proteins, thus impacting their function [1]. One of such proteins is immunoglobulin A (IgA), a heavily glycosylated antibody which plays a key role in the immune system. Glycans modulate its various roles, including antigen binding and the antibody effector functions [2]. Despite its significance, N-glycosylation of IgA remains largely underexplored in autoimmune diseases, including type 1 diabetes (T1D). Former studies on IgA in T1D have focused mostly on its serum levels, suggesting a disruption of IgA catabolism [3]. In this study, we analyzed serum IgA N-glycosylation in 86 children at the onset and 63 of their healthy siblings, as well as in 90 adults with T1D and 90 healthy controls using a high-throughput UPLC approach. N-glycome was divided into 30 glycan groups from which 12 derived traits were calculated based on structural similarities. Our study identified IgA N- glycan traits characteristic of T1D and revealed differences in the N-glycosylation patterns of IgA between children at T1D onset and adults with T1D. In children, IgA N-glycoprofile remained mostly unchanged compared to their healthy siblings, with only one oligomannose N-glycan significantly increased. IgA N-glycome in adults exhibited a shift towards complex high-branched, trigalactosylated and trisialylated as well as oligomannosylated glycans, while the most prominent association with T1D was a decrease in total core fucosylation. These findings indicate a transition of IgA N-glycosylation in T1D toward patterns characteristic of total plasma proteins in various inflammatory conditions.

Aims/hypothesis: We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes. Methods: A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183, 546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples. Results: This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3, MGAT5 and ST6GAL1, encoding beta-1, 4-mannosyl-glycoprotein 4-beta-N- acetylglucosaminyltransferase, alpha-1, 6- mannosylglycoprotein 6-beta-N- acetylglucosaminyltransferase and ST6 beta- galactoside alpha-2, 6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3 ; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N- acetylglucosamine (GlcNAc) (pdiscovery=7.65 × 10- 12, preplication=8.33 × 10-6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (pdiscovery=2.88 × 10-10, preplication=3.03 × 10-3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (C3) showed association with the oligomannose plasma protein N-glycan (pdiscovery=2.43 × 10-11, preplication=8.66 × 10-4 for the top associated SNP rs1047286). Conclusions/interpretation: This study identified novel genetic associations driving the distinct N- glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility.

Aims/hypothesis We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes. Methods A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples. Results This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3, MGAT5 and ST6GAL1, encoding beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N-acetylglucosamine (GlcNAc) (pdiscovery=7.65 × 10−12, preplication=8.33 × 10−6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (pdiscovery=2.88 × 10−10, preplication=3.03 × 10−3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (C3) showed association with the oligomannose plasma protein N-glycan (pdiscovery=2.43 × 10−11, preplication=8.66 × 10−4 for the top associated SNP rs1047286). Conclusions/interpretation This study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility. Graphical abstract

Objective Previously we have shown that plasma protein N-glycosylation is changed in children at the onset of type 1 diabetes. In this study, we aim to identify N-glycan changes in adults with T1DM, compare them to those in children, and investigate their associations with disease duration, complications, glycaemic status, and smoking. Methods Serum protein N-glycans from 200 adults with type 1 diabetes and 298 healthy controls were analysed using ultra-high performance liquid chromatography and divided into 39 directly measured glycan groups from which 16 derived traits were calculated. Results Compared to healthy controls, subjects with type 1 diabetes showed differences in 19 glycan groups and a decrease in monogalactosylated, an increase in digalactosylated, monosialylated, and antennary fucosylated derived traits, from which changes in monogalactosylation and seven directly measured traits overlapped with previously reported in children. Changes in four directly measured and two derived traits previously seen in children were not detected in adults. HbA1c was positively associated with sialylated and highly branched structures, whereas N-glycome was not influenced by disease duration or diabetic complications. Conclusions Our results suggest potential N-glycome involvement in different stages of type 1 diabetes, including processes underlying its development, the disease itself, as well as those occurring after disease establishment.

Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N-glycans. The excellent diagnostic performance of this glycan biomarker in blood plasma N-glycans of individuals with HNF1A-MODY has been demonstrated using liquid chromatography methods. Here, we have developed a high-throughput exoglycosidase plate-based assay to measure α1-3,4 fucosylation levels in blood plasma samples. The assay has been optimized and its validity tested using 1000 clinical samples from a cohort of individuals with young-adult onset diabetes including cases with HNF1A-MODY. The α1-3,4 fucosylation levels in blood plasma showed a good differentiating power in identifying cases with damaging HNF1A variants, as demonstrated by receiver operating characteristic curve analysis with the AUC values of 0.87 and 0.95. This study supports future development of a simple diagnostic test to measure this glycan biomarker for application in a clinical setting.

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.

Aims/hypothesisInflammation is important in development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. Until now, the association of IgG N-glycosylation with type 2 diabetes complications has not been extensively investigated. We hypothesized that N-glycosylation of IgG may be related to development of complications of type 2 diabetes.MethodsIn three independent type 2 diabetes cohorts, IgG N-glycosylation was measured by UPLC (DiaGene n=1815, GenodiabMar n=640) and mass spectrometry (DCS n=1266). We investigated the associations of IgG N-glycosylation (fucosylation, galactosylation, sialylation and bisection) with incident and prevalent nephropathy, retinopathy and macrovascular disease using Cox- and logistic regression, followed by meta-analyses. The models were adjusted for age, sex and additionally for clinical risk factors.ResultsIgG galactosylation was negatively associated with prevalent and incident nephropathy after adjustment for clinical risk factors. Sialylation was negatively associated with incident diabetic nephropathy. For retinopathy, similar associations were found for galactosylation in the basic model. For macrovascular complications, negative associations with galactosylation and sialylation were confined to the cross-sectional analyses.ConclusionsWe showed that IgG N-glycosylation traits are associated with higher prevalence and future development of nephropathy, after correction for clinical risk factors. For other complications, IgG N-glycosylation was associated with their prevalence only, possibly reflecting ongoing vascular inflammation. These findings indicate the predictive potential of IgG N-glycosylation in nephropathy.

Aims: We previously demonstrated that antennary fucosylated N-glycans on plasma proteins are regulated by HNF1A and can identify cases of Maturity-Onset Diabetes of the Young caused by HNF1A variants (HNF1A-MODY). Based on literature data, we further postulated that N-glycans with best diagnostic value mostly originate from alpha-1-acid glycoprotein (AGP). In this study we analyzed fucosylation of AGP in subjects with HNF1A-MODY and other types of diabetes aiming to evaluate its diagnostic potential. Methods: A recently developed LC-MS method for AGP N-glycopeptide analysis was utilized in two independent cohorts: a) 466 subjects with different diabetes subtypes to test the fucosylation differences, b) 98 selected individuals to test the discriminative potential for pathogenic HNF1A variants. Results: Our results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90–0.99). Conclusions: A glycopeptide based diagnostic tool would be beneficial for patient stratification by providing information about the functionality of HNF1A. It could assist the interpretation of DNA sequencing results and be a useful addition to the differential diagnostic process. publishedVersion

Aberrant plasma protein N-glycosylation has been reported in children newly diagnosed with type 1 diabetes (T1D) and N-glycosylation has also been associated with poor glycaemic control and nephropathy in T1D. N-glycosylation features distinct for adult patients with T1D, however, remain to be assessed. In this study we compared the serum protein N-glycosylation between adult patients and corresponding healthy controls as well as examined the association of N- glycans with common diabetic complications, duration of the disease, glycaemic control, and smoking. Serum N-glycans were analysed by UPLC in 201 patients with T1D (18-70) and 298 healthy controls (18-79). N-glycome was divided into 39 glycan groups and 16 derived traits calculated based on structural similarities. Serum N-glycome in T1D subjects exhibited significant decrease in monogalactosylated and an increase in digalactosylated, monosialylated and antennary fucosylated structures, as well as changes in 19 directly measured N- glycan traits. None of the N-glycosylation features, however, correlated with the duration of the disease nor with the development or progression of microvascular complications, similarly to glycaemic parameters, excluding HbA1c. HbA1c was positively associated with sialylated and highly branched glycans, while the effect of smoking was observed through increased complexity of plasma N-glycome.This study shows that T1D associates with a multitude of changes in the N-glycome, reflecting the complexity of the disease. Some of the changes were previously described in newly diagnosed children, but a number of new are found to be present only in the adult patients: e.g., an increase in antennary-fucosylated structures commonly originating from alpha-1-acid glycoprotein, or a decrease in oligomannose structures associated with immunoglobulin M. Interestingly, glycans did not associate with the duration of the disease or with microvascular complications, while conversely poor glycaemic control and smoking were strongly reflected in the N-glycome. In general, this study provides a more comprehensive portrayal of N-glycan changes in adult patients with T1D and highlights changes in certain plasma proteins, but nevertheless warrants further research on N-glycosylation in T1D.

