Presentation number: MG 37
N-GLYCANS OF COMPLEMENT COMPONENT C3 ARE A MARKER OF EARLY ONSET TYPE 1 DIABETES MELLITUS
Dinko Šoić1, Toma Keser1, Jerko Štambuk2, Domagoj Kifer1, Flemming Pociot3,4, Gordan Lauc1,2, Mislav Novokmet2, Grant Morahan5, Olga Gornik Kljaić1
1Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3Steno Diabetes Center Copenhagen, Herlev, Denmark, 4Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 5Centre for Diabetes Research, University of Western Australia, Perth, WA, Australia
Previously it was shown that children at the onset of type 1 diabetes have a higher proportion of oligomannose glycans in plasma N-glycome compared to their healthy siblings. The most abundant complement component, glycoprotein C3, contains two N-glycosylation sites occupied by this type of glycans. Also, C3 gene was recently associated with plasma N-glycosylation in type 1 diabetes population. Using our high-throughput workflow for human C3 N-glycosylation analysis, we wanted to see whether C3 is the carrier of aforementioned changes in plasma N-glycome. C3 enrichment from human plasma was done in a 96-well format using Concanavalin A lectin affinity matrix. We studied plasma samples from 61 children/adolescents (1-16 years) newly diagnosed with type 1 diabetes and 84 of their unaffected siblings (4-22 years). A glycan-based discriminative model was built using logistic mixed model elastic net regression. C3 N-glycan profiles were significantly changed in type 1 diabetes children compared to healthy siblings. Type 1 diabetes was associated with an increase in the proportion of unprocessed glycan structures with more mannose units. A model including C3 N-glycans showed notable discriminative power between children with type 1 diabetes and healthy siblings with AUC of 0.879. There are significant changes of C3 N-glycosylation accompanying the onset of type 1 diabetes, indicating that C3 is the carrier of the previously reported high-mannose glycan changes in the total plasma N-glycome. Our C3 glycan-based descriminative model could be valuable in assessment of type 1 diabetes risk in children.
Key words: C3 glycoprotein, glycopeptides, LC-MS, N-glycosylation, Type 1 diabetes onset
Presentation number: MG 38
CELL AGING AFFECTS GLYCOSYLATION OF IMMUNOGLOBULIN G SECRETED FROM MODEL CELL LINE FREESTYLE™ 293-F
Fran Lukšić1, Anika Mijakovac1, Jasminka Krištić2, Vedrana Vičić Bočkor1, Ana Cindrić2, Gordan Lauc2,3, Vlatka Zoldoš1
1University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
Glycosylation of the Fc fragment of immunoglobulin G (IgG) affects the role of this antibody in the adaptive immune system. Aging is associated with changes in IgG glycosylation, primarily galactosylation, which leads to an increased proportion of proinflammatory IgG antibodies in human plasma. FreeStyle ™ 293-F is a model cell line used for production of recombinant IgG and is thus appropriate for studies of IgG glycosylation. In addition, glycome of IgG secreted from FreeStyle ™ 293-F cells is similar to IgG glycome from human plasma. The aim of this study was to investigate if the aging of the model cell line affects IgG glycome and, if so, are these changes similar to the changes observed on IgG from human plasma in older people. Ultra-high performance liquid chromatography revealed that cell aging, monitored during 90 days, indeed led to changes of IgG glycome. The most significant changes were an increase in the proportion of agalactosylated and a decrease in the proportion of fucosylated glycan structures. Proportion of high-mannose glycans also increased significantly, while proportions of sialylated glycans and glycans with bisecting N-acetylglucosamine remained stable during the time course experiment. Next, we investigated if glycan changes resulted from differential expression of glycosyltransferases responsible for individual steps in the IgG glycosylation pathway. This analysis revealed that a decrease of core fucosylation was associated with changes in FUT8 expression, while changes in galactosylation were not a direct consequence of altered B4GALT1 expression. An increase in the proportion of high-mannose glycans was in correlation with reduced MGAT1 and MGAT2 transcriptional activity, and the downregulation of these genes could also explain the decrease of complex IgG glycan structures. Overall, changes of IgG glycome caused by FreeStyle ™ 293-F cell aging were similar to those observed during human aging, most notably changes of IgG galactosylation. Interestingly, not all of the detected changes could be explained by differential expression of the corresponding glycosyltransferases.
