Differences In Glycosylation Research Articles

Integrative Glycoproteomic and Phosphoproteomic Analyses to reveal Vascular Alterations in Response to a Hyperglycemic Microenvironment

Hyperglycemia‐induced vascular complications are preeminent factors for a decreased life expectancy in patients with diabetes. At a molecular level, increased concentrations of blood glucose is known to accelerate endothelial dysfunction through several negative intracellular signaling events. We hypothesize that these changes are not independent of spontaneous cell surface glycosylation. The cytosolic phosphoproteome and cell surface glycoproteome (both N‐and O‐glycoproteome) were isolated from rat microvascular endothelial cells (RMVECs) cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks. This was followed by tandem mass spectrometry (MS/MS) and integrative bioinformatics analysis. Functional assays, namely tube formation, ROS production, and proliferation assays were performed on RMVECs in the presence or absence of HG conditions. The deleterious impact of hyperglycemic conditions on RMVEC function was confirmed by their inability to form robust tubes in vitro (n=3, p<0.05), and a diminished response to acetylcholine ex vivo (n=3, p<0.05). Hyperglycemic states resulted in differential glycosylation of several receptors, ion channels and cell surface antigens that are critical for endothelial functioning. Tandem MS/MS analysis revealed 87 proteins significantly increased in N‐glycosylation (65 unique) and 143 proteins significantly increased in O‐glycosylation (78 unique) in HG treated versus NG RMVECs (p<0.05). Targets included AT1R (N‐glycosylation: Unique HG, p<0.05), JAM‐A (O‐glycosylation: 2.05 fold, p<0.05) and CD59 (N‐glycosylation: 2.02 fold, p<0.05 and O‐glycosylation: Unique HG, p<0.05). Phosphopeptide pathway enrichment analysis revealed a 25% decrease in phosphorylation status, when filtered for scans (≥ 5) and p‐value (<0.05), in HG treated versus NG RMVECs. Pronounced alterations were observed in several components of insulin (INSR: 1.1 fold, p<0.05 and PKCβ: 2.3 fold, p<0.05), angiogenic (S1PR2: unique HG, p<0.05), and apoptotic (BCL2: −3.75 fold, p<0.05) signaling pathways. Furthermore, elimination of glucose moieties by the treating HG RMVECs with glycosidases, resulted in a normalization of several endothelial functional phenotypes. This study identified a multitude of changes in the glycosylation and phosphorylation status of several surface receptors and cytosolic proteins that are integral to vascular homeostasis. Since glycosidases reversed several of the impairments brought about by a hyperglycemic insult on the endothelium, reversion of dysfunctional endothelial cells to a healthy phenotype by negating a build‐up of glucose moieties on cell surface proteins may be a potential new avenue for the treatment of progressive cardiovascular complications in diabetes.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ample glycosylation in membrane and cell envelope proteins may explain the phenotypic diversity and virulence in the Mycobacterium tuberculosis complex

Multiple regulatory mechanisms including post-translational modifications (PTMs) confer complexity to the simpler genomes and proteomes of Mycobacterium tuberculosis (Mtb). PTMs such as glycosylation play a significant role in Mtb adaptive processes. The glycoproteomic patterns of clinical isolates of the Mycobacterium tuberculosis complex (MTBC) representing the lineages 3, 4, 5 and 7 were characterized by mass spectrometry. A total of 2944 glycosylation events were discovered in 1325 proteins. This data set represents the highest number of glycosylated proteins identified in Mtb to date. O-glycosylation constituted 83% of the events identified, while 17% of the sites were N-glycosylated. This is the first report on N-linked protein glycosylation in Mtb and in Gram-positive bacteria. Collectively, the bulk of Mtb glycoproteins are involved in cell envelope biosynthesis, fatty acid and lipid metabolism, two-component systems, and pathogen-host interaction that are either surface exposed or located in the cell wall. Quantitative glycoproteomic analysis revealed that 101 sites on 67 proteins involved in Mtb fitness and survival were differentially glycosylated between the four lineages, among which 64% were cell envelope and membrane proteins. The differential glycosylation pattern may contribute to phenotypic variabilities across Mtb lineages. The study identified several clinically important membrane-associated glycolipoproteins that are relevant for diagnostics as well as for drug and vaccine discovery.

