Level Of Glycosylation Research Articles

N-glycan profiles of acute myocardial infarction patients reveal potential biomarkers for diagnosis, severity assessment and treatment monitoring.

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity worldwide. Diagnostic challenges remain in this highly time-sensitive condition. Using capillary electrophoresis-laser-induced fluorescence, we analyzed the blood plasma N-glycan profile in a cohort study comprising 103 patients with AMI and 69 controls. Subsequently, the data generated was subjected to classification modeling to identify potential AMI biomarkers. An area under the Receiving Operating Characteristic curve (AUCROC) of 0.81 was obtained when discriminating AMI vs. non-MI patients. We postulate that the glycan profile involves a switch from a pro- to an anti-inflammatory state in the AMI pathophysiology. This was supported by significantly decreased levels in galactosylation, alongside increased levels in sialylation, afucosylation and GlcNAc bisection levels in the blood plasma of AMI patients. By substantiating the glycomics analysis with immunoglobulin G (IgG) protein measurements, robustness of the glycan-based classifiers was demonstrated. Changes in AMI-related IgG activities were also confirmed to be associated with alterations at the glycosylation level. Additionally, a glycan-biomarker panel derived from glycan features and current clinical biomarkers performed remarkably (AUCROC=0.90, sensitivity=0.579 at 5% false positive rate) when discriminating between patients with ST-segment elevation MI (n=84) and non-ST-segment elevation MI (n=19). Moreover, by applying the model trained using glycomics information, AMI and controls can still be discriminated at 1 and 6months after baseline. Thus, glycomics biomarkers could potentially serve as a valuable complementary test to current diagnostic biomarkers. Additional research on their utility and associated biomechanisms via a large-scale study is recommended.

Use of Psychoeducation for Psychotic Disorder Patients Treated With Modern, Long-Acting, Injected Antipsychotics.

Introduction: There is an increased risk of adverse metabolic effects of some modern antipsychotic drugs, and concern that long-acting, injected preparations of them may increase such risk. We now report on clinical and metabolic outcomes in patient-subjects diagnosed with affective and non-affective psychotic disorders following exposure to psychoeducation on metabolic risks of modern antipsychotics followed by treatment with long-acting atypical injected antipsychotics over 6-months.Materials and Methods: 85 psychotic disorder outpatients (42 affective [AP]; 43 non-affective [NAP]) at the University of Foggia were treated with long-acting, injected, second-generation antipsychotics in association with a set of psychoeducational sessions concerning general health and potential effects of antipsychotic drug treatments. They were evaluated at baseline and six months.Results: Initially, NAP subjects reported higher ratings of positive and negative symptoms than AP subjects, were more likely to receive risperidone or paliperidone, with higher CPZ-eq doses of antipsychotics (294.0 ± 77.8 vs. 229.3 ± 95.8 mg/day), and shorter QTc electrocardiographic recovery intervals. During the 6-month follow-up, ratings of treatment-adherence improved through overall (+8.75%), and symptom-ratings decreased (−7.57%) as did Body-Mass Index (−2.40%; all p ≤ 0.001). Moreover, serum levels of fasting glucose, hemoglobin glycosylation, cholesterol and prolactin concentrations all decreased, with little difference between subjects with AP vs. NAP.Discussion and Conclusions: A psychoeducational program was associated with consistent improvement in psychotic symptoms and several metabolic and physiological measures, as well as with treatment-adherence during six months of treatment with long-acting, injected, second-generation antipsychotics, in association with both affective and non-affective psychotic disorders.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei.

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC’s diversity and multiple immune activities.

N-glycosylation modification of heat shock protein gp96 affects its immunological function

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Abstract 10777: Circulating and Adipose Tissue Levels of CD147 Are Linked to Vascular Dysfunction in Obese Diabetic Adults

