Post-translational modifications represent a critical layer of protein regulation, yet their genetic control and population variation remain poorly characterized. Here we present a comprehensive analysis integrating population-scale glycoproteomics with genome-wide association study (GWAS) to uncover genetic regulatory networks controlling N-glycosylation of complement component C3. Through LC–MS glycopeptide analysis of 816 Croatian adults, we characterize site-specific N-glycosylation at two C3 sites (N63 and N917), confirming an unusual phenotype consisting of exclusive high-mannose structures rather than complex glycans typical of secreted proteins. GWAS identifies six genetic loci significantly associated with C3 N-glycosylation patterns, including genes encoding proteins involved in maintenance of the protein secretory pathway, proteins involved in the complement pathway or regulation of complement, and regulators of gene expression. Using colocalization analysis, we discover shared causal variants between C3 glycosylation and immune diseases, particularly rheumatoid arthritis and inflammatory bowel disease, suggesting glycosylation as a mechanistic link between genetic variation and disease susceptibility. C3 N-glycoprofiling reveals significant associations with sex, age, and metabolic parameters, indicating integration of genetic and environmental factors. Structural modeling provides mechanistic insights, revealing how protein architecture constrains glycan processing and enables functional glycan-mediated interactions. Our multiomic approach establishes a framework for understanding how genetic variation shapes post-translational modifications at population scale, demonstrating C3 N-glycosylation as both a genetically and environmentally regulated checkpoint in complement activation with implications for precision medicine approaches in immune and metabolic diseases.

BACKGROUND:Nonseminomatous testicular germ cell tumors (NSE) present significant diagnostic challenges, especially for the early detection of serum tumor marker (STM) negative cases. Current diagnostic tools are limited, highlighting the need for innovative approaches. This study investigates a novel multi-analyte approach combining circulating cell-free DNA (cfDNA) and N-glycan profiling in both blood and seminal plasma to improve NSE diagnostics. METHODS:The study included 41 NSE patients and 114 healthy controls. Diagnostic potential of cfDNA parameters (quality and fragmentation), cfDNA methylation (RASSF1A, PRSS21, and LINE-1) and N-glycan alterations in blood plasma and seminal plasma samples was investigated using logistic regression models. Pre- and post-radical orchidectomy longitudinal samples from NSE patients were analyzed to assess surgical treatment response and disease monitoring utility. RESULTS:Blood plasma analysis of combined cfDNA and N-glycan profiling demonstrated high diagnostic precision, with an AUC of 0.96, identifying 85% of STM-negative patients and all pure-form teratomas. Post-operative blood plasma analysis showed that LINE-1 cfDNA methylation levels returned to those of healthy controls. Seminal plasma analysis revealed an increased cfDNA fragmentation index (CFI) and cfDNA methylation changes in LINE-1 and PRSS21, with an AUC of 0.83 and identifying 85% of STM-negative patients. CONCLUSION:The proposed multi-analyte approach significantly improves early diagnostics of NSE, particularly for STM-negative cases and teratomas. LINE-1 cfDNA methylation is a promising biomarker for NSE detection and treatment monitoring. These findings could transform diagnostic strategies and patient management in testicular germ cell tumors, with potential applications in reducing overtreatment and improving outcomes .

Fibrinogen is a major plasma glycoprotein involved in blood coagulation and inflammatory responses. Alterations in its glycosylation have been implicated in various pathological conditions; yet, its site-specific N-glycosylation profile remains largely unexplored in a clinical context. Here, we present a high-throughput LC-MS workflow for site-specific analysis of fibrinogen N-glycosylation using a cost-effective ethanol precipitation enrichment method. The method demonstrated good intra- and interplate repeatability (CV: 5% and 12%, respectively) and was validated through the first assessment of intraindividual temporal stability in healthy individuals, revealing consistent glycosylation patterns within individuals. Application to 181 atrial fibrillation (AF) patients and 52 healthy controls identified three gamma chain glycoforms significantly associated with AF. Most notably, increased levels of the asialylated N4H5, known to enhance fibrin bundle thickness and promote clot formation, suggest a potential mechanism linking glycosylation changes to the prothrombotic state in AF. Furthermore, fibrinogen sialylation showed strong associations with cardiovascular risk factors, including triglycerides, BMI, and glucose levels. Longitudinal analysis of 108 AF patients six months postcatheter ablation showed stability in the AF-associated glycan profile. Our findings establish fibrinogen glycosylation as a potential biomarker for cardiovascular conditions and demonstrate the utility of site-specific glycosylation analysis for clinical applications.