Key words: immunoglobulin G, N-glycosylation, IgG glycome, HEK293 FreeStyle, in vitro cell aging
Presentation number: MG 39
MAPPING THE ESTRADIOL SIGNALLING NETWORK THAT REGULATES IMMUNOGLOBULIN G GLYCOSYLATION USING CRISPR/dCas9 BASED FREESTYLE293-F TRANSIENT EXPRESSION SYSTEM
Anika Mijakovac1, Karlo Miškec1, Jasminka Krištić2, Azra Frkatović2, Gordan Lauc2,3, Aleksandar Vojta1, Vlatka Zoldoš1
1University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Genos Ltd., Zagreb, Croatia, 3University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, Zagreb, Croatia
Immunoglobulin G (IgG) is a glycoprotein with a central role in adaptive immunity. Glycosylation of Fc domain defines IgG function and different studies link the change in IgG glycosylation with disease and aging. In healthy women, the most prominent change coincides with perimenopause, and a recent study has revealed that estradiol (E2) is involved in regulation of IgG glycosylation. Analysis of the Signaling Pathway Projects (SPP) web knowledgebase revealed that E2 affects expression of four genes with yet unknown role in IgG glycosylation, three of them being associated in previous genome-wide asLCKsociation studies (GWAS) with galactosylation (RUNX1, RUNX3, SPINK4) and one with sialylation (ELL2). To map downstream pathways linking E2 signaling and IgG glycosylation we utilized our FreeStyleTM293-F transient system, expressing IgG antibodies, for targeted manipulation of candidate loci. This system exploits stably integrated CRISPR dCas9-VPR or dCas9-KRAB expression cassettes for targeted activation or silencing of genes via transient transfection of cells with plasmids carrying specific gRNAs and recombinant IgG. Using this cell system we upregulated and downregulated RUNX1, RUNX3, SPINK4 and ELL2 loci but only upregulation of RUNX3 (Runt-related factor 3) and SPINK4 (Serine Peptidase Inhibitor Kazal Type 4) resulted in alternative IgG glycosylation. Upregulation of RUNX3 resulted in a significant decrease of galactosylated glycans accompanied with an increase of agalactosylated glycans. Upregulation of SPINK4 was also accompanied with a decrease of galactosylated glycans, but the ratio of agalactosylated glycans was unchanged. We hypothesized that RUNX3 acts as B4GALT1 repressor considering its role as a transcription factor that can either activate or suppress transcription. However, following RUNX3 upregulation, expression of B4GALT1 gene remained stable. To further investigate RUNX3 signaling, possibly involved in alternative IgG glycosylation, the total cell transcriptome was analyzed following RUNX3 overexpression. In sum, the results suggest a novel mechanism through which E2 could regulate IgG glycosylation, specifically galactosylation. Moreover, this was the first in vitro functional validation of RUNX3 and SPINK4, the GWAS hits associated with IgG glycosylation.
Key words: IgG glycosylation, estrogen, gene regulation, RUNX3, CRISPR/dCas9
Presentation number: MG 40
EFFECTS OF LOW-CALORIE AND DIFFERENT WEIGHT-MAINTENANCE DIETS ON PLASMA N-GLYCOME COMPOSITION
Helena Deriš1, Petra Tominac1, Frano Vučković1, Nina Briški1, Arne Astrup2, Ellen Blaak3, Gordan Lauc1,4, Ivan Gudelj1,5
1Genos Ltd, Glycoscience Research Laboratory, Zagreb, Croatia, 2Obesity Science Dept, The Novo Nordisk Foundation, Hellerup, Denmark, 3Maastricht University, Department of Human Biology, Maastricht, Netherlands, 4University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, 5University of Rijeka, Department of Biotechnology, Rijeka, Croatia
Over half of all proteins are altered by covalently bound glycans that are crucial for maintaining a normal physiological role of glycoproteins. Aberrant glycosylation is associated with a wide range of diseases, including diabetes, and cardiovascular and immunological disorders. Alterations in sialylation and fucosylation of circulating glycoproteins have recently been shown to be affected by the diet, however, this is the first study that considered plasma proteins’ susceptibility to different dietary regimes for weight control after the initial weight loss. To investigate plasma protein glycosylation alterations due to weight loss and successive weight-maintenance diets, 1850 glycomes from participants of the Diogenes study were analyzed using Ultra-High-Performance Liquid Chromatography (UHPLC). Diogenes study is a large dietary intervention study in which participants were subjected to a low-calorie diet (LCD) followed by one of five weight-maintenance diets (low protein/low glycaemic index, low protein/high glycaemic index, high protein/low glycaemic index, high protein/high glycaemic index and control) in a period of 6 months when the participants were at risk of regaining the formerly lost weight. The most notable alteration of the plasma glycome was 8 weeks after the subjects engaged in the LCD; a significant increase of low-branched glycan structures, accompanied by a decrease of high-branched glycan structures. After the LCD period, there was also a significant rise in fucosylated N-glycan structures, both core and antennary. Moreover, we have observed a significant decrease in trigalactosylated and trisialylated glycans and a concomitant increase in tetragalactosylated and tetrasialylated glycan structures. Interestingly, we did not observe significant changes between different diets, and almost all effects we have observed immediately after the LCD period were annulled during the weight maintenance diets.