Background: Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) is a transmembrane glycoprotein that stimulates the production of matrix metalloproteinases (MMPs). These MMPs mediate vascular remodeling and are considered biomarkers for cardiovascular risk. The activity of CD147 depends mainly on its glycosylation, which varies significantly among different conditions. However, it is unclear if CD147 glycosylation or its role in MMP upregulation is influenced by the diabetic milieu characterized by high glucose levels and abundant glycation end products (AGEs). In this study, we sought to investigate the differential expression and glycosylation levels of CD147 in obese, diabetic (OB-T2D) individuals compared to non-OB controls. Methods: Blood and adipose tissue (AT) samples were obtained from OB-T2D individuals and controls (n=40, each). DEXA scanning was used for body composition analyses. Flow-induced dilation (FID) was assessed in vivo in the brachial artery and ex vivo in AT-isolated arterioles. Other cardiometabolic risk factors related to glucose and lipid metabolism and systemic inflammation were measured. Results: Circulating CD147 was considerably greater in OB individuals, mirroring the glycosylated CD147 and the N-acetylglucosaminyltransferase, MGAT5 protein levels in the AT. Within the OB-T2D group, AT and circulating CD147 correlated with higher body mass index, total and visceral fat percentage, fasting plasma glucose and insulin, insulin resistance measurement (HOMA-IR), glycosylated hemoglobin, systolic BP, and plasma levels of MMP2/9, AGEs (Carboxymethyl lysine and methyl-glyoxal-lysine dimer), C-reactive protein, interleukin 6, and tumor necrosis factor-alpha. AT-arteriolar FID was significantly lower in the OB-T2D individuals across all pressure gradients, while carotid pulse wave velocity (PWV) was higher, indicating arterial stiffness. CD147 correlated negatively with brachial artery and AT-arteriolar FID and flow-induced nitric oxide production and positively with PWV. Conclusion: Our findings imply that vascular dysfunction in OB-T2D patients is linked to higher levels of CD147 and its glycosylated fraction, which in turn is associated with other cardiometabolic risk factors.

Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein.

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients.Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction.In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. DisclosuresBianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

Elevated Fab glycosylation of anti-hinge antibodies

Objective Rheumatoid arthritis (RA) is characterized by systemic inflammation and the presence of anti-citrullinated protein antibodies (ACPAs), which contain remarkably high levels of Fab glycosylation. Anti-hinge antibodies (AHAs) recognize immunoglobulin G (IgG) hinge neoepitopes exposed following cleavage by inflammation-associated proteases, and are also frequently observed in RA, and at higher levels compared to healthy controls (HCs). Here, we investigated AHA specificity and levels of Fab glycosylation as potential immunological markers for RA. Method AHA serum levels, specificity, and Fab glycosylation were determined for the IgG1/4-hinge cleaved by matrix metalloproteinase-3, cathepsin G, pepsin, or IdeS, using enzyme-linked immunosorbent assay and lectin affinity chromatography, in patients with early active RA (n = 69) and HCs (n = 97). Results AHA reactivity was detected for all hinge neoepitopes in both RA patients and HCs. Reactivity against CatG-IgG1-F(ab´)2s and pepsin-IgG4-F(ab´)2s was more prevalent in RA. Moreover, all AHA responses showed increased Fab glycosylation levels in both RA patients and HCs. Conclusions AHA responses are characterized by elevated levels of Fab glycosylation and highly specific neoepitope recognition, not just in RA patients but also in HCs. These results suggest that extensive Fab glycosylation may develop in response to an inflammatory proteolytic microenvironment, but is not restricted to RA.

The Utility of Differential Scanning Calorimetry Curves of Blood Plasma for Diagnosis, Subtype Differentiation and Predicted Survival in Lung Cancer.

Simple SummaryLung cancer (LC) is the most common malignancy and the leading cause of cancer deaths in the world. Limitations of current screening approaches, such as substantial cost, radiation exposure, and high-false positive rates as well as increasing numbers of LC diagnoses in people without known risk factors, indicate the need for the development of new screening strategies. The aim of our study was to evaluate the utility of differential scanning calorimetry (DSC) for LC patients’ diagnosis. We found that DSC curves could be useful in differentiation of LC patients from control individuals and some changes were subtype or/and stage-dependent. Moreover, some DSC curve features correlated with patients’ overall/progression-free survival. Although the utility of the DSC technique still needs to be confirmed in a clinical setting, with further optimization and development of the classification method, this technique could provide an accurate, non-invasive, radiation-free strategy for LC screening and diagnosis.Early detection of lung cancer (LC) significantly increases the likelihood of successful treatment and improves LC survival rates. Currently, screening (mainly low-dose CT scans) is recommended for individuals at high risk. However, the recent increase in the number of LC cases unrelated to the well-known risk factors, and the high false-positive rate of low-dose CT, indicate a need to develop new, non-invasive methods for LC detection. Therefore, we evaluated the use of differential scanning calorimetry (DSC) for LC patients’ diagnosis and predicted survival. Additionally, by applying mass spectrometry, we investigated whether changes in O- and N-glycosylation of plasma proteins could be an underlying mechanism responsible for observed differences in DSC curves of LC and control subjects. Our results indicate selected DSC curve features could be useful for differentiation of LC patients from controls with some capable of distinction between subtypes and stages of LC. DSC curve features also correlate with LC patients’ overall/progression free survival. Moreover, the development of classification models combining patients’ DSC curves with selected plasma protein glycosylation levels that changed in the presence of LC could improve the sensitivity and specificity of the detection of LC. With further optimization and development of the classification method, DSC could provide an accurate, non-invasive, radiation-free strategy for LC screening and diagnosis.