Diabetes mellitus is a group of metabolic disorders characterized by the presence of hyperglycaemia. Due to its high prevalence and substantial heterogeneity, many studies have been investigating markers that could identify predisposition for the disease development, differentiate between the various subtypes, establish early diagnosis, predict complications or represent novel therapeutic targets. N-glycans, complex oligosaccharide molecules covalently linked to proteins, emerged as potential markers and functional effectors of various diabetes subtypes, appearing to have the capacity to meet these requirements. For instance, it has been shown that N-glycome changes in patients with type 2 diabetes and that N-glycans can even identify individuals with an increased risk for its development. Moreover, genome-wide association studies identified glycosyltransferase genes as candidate causal genes for both type 1 and type 2 diabetes. N-glycans have also been suggested to have a major role in preventing the impairment of glucose-stimulated insulin secretion by modulating cell surface expression of glucose transporters. In this chapter we aimed to describe four major diabetes subtypes: type 1, type 2, gestational and monogenic diabetes, giving an overview of suggested role for N-glycosylation in their development, diagnosis and management.

OBJECTIVES: Glycosylation of immunoglobulin G (IgG) is an important regulator of the immune system and has been implicated in prevalent hypertension.The aim of this study is to investigate whether the IgG glycome begins to change prior to hypertension diagnosis by analysing the IgG glycome composition in a large population-based female cohort with two independent replication samples. METHODS: We included 989 unrelated cases with incident hypertension and 1628 controls from the TwinsUK cohort (mean follow-up time of 6.3 years) with IgG measured at baseline by ultra-performance liquid chromatography and longitudinal BP measurement available. We replicated our findings in 106 individuals from the 10 001 Dalmatians and 729 from KORA S4. Cox regression mixed models were applied to identify changes in glycan traits preincident hypertension, after adjusting for age, mean arterial pressure, BMI, family relatedness and multiple testing (FDR

IntroductionPrediction of type 2 diabetes mellitus (T2DM) and its preceding factors, such as insulin resistance (IR), is of great importance as it may allow delay or prevention of onset of the disease. Plasma protein N-glycome has emerged as a promising predictive biomarker. In a prospective longitudinal study, we included patients with a first diagnosis of impaired glucose metabolism (IR or T2DM) to investigate the N-glycosylation’s predictive value years before diabetes development.Research design and methodsPlasma protein N-glycome was profiled by hydrophilic interaction ultra-performance liquid chromatography in 534 TwinsUK participants free from disease at baseline. This included 89 participants with incident diagnosis of IR or T2DM during the follow-up period (7.14±3.04 years) whose last sample prior to diagnosis was compared using general linear regression with 445 age-matched unrelated controls. Findings were replicated in an independent cohort. Changes in N-glycome have also been presented in connection with time to diagnosis.ResultsEight groups of plasma N-glycans were different between incident IR or T2DM cases and controls (pConclusionsIn addition to previous studies, we showed the diagnostic potential of plasma N-glycome in the prediction of both IR and T2DM development years before the clinical manifestation and indicated the continuous deterioration of N-glycome toward the diagnosis.