Differential Glycosylation Expression in Injured Rat Spinal Cord Treated with Immunosuppressive Drug Cyclosporin-A.

Glycosylation is ubiquitous throughout the central nervous system and altered following spinal cord injury (SCI). The glial scar that forms following SCI is composed of several chondroitin sulfate proteoglycans, which inhibit axonal regrowth. Cyclosporin-A (CsA), an immunosuppressive therapeutic, has been proposed as a potential treatment after SCI. We investigated CsA treatment in the spinal cord of healthy, contusion injured, and injured CsA-treated rats. Lectin histochemistry using fluorescently labeled lectins, SBA, MAA, SNA-I, and WFA, was performed to identify the terminal carbohydrate residues of glycoconjugates within the spinal cord. SBA staining decreased in gray and white matter following spinal cord injury, whereas staining was increased at the lesion site in CsA-treated animals, indicating an increase in galactose and N-acetylgalactosamine terminal structures. No significant changes in MAA were observed. WFA staining was abundant in gray matter and observed to increase at the lesion site, in agreement with increased expression of chondroitin sulfate proteoglycans. SNA-I-stained blood vessels in all spinal cord regions and dual staining identified a subpopulation of astrocytes in the lesion site, which expressed α-(2,6)-sialic acid. Glycosylation were altered in injured spinal cord treated with CsA, indicating that glycosylation and alteration of particular carbohydrate structures are important factors to consider in the examination of the environment of the spinal cord after injury.

Alterations in the glycome after HDAC inhibition impact oncogenic potential in epigenetically plastic SW13 cells

BackgroundDefects in the type and degree of cellular glycosylation impact oncogenesis on multiple levels. Although the type of glycosylation is determined by protein sequence encoded by the genome, the extent and modifications of glycosylation depends on the activity of biosynthetic enzymes and recent data suggests that the glycome is also subject to epigenetic regulation. This study focuses on the ability of HDAC inhibition to alter glycosylation and to lead to pro-oncogenic alterations in the glycome as assessed by metastatic potential and chemoresistance.MethodsEpigenetically plastic SW13 adrenocortical carcinoma cells were treated with FK228, an HDAC inhibitor with high affinity for HDAC1 and, to a lesser extent, HDAC2. In comparing HDAC inhibitor treated and control cells, differential expression of glycome-related genes were assessed by microarray. Differential glycosylation was then assessed by lectin binding arrays and the ability of cellular proteins to bind to glycans was assessed by glycan binding arrays. Differential sensitivity to paclitaxel, proliferation, and MMP activity were also assessed.ResultsTreatment with FK228 alters expression of enzymes in the biosynthetic pathways for a large number of glycome related genes including enzymes in all major glycosylation pathways and several glycan binding proteins. 84% of these differentially expressed glycome-related genes are linked to cancer, some as prognostic markers and others contributing basic oncogenic functions such as metastasis or chemoresistance. Glycan binding proteins also appear to be differentially expressed as protein extracts from treated and untreated cells show differential binding to glycan arrays. The impact of differential mRNA expression of glycosylation enzymes was documented by differential lectin binding. However, the assessment of changes in the glycome is complicated by the fact that detection of differential glycosylation through lectin binding is dependent on the methods used to prepare samples as protein-rich lysates show different binding than fixed cells in several cases. Paralleling the alterations in the glycome, treatment of SW13 cells with FK228 increases metastatic potential and reduces sensitivity to paclitaxel.ConclusionsThe glycome is substantially altered by HDAC inhibition and these changes may have far-reaching impacts on oncogenesis.