OGT upregulates myogenic IL-6 by mediating O-GlcNAcylation of p65 in mouse skeletal muscle under cold exposure.

Cold exposure is an unavoidable and severe challenge for people and animals residing in cold regions of the world, and may lead to hypothermia, drastic changes in systemic metabolism, and inhibition of protein synthesis. O-linked-N-acetylglucoseaminylation (O-GlcNAcylation) directly regulates the activity and function of target proteins involved in multiple biological processes by acting as a stress receptor and nutrient sensor. Therefore, our study aimed to examine whether O-GlcNAcylation affected myogenic IL-6 expression, regulation of energy metabolism, and promotion of survival in mouse skeletal muscle under acute cold exposure conditions. Total protein was extracted from C2C12 cells that had been cultured at 32°C for 3, 6, 9, and 12 h. Western blot analysis showed that mild hypothermia enhanced O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) expression. Furthermore, global OGT-dependent glycosylation and interleukin-6 (IL-6) levels peaked 3 h after induction of mild hypothermia. Enhanced activation of the NF-κB pathway was also observed in response to mild hypothermia. Alloxan and Thiamet G were used to reduce and increase global OGT glycosylation levels in C2C12 cells, respectively. Increased O-GlcNAcylation was associated with significant upregulation of IL-6 expression, as well as enhanced activity and nuclear translocation of p65, while decreased O-GlcNAcylation had the opposite effect. In addition, increased O-GlcNAcylation was associated with significantly increased glucose metabolism, and OGT-mediated O-GlcNAcylation of p65. We generated skeletal muscle-specific OGT knockout mice and exposed them to cold at 4°C for 3 h per day for 1 week. OGT deficiency attenuated the O-GlcNAcylation, activity, and nuclear translocation of p65, resulting in downregulation of IL-6 in mouse skeletal muscle of mice exposed to cold conditions. Taken together, our data suggested that O-GlcNAcylation of p65 enhanced p65 activity and nuclear translocation leading to the upregulation of IL-6, which maintained energy homeostasis and promotes cell survival in mouse skeletal muscle during cold exposure.

Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation, and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements, is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series of genetic and process engineering strategies to increase recombinant N-linked glycosylation, mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency.ConclusionsIn this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present.

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein’s surface. Here we used IBV propagated in embryonated hens’ eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Mannose-Binding Lectin and Complement Mediate Follicular Localization and Enhanced Immunogenicity of Diverse Protein Nanoparticle Immunogens

Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigated effects of MBL and complement on NP forms of HIV and other clinically-relevant antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increased antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediated follicular trafficking and enhanced GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determined that mannose patches at a minimal density of 2.1x10-3 mannose patches/nm2 were required to trigger follicular targeting, which increased with increasing glycan density up to at least ~8.2×10-3 mannose patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

Longitudinal Changes of Serum N-Glycan Levels for Earlier Detection of Pancreatic Cancer in High-Risk Individuals

