Ivo Ugrina

The Association Between Glycosylation of Immunoglobulin G and Hypertension: A Multiple Ethnic Cross-Sectional Study

AbstractMore than half of all known proteins, and almost all membrane and extra-cellular proteins have oligosaccharide structures or glycans attached to them. Defects in glycosylation pathways are directly involved in at least 30 severe human diseases.A multiple center cross-sectional study (China, Croatia, and Scotland) was carried out to investigate the possible association between hypertension and IgG glycosylation. A hydrophilic interaction chromatography of fluorescently labeled glycans was used to analyze N-glycans attached to IgG in plasma samples from a total of 4757 individuals of Chinese Han, Croatian, and Scottish ethnicity.Five glycans (IgG with digalactosylated glycans) significantly differed in participants with prehypertension or hypertension compared to those with normal blood pressure, while additional 17 glycan traits were only significantly differed in participants with hypertension compared to those of normal blood pressure. These glycans were also significant correlated with systolic blood pressure (SBP) or diastolic blood pressure (DBP).The present study demonstrated for the 1st time an association between hypertension and IgG glycome composition. These findings suggest that the individual variation in N-glycosylation of IgG contributes to pathogenesis of hypertension, presumably via its effect on pro- and/or anti-inflammatory pathways.

High-throughput glycomics: optimization of sample preparation.

Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. A wide range of analytical methods is available for glycan analysis of antibodies (mainly IgG), but analysis of plasma glycans is less established due to additional challenges encountered with higher complexity of the sample. Here we describe development and optimization of a high-throughput sample preparation method for hydrophilic interaction liquid chromatography and ultra-performance liquid chromatography analysis of plasma N-glycans. Clean-up of labeled glycans was found to be the largest source of variation, and we tested cellulose, silica gel, Bio-Gel, and hydrophilic GHP filter as stationary phases for solid-phase extraction. All stationary phases were shown to be suitable for purification of labeled glycans, but GHP filter plate in combination with cold 96% acetonitrile had the highest reproducibility and was easiest to work with. The method was further optimized with Plackett—Burman screening design and validated in terms of analysis of major step variation and between-day and between-person variation. The developed method is fast, cost-effective, and easy to perform, and it has very good reproducibility during long period of time, enabling the detection of biological variability of the plasma N-glycome.

Evaluation of Android Malware Detection Based on System Calls

With Android being the most widespread mobile platform, protecting it against malicious applications is essential. Android users typically install applications from large remote repositories, which provides ample opportunities for malicious newcomers. In this paper, we evaluate a few techniques for detecting malicious Android applications on a repository level. The techniques perform automatic classification based on tracking system calls while applications are executed in a sandbox environment. We implemented the techniques in the maline tool, and performed extensive empirical evaluation on a suite of around 12,000 applications. The evaluation considers the size and type of inputs used in analyses. We show that simple and relatively small inputs result in an overall detection accuracy of 93% with a 5% benign application classification error, while results are improved to a 96% detection accuracy with up-sampling. This indicates that system-call based techniques are viable to be used in practice. Finally, we show that even simplistic feature choices are effective, suggesting that more heavyweight approaches should be thoroughly (re)evaluated.

Glycosylation Profile of IgG in Moderate Kidney Dysfunction

Glycans constitute the most abundant and diverse form of the post-translational modifications, and animal studies have suggested the involvement of IgG glycosylation in mechanisms of renal damage. Here, we explored the associations between IgG glycans and renal function in 3274 individuals from the TwinsUK registry. We analyzed the correlation between renal function measured as eGFR and 76 N-glycan traits using linear regressions adjusted for covariates and multiple testing in the larger population. We replicated our results in 31 monozygotic twin pairs discordant for renal function. Results from both analyses were then meta-analyzed. Fourteen glycan traits were associated with renal function in the discovery sample (P<6.5×10(-4)) and remained significant after validation. Those glycan traits belong to three main glycosylation features: galactosylation, sialylation, and level of bisecting N-acetylglucosamine of the IgG glycans. These results show the role of IgG glycosylation in kidney function and provide novel insight into the pathophysiology of CKD and potential diagnostic and therapeutic targets.