Key words: plasma N-glycans, weight loss, low-calorie diet, glycoproteins
Presentation number: MG 41
N-GLYCOSYLATION OF IgG IS NOT INFLUENCED BY THE LEVEL OF RENAL COMPLICATIONS OR RETINOPATHY IN TYPE 1 DIABETES MELLITUS
Branimir Plavša1, Ivona Kološnjaj1, Tomislav Bulum2, Martina Tomić3, Sandra Vučković Rebrina2, Marijana Vučić Lovrenčić4, Lea Duvnjak2, Olga Gornik1
1University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, 2Merkur University Hospital, Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Department of Endocrinology, Zagreb, Croatia, 3Merkur University Hospital, Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Department of Ophthalmology, Zagreb, Croatia, 4Merkur University Hospital, Department of Laboratory Medicine, Zagreb, Croatia
N-glycosylation of immunoglobulin G (IgG) is known to influence the antibody function. Changes in IgG N-glycome associate with many inflammatory conditions and were reported in early stages of type 1 and type 2 diabetes mellitus (DM). IgG N-glycosylation was also studied in relation to disease progression in type 2 DM and was found to associate with diabetes complications. Although type 1 and type 2 DM share complications, these findings may not apply to type 1 DM due to different pathophysiology involved and a longer disease duration prior to complication manifestation. In this study we investigated IgG N-glycosylation of 190 patients (age 18-70, median 46, 81 M, 109 F) with type 1 complications. Complications included: hypertension, albuminuria and retinopathy. Patients were differentiated by the levels of complications but were all in later stages of disease progression. N-linked glycans from IgG were released, fluorescently labelled and analysed using HILIC-UPLC. Twenty-four glycan structures were identified and relatively quantified. In addition, nine derived traits, corresponding to different structural characteristics of glycans were calculated. Data was analysed using multiple linear regression with age and sex as covariates. Glycan traits were log transformed prior to analysis. We observed no statistically significant changes of IgG N-glycosylation with respect to type of complication or severity level. Previously reported changes with respect to age, sex and lifestyle aspects, e.g., smoking were replicated in this study, confirming their influence on the glycome. IgG N-glycosylation changes previously reported as connected to the severity of complications in type 2 DM were not replicated for type 1 DM patients in our study. This can be explained by the fact that pathophysiological changes leading to type 1 DM and influencing the N-glycome occur much earlier in life and are not further influenced by disease progression and development of complications in the adult age. Absence of differences with respect to type of complications may also be due to fact that they share some common pathophysiological processes which prevent their distinction based on IgG N-glycosylation profile.