Background: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities is challenging. Alternatively, individuals with lesions progressing to malignancy can be recognized by molecular biomarkers. We aimed to identify changes in serum protein glycosylation levels over time to earlier detect PDAC in high-risk individuals undergoing surveillance. Methods: Individuals with a hereditary predisposition to develop PDAC were followed in two surveillance programs and we included those of which at least two consecutive serum samples were available. Mass spectrometry analysis was performed to determine total N-glycome for each consecutive sample. Potentially discriminating N-glycans were selected based on our previous cross-sectional analysis (PDAC cases versus controls) and relative abundances were calculated for each glycosylation feature. Findings: 165 individuals (“FPC-cohort” N=119; Leiden cohort N=46) were included. In total, 97 (59%) individuals had a genetic predisposition (77 CDKN2A, 15 BRCA1/2, 5 STK11) and 68 (41%) a family history of PDAC without a known genetic predisposition (estimated >10-fold increased risk of developing PDAC). From each individual, a median number of 3 serum samples (IQR 3) was collected.Ten individuals (6%) developed PDAC during 35 months of follow-up. Upon comparison of these patients with all other individuals, several glycosylation characteristics were increased, namely fucosylation, tri- and tetra-antennary structures, and specific sialic linkage types. Other glycosylation characteristics decreased, such as complex-type diantennary and bisected glycans. The largest change over time was observed for tri-antennary fucosylated glycans, which were able to differentiate cases from controls with a specificity of 92%, sensitivity of 49% and accuracy of 90%. Interpretation: This study explores the applicability of serum N-glycan longitudinal monitoring for early detection in a pancreas surveillance program. Funding: The current study was supported by ZonNW project number 531002011 and Genootschap Bollenstreek – Bollenstreekfonds, Lisse-Hillegom, The Netherlands. Declaration of Interest: F.P. Vleggaar: Boston Scientific (Consultant) H.F., M.J Bruno: Boston Scientific (Consultant, support for industry and investigator-initiated studies), Cook Medical (Consultant, support for industry and investigator-initiated studies), Pentax Medical (Consultant, support for investigator-initiated studies), Mylan (Support for investigator-initiated studies), ChiRoStim (Support for investigator-initiated studies), all other authors have nothing to declare. Ethical Approval: The institutional ethical review boards of participating centers (2007_024, Amsterdam University Medical Center; MEC-2021-448 EMC; MEC P00.107 LUMC) have approved the study

Abstract 3045: Investigating the influence of glycosylation using cellular models of neuroblastoma

Abstract Background: Neuroblastoma is a common childhood malignancy with approximately 800 new cases diagnosed each year in the United States. Treatment of neuroblastoma directly depends on patient risk categories and includes combinations of surgery, radiation therapy, chemotherapy, stem cell therapy, immunotherapy, and retinoid therapy. Various characteristics of cancer in general and some specific to neuroblastoma cell lines contribute to a difficulty in delivering clinically relevant concentrations of therapeutic agents to neuroblastoma masses. Amplification of the MYCN gene in certain neuroblastoma cell lines correlates with increased cellular surface glycosylation levels as well as a worse prognosis for patients. Overexpression of the MUC1 gene is also associated with increased glycosylation, specifically type-O, in various cancers, but the role of MUC1 in neuroblastoma has not been thoroughly defined. The objective of this study was to evaluate the influence of type-O glycosylation on therapeutic uptake in different neuroblastoma cell lines. Methods: The neuroblastoma cell line SK-N-DZ, a MYCN-amplified line, was compared to SK-N-SH, a non-MYCN-amplified line (negative control) and BT-20, a highly glycosylated breast cancer cell line (positive control). Benzyl-α-GalNAc, an O-glycosylation inhibitor, was introduced to each cell line. To determine glycosylation levels in the respective cell lines, fluorescence assays were performed using CD227 (anti-MUC1) antibody. In vitro cytotoxicity assays were performed to determine maximum non-toxic concentrations of the glycosylation inhibitor (benzyl-α-GalNAc) and antibody (CD227) and to subsequently evaluate the functional role of glycosylation using cellular models of neuroblastoma. Results: The maximum non-toxic concentrations per 10,000 cells for each neuroblastoma cell line was 0.05 mg/ml of benzyl-α-GalNAc, and 6 µl of CD227 stock preparation. Preliminary results from fluorescence detection studies showed an increase in CD227-FITC antibody uptake by the MYCN-amplified neuroblastoma cells (SK-N-DZ) following exposure to the O-glycosylation inhibitor (benzyl-α-GalNAc). Cellular uptake of CD227-FITC by non-MYCN-amplified cell line (SK-N-SH) was less by comparison. Conclusion: Preliminary findings suggest that O-glycosylation is a barrier capable of limiting cellular uptake of agents in MYCN-amplified neuroblastoma cell lines. Ongoing studies are focused on investigating the influence of glycosylation inhibition on cytotoxicity of FDA-approved therapeutic agents. Citation Format: Meghan Cook, Alma Garcia, Katie Konieczny, Meghan Ferguson, Robert Campbell. Investigating the influence of glycosylation using cellular models of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3045.