Changes in total plasma and serum N-glycome composition and patient-controlled analgesia after major abdominal surgery

Systemic inflammation participates to the complex healing process occurring after major surgery, thus directly affecting the surgical outcome and patient recovery. Total plasma N-glycome might be an indicator of inflammation after major surgery, as well as an anti-inflammatory therapy response marker, since protein glycosylation plays an essential role in the inflammatory cascade. Therefore, we assessed the effects of surgery on the total plasma N-glycome and the association with self-administration of postoperative morphine in two cohorts of patients that underwent major abdominal surgery. We found that plasma N-glycome undergoes significant changes one day after surgery and intensifies one day later, thus indicating a systemic physiological response. In particular, we observed the increase of bisialylated biantennary glycan, A2G2S[3,6]2, 12 hours after surgery, which progressively increased until 48 postoperative hours. Most changes occurred 24 hours after surgery with the decrease of most core-fucosylated biantennary structures, as well as the increase in sialylated tetraantennary and FA3G3S[3,3,3]3 structures. Moreover, we observed a progressive increase of sialylated triantennary and tetraantennary structures two days after surgery, with a concomitant decrease of the structures containing bisecting N-acetylglucosamine along with bi- and trisialylated triantennary glycans. We did not find any statistically significant association between morphine consumption and plasma N-glycome.

Effects of allergic diseases and age on the composition of serum IgG glycome in children

It is speculated that immunoglobulin G (IgG) plays a regulatory role in allergic reactions. The glycans on the Fc region are known to affect IgG effector functions, thereby possibly having a role in IgG modulation of allergic response. This is the first study investigating patients’ IgG glycosylation profile in allergic diseases. Subclass specific IgG glycosylation profile was analyzed in two cohorts of allergen sensitized and non-sensitized 3- to 11-year-old children (conducted at University of Aberdeen, UK and Children’s Hospital Srebrnjak, Zagreb, Croatia) with 893 subjects in total. IgG was isolated from serum/plasma by affinity chromatography on Protein G. IgG tryptic glycopeptides were analyzed by liquid chromatography electrospray ionization mass spectrometry. In the Zagreb cohort IgG glycome composition changed with age across all IgG subclasses. In both cohorts, IgG glycome composition did not differ in allergen sensitized subjects, nor children sensitized to individual allergens, single allergen mean wheal diameter or positive wheal sum values. In the Zagreb study the results were also replicated for high total serum IgE and in children with self-reported manifest allergic disease. In conclusion, our findings demonstrate no association between serum IgG glycome composition and allergic diseases in children.

Comparative Analysis and Validation of Different Steps in Glycomics Studies

Large-scale glycomics studies enable identification of aberrant glycosylation patterns in disease and provide information about functional relevance of individual glycans through genome-wide association studies. Developed high-throughput methodologies have to be sensitive, robust, and stable during long periods of time (few months) to be able to reliably detect small biological variations in glycosylation. Here, we describe a simple, robust, and affordable protocol for immunoglobulin G N-glycan analysis by hydrophilic interaction liquid chromatography-ultra-performance liquid chromatography (HILIC-UPLC), as well as useful strategies for method optimization: Plackett-Burman screening design and analysis of source of variation. We put our focus on experimental design for high-throughput glycan analysis, critical steps in sample preparation procedure for obtaining high-quality data, and propose a validation protocol relevant for high-throughput methods in terms of their long-term robustness and ability to detect biologically relevant changes in glycosylation. The quality of the procedure was assessed by employing appropriate experimental designs and subsequent statistical techniques.

IgG glycosylation and DNA methylation are interconnected with smoking

BACKGROUND Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic. METHODS With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed. RESULTS The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites. CONCLUSION Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures. GENERAL SIGNIFICANCE An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.IgG glycosylation is an important factor in immune function, yet the molecular details of protein glycosylation remain poorly understood. The data-driven approach presented here uses large-scale plasma IgG mass spectrometry measurements to infer new biochemical reactions in the glycosylation pathway.

Prognostic significance of constitutive phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase phosphorylation in acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor clinical course and outcome. There is a constant need for new prognostic factors that could facilitate patient risk stratification. The aim of our research was to determine the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways in leukemic cells, their relation to P-glycoprotein (P-gp) expression/activity and their prognostic significance in adult de novo AML. A total of 118 patients with AML were enrolled in the study. In a multivariate Cox regression analysis we found that P-gp activity and Akt phosphorylation were independent poor prognostic factors of overall survival (OS). In contrast, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) represented a favorable prognostic factor of OS and relapse-free survival (RFS). A negative correlation between P-gp activity and p38 phosphorylation level was found, implying a possible role of this MAPK pathway in P-gp regulation. In addition, we found correlation between Akt and p38 phosphorylation levels, indicative of co-activation of two signaling cascades in AML.