Key words: glycosylation, N-glycosylation, IgG, diabetes, type 1
Presentation number: MG 42
N-GLYCOSYLATION OF TOTAL SERUM PROTEINS IN ADULTS WITH TYPE I DIABETES MELLITUS
Matej Nemčić1, Marko Tijardović1, Tomislav Bulum2, Martina Tomić3, Branimir Plavša1, Sandra Vučković Rebrina2, Renata Zadro4, Dragan Primorac4,5,6,7,8,9,10,11,12,13, Gordan Lauc1,14, Grant Morahan15,16, Marijana Vučić Lovrenčić17, Lea Duvnjak2, Olga Gornik1
1Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, Zagreb, Croatia, 2Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Merkur University Hospital, Department of Endocrinology, Zagreb, Croatia, 3Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Merkur University Hospital, Department of Ophtalmology, Zagreb, Croatia, 4St. Catherine Hospital, Zagreb, Croatia, 5University of Split, School of Medicine, Split, Croatia, 6University of Split, University Department of Forensic Sciences, Split, Croatia, 7University of Osijek, Faculty of Medicine, Osijek, Croatia, 8University of Osijek, Faculty of Dental Medicine and Health, Osijek, Croatia, 9University of Rijeka, School of Medicine, Rijeka, Croatia , 10Pennsylvania State University, Eberly College of Science, University Park, PA, USA, 11University of New Haven, Henry C. Lee College of Criminal Justice and Forensic Sciences, West Haven, CT, USA, 12Medical School REGIOMED, Coburg, Germany, 13The National Forensic Sciences University, Gandhinagar, Gujarat, India, 14Genos Glycoscience Research Laboratory, Zagreb, Croatia,15The Harry Perkins Institute for Medical Research, Centre for Diabetes Research, Perth, WA, Australia, 16University of Melbourne, Melbourne, VIC, Australia 17Merkur University Hospital, Department of Laboratory Medicine, Zagreb, Croatia
Previous studies have shown that aberrant plasma protein N-glycosylation exists in children newly diagnosed with T1D, but there is little information on the N-glycome in the adult life of patients. N-glycosylation has been associated with poor glycaemic control and nephropathy, but other common diabetic complications remain to be assessed. Therefore, we compared the serum protein N-glycosylation between patients and controls and analysed its changes in regard to complication status, duration of the disease, glycaemic control and biochemical parameters. Total serum protein N-glycans were analysed in 200 patients with T1D (aged 18-70) and 298 healthy controls (aged 18-79). N-glycome was divided in 39 glycan groups and 16 derived traits calculated based on structural similarities. Patients were further divided based on the status of hypertension, albuminuria and retinopathy. Smoking, a major risk factor for the development of diabetic complications, was noted. Patients with T1D exhibited significant differences in N-glycosylation compared to healthy controls (19/39 glycan groups and 4/16 derived traits), e.g., monogalactosylated glycans were lowered, while digalactosylated, monosialylated and antennary fucosylated were elevated. The duration of the disease or biochemical parameters, other than HbA1c, showed no correlation with N-glycome. HbA1c was positively associated with sialylated and highly branched glycans. The effect of smoking was observed through the increase of high-branched, trigalactosylated and trisialylated glycans and the decrease of low-branched, a- and monogalactosylated, asialylated, bisecting and core-fucosylated glycans. Presence of T1D in adults associates with major changes in total serum N-glycome, some of which were previously reported in children. The duration of T1D, as well as the development of diabetic complications, appear not to further affect these changes. Poor glycaemic control and smoking status are strongly reflected in the N-glycome.
Key words: Type 1 diabetes mellitus, N-glycosylation, Serum protein N-glycosylation
Presentation number: MG 43
AUTOMATED IgG N-GLYCAN SAMPLE PREPARATION METHOD FOR HIGH THROUGHPUT ANALYSIS
Borna Rapčan1,2, Jelena Šimunović2, Branimir Plavša1,2, Jerko Štambuk2, Gordan Lauc1,2, Genadij Razdorov2
1Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, 2Genos Ltd, Glycoscience Research Laboratory, Zagreb, Croatia
Role of protein N-glycosylation and especially changes in IgG N-glycosylation pattern has repeatedly been confirmed as crucial in different physiological and pathological processes. Expanding knowledge requires more affordable, reliable, and higher throughput methods. New approaches to glycosylation analysis would allow its optimization and application in the biopharmaceutical industry, epidemiology as well as advance clinical diagnostics that would rely on glycan biomarkers. Though there were successful attempts to develop new automated strategies for glycan research, many laboratories still leverage manual sample preparation protocols that limit their throughput. In order to help address this issue, automated methods for glycosylation analysis should be developed further. This research proposes one possible approach to automation of IgG glycan sample preparation using the Tecan Freedom Evo liquid handling automated platform. To improve throughput and robustness, the manual method was substituted by the automated system that was equipped with a liquid handling arm (LiHa) and a robotic manipulator arm (RoMa), while the vacuum manifold operations were replaced with A200, a positive pressure unit which allows for higher pressure and flow-through. The adapted automated protocol features IgG isolation on a protein G plate, IgG deglycosylation, and glycan 2-AB labeling. This automated protocol allows the samples to be analyzed using ultra-high performance liquid chromatography (UPLC). UPLC analysis showed that peaks’ number, retention time and peak area corresponded with the manual method. Peaks with the largest area showed small variation, while the smaller peaks exhibited larger variation. Method has yielded promising results and with development could encourage additional automation efforts in other protocols.
Key words: liquid handling, lab automation, glycomics, UPLC