Changes of Serum IgG Glycosylation Patterns in Primary Biliary Cholangitis Patients.

ObjectivePrimary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease whose diagnosis is based significantly on autoantibody detection. This study aims to investigate the glycosylation profile of serum IgG in PBC patients using high-throughput lectin microarrays technology.MethodLectin microarray containing 56 lectins was used to detect and analyze the expression of serum IgG glycosylation in 99 PBC patients, 70 disease controls (DCs), and 38 healthy controls (HCs). Significant differences in PBC from control groups as well as across PBC subgroups positive for various autoantibodies were explored and verified by lectin blot technique.ResultsLectin microarray detection revealed that compared to DC and HC groups, the specific glycan level of serum IgG sialic acid in PBC patients was increased. For each PBC subgroup, glycan levels of IgG mannose and galactose were decreased in AMA-M2 positive PBC patients compared to the AMA-M2 negative group. IgG N-Acetylgalactosamine (GalNAc) and fucose were decreased in anti-sp100 positive patients. IgG galactose was increased in anti-gp210 positive patients. IgG mannose was decreased in ACA-positive patients. Although the difference in overall sialic acid level was not observed using lectin blot, all results among the above PBC subgroups were consistent with the results of the technique.ConclusionLectin microarray is an effective and reliable technique for analyzing glycan structure. PBC patients positive for different autoantibody exhibits distinct glycan profile. Altered levels of glycosylation may be related to the occurrence and development of the disease, which could provide a direction for new biomarker identification.

Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.Results:As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (KD, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.Conclusions:The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency. DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Wuschko:Octapharma Pharmazeutika Produktionsges.m.b.H.: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

Age-related type I collagen modifications reveal tissue-defining differences between ligament and tendon

Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

The Immune Microenvironment of Hashimoto’s Thyroiditis Regulates the Glycosylation Modification of IgG

Objectives: Elevation of anti-thyroglobulin antibodies that are primarily IgG isotype is a hallmark of Hashimoto’s thyroiditis (HT). As for IgG,it bears two conserved repertoire of N-linked glycans attached to its crystallizable fragment (Fc) at the 297 asparagine residue (Asn297). In our previous study, we found that serum TgAb IgG from HT patients exhibits higher glycosylation levels than those observed from healthy controls. Previous studies confirmed that imbalance of Th1/Th2 and Th17/Treg leading to altered immune microenvironment with elevation of certain cytokines was found in the thyroid tissue of HT, including IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF, APRIL. Thus, the aim of our study was to investigate the influence of the elevated cytokines on the differentiation process of B cells and the glycosylation levels of IgG. Methods: We formed a two-phase culture system in vitro to promote B cells to differentiate to antibody-secreting cells (ASCs). In the process of cell culture, B cells were co-cultured with cytokines as followed: IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF and APRIL. Flow cytometry was performed to identify the percentage of plasmablasts (CD38+CD27high) and plasma cells (CD20-CD138+). ELISA was used to measure the yield of IgG in culture supernatants. The glycosylation levels of secreted IgG under different stimulation conditions were detected by lectin microarray. Results: We found that IL-21, TNF-α and BAFF can significantly promote the differentiation of B cells into ASCs in vitro culture system, and augment the production of IgG to over 4-fold. In addition, cytokines affected the glycosylation modification profile of IgG diversely: 1) IL-21, IL-17A, TNF-α, BAFF significantly increased the glycosylation level of sialic acid of total IgG; 2) IFN-γ significantly increased the level of galactose; 3) IL-21, IL-17A, IFN-γ, BAFF, and APRIL significantly increased the level of mannose; 4) IL-6 significantly decreased the level of sialic acid, galactose and mannose; 5) IL-17A, IFN-γ, TNF-α, BAFF significantly increased the level of GalNAc that was a component of O-Glycan,which only exists in the hinge region of IgG3 subclass. Conclusions: The abnormally elevated cytokines in microenvironment participated in the regulation of B cell terminal differentiation process and glycosylation level of IgG, thereby involving in the pathogenesis of AITD.

Aberrant Immunoglobulin G Glycosylation in Multiple Sclerosis.

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS.