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.

Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis.

Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.

Human complement component C3 N-glycome changes in type 1 diabetes complications.

Aim: Changes in N-glycosylation have been described in numerous diseases and are being considered as biomarkers of ongoing pathological condition. Previous studies demonstrated the interrelation of N-glycosylation and type 1 diabetes (T1D), particularly linking serum N-glycan changes with complications accompanying the disease. Moreover, the role of complement component C3 in diabetic nephropathy and retinopathy has been implicated, and C3 N-glycome was found to be altered in young T1D patients. Therefore, we investigated associations between C3 N-glycan profiles and albuminuria and retinopathy accompanying T1D, as well as glycosylation connection with other known T1D complication risk factors. Research design and methods: Complement component C3 N-glycosylation profiles have been analyzed from 189 serum samples of T1D patients (median age 46) recruited at a Croatian hospital centre. Using our recently developed high-throughput method, relative abundances of all six of the C3 glycopeptides have been determined. Assessment of C3 N-glycome interconnection with T1D complications, hypertension, smoking status, estimated glomerular filtration rate (eGFR), glycaemic control and duration of the disease was done using linear modelling. Results: Significant changes of C3 N-glycome in severe albuminuria accompanying type 1 diabetes were observed, as well as in T1D subjects with hypertension. All except one of the C3 glycopeptides proved to be associated with measured HbA1c levels. One of the glycoforms was shown to be changed in non-proliferative T1D retinopathy. Smoking and eGFR showed no effect on C3 N-glycome. Furthermore, C3 N-glycosylation profile was shown to be independent of disease duration. Conclusion: This study empowered the role of C3 N-glycosylation in T1D, showing value in distinguishing subjects with different diabetic complications. Being independent of the disease duration, these changes may be associated with the disease onset, making C3 N-glycome a potential novel marker of the disease progression and severity.

Integrated glycomics and genetics analyses reveal a potential role for N-glycosylation of plasma proteins and IgGs, as well as the complement system, in the development of type 1 diabetes.

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.

In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis.

Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan's lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separation or detection, and to enhance the sensitivity of the analysis. Released glycan's are usually derivatized using a fluorescent tag, which enables their optical detection in LC profiling. Some fluorescent labels can also promote ionization efficiency, thus facilitating MS detection. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycan's and the need to quantify these contributions to improve glycan analysis methods. In this paper we focused on negative MS mode as these methods are more informative than methods involving positive MS mode, allowing for a less ambiguous elucidation of detailed glycan structures. Additionally, traditional glycan labels in negative mode MS usually result with diminished sensitivity compared to positive mode, thus making selection of appropriate label even more important for successful high-throughput analysis. Therefore, eleven fluorescent labels of different chemo-physical properties were chosen to study the influence of label hydrophobicity and presence of a negative charge on glycan ionization in negative MS mode. N-glycans released from IgG sample were labeled with one of the eleven labels, purified with HILIC-SPE and analyzed with HILIC-UPLC-FLR-MS. To make evaluation of studied labels performance more objective, analysis was performed in two laboratories and at two mobile phase pH (4.4 and 7.4). Although there was a notable trend of more hydrophobic labels having bigger signal intensities in one laboratory, we observed no such trend in the other laboratory. The results show that MS parameters and intrinsic configuration of the spectrometer have even bigger effect on the final ESI response of the labeled-glycan ionization in negative MS mode that the labels themselves. With this in mind, further research and development of fluorophores that will be suitable for high-throughput glycan analysis in the negative MS mode are proposed.

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.