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.

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.

AimChanges 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 methodsComplement 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. ResultsSignificant 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. ConclusionThis 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.

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) ( p discovery =7.65 × 10 −12 , p replication =8.33 × 10 −6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc ( p discovery =2.88 × 10 −10 , p replication =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 ( p discovery =2.43 × 10 −11 , p replication =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

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.

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.

Glycosylation is the most common and complex modification of proteins and as such has a key role in shaping the final properties and functions of glycoproteins. Since glycan synthesis is not template-driven, but is instead strongly affected by environmental factors and pathophysiological processes, the diagnostic potential of glycans is being thoroughly researched. Due to the strong need for simplicity and ease of manipulation, cell lines are widely used as models in biomedicinal research. Detailed characterization of their glycane profile is extremely important for determining the success of the model used in glycome research. This thesis analyzed N-glycans of HepG2 and 1.1B4 cell lines using HILIC-UPLC-FLR-MS/MS method. The results are presented as assigned FLR chromatograms and tables with the structural representation of the glycan peaks. In the HepG2 cell line there were 47 glycane peaks determined, while in the 1.1B4 line there were 46 peaks respectively. The glycan profiles of the cell lines vastly overlapped, with several characteristic structures for each line. Although most structures belonged to a complex type, the most intense peaks contained a high-mannose type of glycans. The obtained glycane profiles differ from those of the plasma precisely due to the large proportion of high-mannose type of N-linked glycans, as well as the presence of the paucimannose structures.

In 1886 pharmacist John Pemberton introduced new medicine called Coca- Cola. Even though today it is not available at the pharmacy shelves, during past 130 years it has become one of the best selling soft carbonated drinks all over the world. The core of its success lays in its natural flavour, obtained mainly from the coca leaves and kola nuts. In this paper a brief history of Coca-Cola is given, accompanied with an overview of herbal drugs used for its preparation Coca (Erythroxylum spp., Erythroxylaceae) and Kola (Cola spp., Sterculiaceae).

Ovaj clanak prikazuje pregled najcesce primjenjivanih biljnih pripravaka u samolijecenju virusnih bradavica. Navedene biljne vrste (Chelidonium maius, Ficus carica, Thuja occidentalis, Allium sativum, Melaleuca alternifolia, Melaleuca quinquenervia, Cinnamomum camphora kemotip cineol, Ricinus communis, Salix alba, Citrus limun, Drosera rotundifolia i Taraxacum officinale) sadrže razlicite kemijske spojeve (flavonoide, fenolne kiseline, etericna ulja, trjeslovine, alkaloide, kumarine, proteoliticke enzime, fenolne heterozide, vitamine, polisaharide itd.) koji doprinose njihovom djelovanju. Mnogi od navedenih biljnih pripravaka se odavno primjenjuju u puckoj medicini, a neki su i klinicki ispitani.

Botanical data, chemical composition and use of the wild asparagus (Asparagus acutifolius L.) are presented in this paper. Wild asparagus is rich in various minerals and vitamins, especially vitamin C and folic acid, as well as many flavonoids, making it suitable for many types of usage. Its fresh sprouts are among the richest sources of glutathione, one of the strongest natural antioxidants, while dried flowers contain high content of flavonoids rutin and quercetin, making wild asparagus beneficial to blood vessel protection. It is commonly used among pregnant women due to its positive effect on lactation and the important role of folic acid in the proper fetal development. Furthermore, wild asparagus is regarded as a strong diuretic, making it appropriate for toxin elimination and detoxification.

This article represents an overview of the most common used medicinal plant preparations in the self-treatment of viral warts. These plants (Chelidonium maius, Ficus carica, Thuja occidentalis, Allium sativum, Melaleuca alternifolia, Melaleuca quinquenervia, Cinnamomum camphora chemotype cineol, Ricinus communis, Salix alba, Citrus limon, Drosera rotundifolia and Taraxacum officinale) contain various chemical compounds (flavonoids, phenolic acids, essential oils, tannins, alkaloids, coumarins, proteolytic enzymes, phenolic heterosides, vitamins, polysaccharides etc.) that contribute to their activity. Many of these herbs have a traditional medicinal use and some have even been tested in clinical trials. Some plants are shown to have proteolytic enzymes that soften the wart tissue, while others have antiviral substances.