Diabetes mellitus is a group of metabolic disorders characterized by the presence of hyperglycaemia. Due to its high prevalence and substantial heterogeneity, many studies have been investigating markers that could identify predisposition for the disease development, differentiate between the various subtypes, establish early diagnosis, predict complications or represent novel therapeutic targets. N-glycans, complex oligosaccharide molecules covalently linked to proteins, emerged as potential markers and functional effectors of various diabetes subtypes, appearing to have the capacity to meet these requirements. For instance, it has been shown that N-glycome changes in patients with type 2 diabetes and that N-glycans can even identify individuals with an increased risk for its development. Moreover, genome-wide association studies identified glycosyltransferase genes as candidate causal genes for both type 1 and type 2 diabetes. N-glycans have also been suggested to have a major role in preventing the impairment of glucose-stimulated insulin secretion by modulating cell surface expression of glucose transporters. In this chapter we aimed to describe four major diabetes subtypes: type 1, type 2, gestational and monogenic diabetes, giving an overview of suggested role for N-glycosylation in their development, diagnosis and management.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels. Supplementary Information The online version contains supplementary material available at 10.1007/s10719-021-09992-w.

Asparagine-linked glycosylation (N-glycosylation) plays a key role in many neurodevelopmental processes, including neural cell adhesion, neurite outgrowth and axon targeting. However, little is known about the dynamics of N-glycosylation during brain development and, in particular, how the N-glycome of the developing neocortex differs from that of the adult. The aim of this study, therefore, was to perform a thorough characterization of N-glycosylation in both the adult and neonatal rat neocortex in order to gain insights into the types of changes occurring in the N-glycome during neurodevelopment. To this end, we used hydrophilic interaction ultraperformance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry to compare the adult neocortical N-glycome with that of 24- and 48-h neonates. We report that the abundance of complex N-glycans is significantly lower in adults compared with neonates. Furthermore, the proportion of charged complex N-glycans is also greatly reduced. This decrease in the abundance of complex N-glycans is offset by a corresponding increase in the proportion of truncated and, to a lesser extent, hybrid N-glycans. Lastly, we report that although the proportion of oligomannose N-glycans remains constant at around 24%, the distribution of high-mannose subtypes shifts from predominantly large subtypes in neonates to smaller subtypes in the adult. In summary, our findings indicate that N-glycan synthesis in the rat neocortex is fundamentally different in neonates compared with adults with a general shift occurring from large, sialylated N-glycans towards smaller, neutral structures as neonates develop into adults, coupled with a parallel shift towards smaller oligomannose structures.

Background Obesity, a major global health problem, is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent posttranslational modification, highly responsive to inflammation and ageing. The prospect of biological age reduction, by changing glycosylation patterns through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort. Methods IgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1680 participants from the TwinsUK cohort. Results Low-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG N-glycome that accompanied progressive weight loss during 1-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG N-glycans (adjusted p value range 7.38 × 10−04–3.94 × 10−02). This IgG N-glycan profile is known to be associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease (adjusted p value 1.79 × 10−02) and an increase in digalactosylation (adjusted p value 5.85 × 10−06). Conclusions Altogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age., Highlights Obesity is associated with inflammation-related agalactosylated and bisected IgG glycoforms. IgG bisected glycoforms decrease after low-calorie diet. IgG galactosylation and sialylation increase after bariatric surgery-induced weight loss. Progressive decrease of BMI is associated with increased IgG galactosylation, implying a reduction of biological age.

BackgroundObesity is a major global health problem, and is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent postranslational modification, highly responsive to numerous pathophysiological conditions and ageing. The prospect of biological age reduction, by reverting glycosylation changes through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort.MethodsIgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1,680 participants from the TwinsUK cohort.ResultsLow-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG glycome that accompanied progressive weight loss during one-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG glycans. In general, this IgG glycan profile is associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease and an increase in digalactosylation.ConclusionsAltogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced biological and immune age.GRAPHICAL ABSTRACTHIGHLIGHTSObesity is associated to inflammation-related agalactosylated and bisected IgG glycoformsIgG galactosylation and sialylation increase after bariatric surgery-induced weight lossProgressive decrease of BMI is associated to increased IgG galactosylation, implying a reduction of biological age

IntroductionInflammatory processes are thought to be involved in kidney function decline in individuals with type 2 diabetes. Glycosylation of immunoglobulin G (IgG) is an important post-translation process affecting the inflammatory potential of IgG. We investigated the prospective relationship between IgG N-glycosylation patterns and kidney function in type 2 diabetes.Research design and methodsIn the DiaGene study, an all-lines-of-care case–control study (n=1886) with mean prospective follow-up of 7.0 years, the association between 58 IgG N-glycan profiles and estimated glomerular filtration rate (eGFR) and albumin-to-creatinine ratio (ACR) per year and during total follow-up was analyzed. Models were adjusted for clinical variables and multiple comparisons.ResultsEleven traits were significantly associated with eGFR change per year. Bisecting GlcNAc in fucosylated and fucosylated disialylated structures and monosialylation of fucosylated digalactosylated structures were associated with a faster decrease of eGFR. Fucosylation of neutral and monogalactosylated structures was associated with less eGFR decline per year. No significant associations between IgG glycans and ACR were found.ConclusionsIn type 2 diabetes, we found IgG N-glycosylation patterns associated with a faster decline of kidney function, reflecting a pro-inflammatory state of IgG. eGFR, but not ACR, was associated with IgG glycans, which suggests these associations may represent renal macroangiopathy rather than microvascular disease.

Glycans influence a variety of aspects of tissue development and pathophysiology, harboring a wealth of biochemical information exploitable for the discovery of new biomarkers. The development of versatile and precise glycoanalytical platforms is crucial to bring to the scientific community reliable tools to unveil the glycan- encoded biochemical information. In this work, a novel platform for brain glycan analysis, named Lysate in-Solution Deglycosylation (LSD), is presented, validated and compared to similar methodologies reported in the literature. Briefly, within the LSD workflow, brain tissue is lysed, proteins are purified via methanol/chloroform extraction, and directly deglycosylated with PNGase F. Released N- glycans are purified using a second round of methanol/chloroform extraction, labelled with fluorescent tags, and cleaned-up prior to LC- FLR/MS analysis. LSD is a quick, sensitive, robust, precise, specific and versatile glycomic workflow, compatible with multiple glyco(proteo)mics analyses of the same sample and eventually applicable to other solid tissues. LSD was devised as a plug-and-play platform for quick glycosylation analysis, and was designed to be compatible with a variety of analytical set-ups (i.e., diverse detection systems), thus enabling the extraction of an additional layer of information from different studies (i.e., proteomics) without the need to collect fresh biological materials. In comparison to analogous N-glycomic workflows, LSD performs reliably on the smallest amount of starting brain tissue and, to the best of the authors' knowledge, is currently the most thoroughly validated neuro-N- glycomic method in the field-related literature, thus representing a significant step forward on the road towards glycan-based biomedical applications.

OBJECTIVE Plasma protein N-glycan profiling integrates information on enzymatic protein glycosylation, which is a highly controlled ubiquitous posttranslational modification. Here we investigate the ability of the plasma N-glycome to predict incidence of type 2 diabetes and cardiovascular diseases (CVDs; i.e., myocardial infarction and stroke). RESEARCH DESIGN AND METHODS Based on the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort (n = 27,548), we constructed case-cohorts including a random subsample of 2,500 participants and all physician-verified incident cases of type 2 diabetes (n = 820; median follow-up time 6.5 years) and CVD (n = 508; median follow-up time 8.2 years). Information on the relative abundance of 39 N-glycan groups in baseline plasma samples was generated by chromatographic profiling. We selected predictive N-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. RESULTS The N-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. N-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 N-glycans improved the accuracy of established and clinically applied risk prediction scores for type 2 diabetes and CVD. CONCLUSIONS Selected N-glycans improve type 2 diabetes and CVD prediction beyond established risk markers. Plasma protein N-glycan profiling may thus be useful for risk stratification in the context of precisely targeted primary prevention of cardiometabolic diseases.

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Plasma proteinBased on the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort (TheSelected

BACKGROUND: Type 2 diabetes results from interplay between genetic and acquired factors. Glycans on proteins reflect genetic, metabolic and environmental factors. However, associations of IgG glycans with type 2 diabetes have not been described. We compared IgG N-glycan patterns in type 2 diabetes with healthy subjects.METHODS: In the DiaGene study, a population-based case-control study, (1886 cases and 854 controls) 58 IgG glycan traits were analyzed. Findings were replicated in the population-based CROATIA-Korcula-CROATIA-Vis-ORCADES studies (162 cases and 3162 controls), and meta-analyzed. AUCs of ROC-curves were calculated using 10-fold cross-validation for clinical characteristics, IgG glycans and their combination.RESULTS: After correction for extensive clinical covariates, 5 IgG glycans and 13 derived traits significantly associated with type 2 diabetes in meta-analysis (after Bonferroni correction). Adding IgG glycans to age and sex increased the AUC from 0.542 to 0.734. Adding them to the extensive model did not substantially improve the AUC. The AUC for IgG glycans alone was 0.729.CONCLUSIONS: Several IgG glycans and traits firmly associate with type 2 diabetes, reflecting a pro-inflammatory and biologically-aged state. IgG glycans showed limited improvement of AUCs. However, IgG glycans showed good prediction alone, indicating they may capture information of combined covariates. The associations found may yield insights in type 2 diabetes pathophysiology.GENERAL SIGNIFICANCE: This work shows that IgG glycomic changes have biomarker potential and may yield important insights into pathophysiology of complex public health diseases, illustrated here for the first time in type 2 diabetes.

Background Numerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N -glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods. Methods The aim of this study was to optimise cloud-point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X-114 and analyse their N -glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries. Results CPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N -glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N -glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material. Conclusions This method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N -glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states. General significance The simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N -glycans released from membrane proteins. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

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., Graphical Abstract, Highlights • 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., In Brief 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.

Background 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. Methods 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s12967-018-1695-0) contains supplementary material, which is available to authorized users.

OBJECTIVE 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. RESEARCH DESIGN AND METHODS 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 HbA1c, albumin-to-creatinine ratio (ACR), and eGFR slope. Models were adjusted for age, sex, duration of type 1 diabetes, and total serum IgG. RESULTS Higher HbA1c 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 P < 3.79 × 10−4). 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 P < 3.79 × 10−4). CONCLUSIONS Higher HbA1c 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.

The authors regret that Nish Chaturvedi's name was spelt incorrectly in the author list. The details given in this correction are correct.

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.

Aims/hypothesisBetter understanding of type 2 diabetes and its prevention is a pressing need. Changes in human plasma N-glycome are associated with many diseases and represent promising diagnostic and prognostic biomarkers. Variations in glucose metabolism directly affect glycosylation through the hexosamine pathway but studies of plasma glycome in type 2 diabetes are scarce. The aim of this study was to determine whether plasma protein N-glycome is changed in individuals who are at greater risk of developing type 2 diabetes.MethodsUsing a chromatographic approach, we analysed N-linked glycans from plasma proteins in two populations comprising individuals with registered hyperglycaemia during critical illness (increased risk for development of type 2 diabetes) and individuals who stayed normoglycaemic during the same condition: AcuteInflammation (59 cases vs 49 controls) and AcuteInflammation Replication (52 cases vs 14 controls) populations. N-glycome was also studied in individuals from FinRisk (37 incident cases of type 2 diabetes collected at baseline vs 37 controls), Orkney Complex Disease Study (ORCADES; 94 individuals with HbA1c > 6.5% [47.5 mmol/mol] vs 658 controls) and Southall and Brent Revisited (SABRE) cohort studies (307 individuals with HbA1c > 6.5% [47.5 mmol/mol] vs 307 controls).ResultsIndividuals with increased risk for diabetes type 2 development (AcuteInflammation and AcuteInflammation Replication populations), incident cases of type 2 diabetes collected at baseline (FinRisk population) and individuals with elevated HbA1c (ORCADES and SABRE populations) all presented with increased branching, galactosylation and sialylation of plasma protein N-glycans and these changes were of similar magnitude.Conclusions/interpretationIncreased complexity of plasma N-glycan structures is associated with higher risk of developing type 2 diabetes and poorer regulation of blood glucose levels. Although further research is needed, this finding could offer a potential new approach for improvement in prevention of diabetes and its complications.KeywordsDiabetes predisposition Hyperglycaemia N-linked glycans Plasma N-glycome Type 2 diabetes