OR32. Differential detection of glycosylation of HLA donor specific antibodies (DSA) using single antigen microbeads

Aim Differential glycosylation of Immunoglobulin G (IgG) is known to affect the functions of IgG by modulating the interactions with its binding proteins, such as C1q and Fc receptors. Our research aim was to develop an assay to analyze glycosylation of donor specific antibodies (DSA) using conventional single antigen microbead technology, and subsequently to analyze glycosylation profiles of kidney transplant patients with DSA and biopsy data for graft function assessment. Methods Conventional Class I and II LABScreen® single antigen beads were pretreated to eliminate glycans on the Class I and II antigens. Patient serum was incubated with the pretreated beads according to the standard assay. Glycans of the DSA bound to the beads were detected with biotinylated lectins known to specifically detect sialic acid, galactose, or fucose residues, along with neutravidin-PE (phycoerithrin). Data acquisition was performed using a LABScan 3D™. Results Pretreated Class I and II single antigen beads maintained DSA binding capabilities comparable to the untreated conventional beads when PAN IgG-PE was used as the detection agent. Using pretreated beads, glycans on the bound DSA were specifically detectable with lectin/neutravidin based reporters with slight modification of conventional LABScreen® single antigen protocol (Figure: Detection of Class I DSA with lectin specific to galactose vs. PAN IgG). Conclusions We have demonstrated a simple and robust assay for detection of glycans on DSA using modifications of conventional LABScreen® single antigen technology. We used lectins as specific glycan probes and neutravidin-PE as a reporter. Further research is in progress to assess the clinical validity of the assay and to assess the predictive value of glycosylation profiles of Class I and II DSA on the outcome of kidney transplant recipients. Download : Download high-res image (109KB) Download : Download full-size image

A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications.

The cell surface of the oral pathogen Tannerella forsythia is heavily glycosylated with a unique, complex decasaccharide that is O-glycosidically linked to the bacterium’s abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of T. forsythia and there is evidence that protein O-glycosylation underpins the bacterium’s pathogenicity. To elucidate the protein O-glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released O-glycans, we show that the gene cluster encodes the species-specific part of the T. forsythia ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the Bacteroidetes phylum, to which T. forsythia is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein O-glycosylation among Tannerella sp., the publicly available genome sequences of six T. forsythia strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated Tannerella isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts T. forsythia’s overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the O-glycan is pivotal to modulating DC effector functions, with the T. forsythia-specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein O-glycosylation is a hallmark of pathogenic T. forsythia strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.

Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells

Site specific glycosylation of immunoglobulin G (IgG) occurs at Asn297 in the Fc region. The heterogeneous ensemble of glycoform occurs due to the degree of terminal galactosylation and sialylation, and these differences in glycosylation affect both the pharmacokinetic behavior and effector functions of the IgG, such as complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this study, the differential glycosylation of IgG was compared and environmental physical and chemical parameters were evaluated in an attempt to promote glycosylation of recombinant antibodies, thereby creating more humanized glycoform antibodies and increasing their in vivo efficacy as therapeutic drugs. It was shown that cells at late stationary growth phase in batch cultures, cells with increased passage number, and the culture conditions of lowered temperature and pH promoted galactosylation and sialylation of antibodies. Galactose, fructose and mannose were found to elicit galactosylation and sialylation when they were used alone as a substitute of glucose. Mannose showed synergistic effects on glycosylation when used with other sugars, such as glucose and galactose. However when fructose was used with other sugars, the degree of galactosylation mechanism appeared to be decreased. These results support understandings of the glycosylation mechanisms in glycoprotein, particularly recombinant antibodies for therapeutics.

Immunoglobulin G glycosylation in aging and diseases

The Immunoglobulin G (IgG) glycome is well known for its heterogeneity and shows a significant degree of variation within populations. IgG glycome composition is influenced both by genes and by environment, making it an excellent biomarker of a person's general health state, i.e. biological age. IgG glycosylation appears to be highly regulated, both during homeostasis and in cases of its disturbance. Changes in IgG glycosylation patterns have been observed in aging and in various diseases. Differential IgG glycosylation is known to modulate IgG effector functions and is involved in disease development and progression, representing both a predisposition and a functional mechanism involved in disease pathology. This makes IgG glycosylation analysis a promising add-on to improve existing disease biomarkers.

Ramifications of secreted mucin MUC5AC in malignant journey: a holistic view

Heavily glycosylated secreted mucin MUC5AC, by the virtue of its cysteine-rich repeats, can form inter- and intramolecular disulfide linkages resulting in complex polymers, which in turn craft the framework of the polymeric mucus gel on epithelial cell surfaces. MUC5AC is a molecule with versatile functional implications including barrier functions to epithelial cells, host-pathogen interaction, immune cell attraction to sites of premalignant or malignant lesions and tumor progression in a context-dependent manner. Differential expression, glycosylation and localization of MUC5AC have been associated with a plethora of benign and malignant pathologies. In this era of robust technologies, overexpression strategies and genetically engineered mouse models, MUC5AC is emerging as a potential diagnostic, prognostic and therapeutic target for various malignancies. Considering the clinical relevance of MUC5AC, this review holistically encompasses its genomic organization, domain structure, glycosylation patterns, regulation, functional and molecular connotation from benign to malignant pathologies. Furthermore, we have here explored the incipient and significant experimental tools that are being developed to study this structurally complex and evolutionary conserved gel-forming mucin.

Differential Glycosylation and Modulation of Camel and Human HSP Isoforms in Response to Thermal and Hypoxic Stresses.

Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances.

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer.

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65-21-92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (n = 44 cases and n = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development.

Structural and functional studies of differentially O‐glycosylated analogs of a thrombin inhibitory peptide – variegin

Variegin is a 32-amino acid long thrombin inhibitory peptide isolated from the salivary gland extract of tropical bont tick Amblyomma variegatum. It was identified to be O-glycosylated on its Thr-14 side chain, and this glycosylated form was 14-fold more potent than that of its non-glycosylated form. However, as the identity of this glycosylation remained elusive, the mechanistic details underlying its functional impact are not yet known. In this report, we synthesized four different O-glycosylated analogs of variegin bearing physiologically relevant sugars on its Thr-14. Functional characterization of these analogs by enzyme inhibitory kinetics and surface plasmon resonance methods showed that all the synthesized glycopeptides are strong thrombin inhibitors. Structural studies by macromolecular docking identified that the sugar moiety of these peptides can potentially mediate favorable interactions with amino acids at the base of thrombin's autolysis loop. This report, for the first time, describes the impact of differential glycosylation on the function of a thrombin inhibitory peptide and tries to provide structural insights into the relevance of peptide glycosylation in thrombin inhibition. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity?

A loss of humoral tolerance is a hallmark of many autoimmune diseases and the detection of self-reactive antibodies (autoantibodies) of the immunoglobulin G (IgG) isotype is widely used as a biomarker and diagnostic tool. However, autoantibodies might also be present in individuals without autoimmune disease, thus limiting their usefulness as a sole indicator of disease development. Moreover, while clear evidence exists of the pathogenic effects of autoantibodies in mouse model systems, the contribution of autoantibodies to the pathology of many autoimmune diseases has yet to be established. In this Review, the authors discuss the changes in total serum IgG and autoantibody glycosylation that occur during autoimmune disease and how these changes might help to predict disease development in the future. Furthermore, current knowledge of the signals regulating antibody glycosylation and how individual antibody glycoforms could be used to optimize current treatment approaches will be discussed.

Abstract 4588: Novel anti-Trop-2 monoclonal antibodies with unique binding specificities show therapeutic synergy against most human cancers

Abstract Trop-2 is an epithelial transmembrane glycoprotein that transduces a calcium signal and activates a growth-signaling network that converges on AKT. Trop-2 is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation, and in its mature, glycosylated/functionally-competent form associates with worse prognosis. Trop-2 extracellular domain contains an N-terminal cysteine-rich globular region followed by a cysteine-less region as a connecting “stem” to the transmembrane domain. Trop-2 molecules engage in homophylic interactions between adjacent cells and establish multimeric complexes with tight junction proteins, which may hinder accessibility by therapeutic antibodies. Up to now, Trop-2-targeted approaches have employed anti-Trop-2 monoclonal antibodies (mAb) which essentially recognize a single immunodominant epitope poised between the globular and stem regions. Such mAb have limited or no therapeutic efficacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specificity towards the globular versus stem regions. Hybridoma diversity was maximized by immunization with soluble human Trop-2 extracellular region produced in different transformed mammalian cell lines (human 293 and murine L) and in insect cells/baculovirus expressing system. These were expected to provide native folding of Trop-2 together with a broad spectrum of differential glycosylation. Trop-2-binding hybridomas were further selected by multiple rounds of flow cytometry analysis using live 293 cells expressing different Trop-2 extracellular portions. Two classes of mAb were identified, that bound the stem versus the globular region. These mAb efficiently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. In vivo the naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of distinct tumors, including colon, ovary and prostate cancers. Notably, they showed differential efficacy for established tumors versus isolated-cell models of metastatic dissemination, consistent with our strategy of maximizing differential accessibility of Trop-2 according to growth mode. Most remarkably, we demonstrated in vivo synergy of these anti-Trop-2 mAb, paving the way for game-changing anti-cancer mAb therapy. The differential efficacy of the OX-G64 and OX-S55 anti-Trop2 mAb against different tumor histotypes and growth stages further allows to exploit their cancer-killing potential in pathological stage-tailored therapeutic approaches. Citation Format: Emanuela Guerra, Marco Trerotola, Valeria Relli, Chiara Pedicone, Antonella D' Amore, Francesca Dini, Silvia Fratarcangeli, Saverio Alberti. Novel anti-Trop-2 monoclonal antibodies with unique binding specificities show therapeutic synergy against most human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2017-4588

Hypoxia induced HIF1a-mediated O-GalNAc glycosylation of cytosolic O-GlcNAcylated proteins to regulate signaling pathways in pancreatic cancer.

e15739 Background: Hypoxia induced reprogramming of cell energy metabolism and changes in glycosylation are hallmarks of cancer promoting the induction of an invasive and treatment-resistant phenotype, triggering metastases at an early stage of tumor development. We examined the impact of hypoxia on O-GalNAc glycosylation in human HEK293, PDAC cell lines and clinical specimens. Methods: We profiled the expression of 88 glycosylation related genes by qPCR in HEK293 cells subjected to hypoxia either induced by 1% O2 or 200 mm CoCl2 identifying key O-GalNAc glycosyltransferases downregulated. Functional assays and glycoprotein analysis displayed a pronounced rate of O-GalNAc modified cytosolic proteins derived from hypoxia treated cells and PDAC specimens. Glycosidase assays could validate specificity of detection method used. Aberrant glycotype could be induced by HIF pathway activator ML 228 and inhibited using Echinomycin. PTK and STK analysis of cell lysates displayed correlation between phosphorylation and O-glycosylation in hypoxic samples. Results: Mechanistically we could show, that hypoxia induced decreased levels of C1GALT1C1 results in reduced T-Synthase activity with subsequent expression of truncated O-glycans (Tn antigen). Differential O-GalNAc glycosylation is inducible using HIF pathway activator ML228 under normoxia and the effect is reversed using 5 µM Echinomycin under hypoxia underscoring the role of HIF1a regulated transcription. Interestingly, the pattern of Tn antigen modified proteins derived from hypoxic samples differs significantly from engineered COSMC deficient cells, displaying O-GalNAc moieties in addition to O-GlcNAc in cytosolic protein fractions. Conclusions: Our findings point to a novel crosstalk of O-GalNAc and O-GlcNAcylation under hypoxia extending the knowledge base of differential O-GalNAc glycosylation in pancreatic cancer.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin, C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids.

Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine proteases. KLK6 is synthesized as a preproenzyme, mainly in tissues of the central nervous system (CNS), and secreted as an inactive precursor. Serum KLK6 is a biomarker of unfavorable prognosis for ovarian cancer, but its sensitivity for early detection is relatively low. Differential glycosylation of KLK6 has been identified in ascites fluid obtained from ovarian cancer patients, suggesting the presence of unique KLK6 isoforms in biological samples. In the present study, we applied a two-step enrichment approach for KLK6 in ovarian cancer ascites, followed by mice immunization and production of monoclonal antibodies. Immunoaffinity techniques coupled to mass spectrometric methods were employed for hybridoma screening and target antigen identification. We found that the main target of the newly-generated monoclonal antibodies target was the serine protease inhibitor α1-antitrypsin (A1AT). Additional experiments confirmed that A1AT is the main inhibitor of KLK6 in biological fluids. One new antibody (24ED138) was chosen to build a hybrid assay for the accurate quantification of the A1AT-KLK6 complex in biological samples. The aforementioned assay was evaluated with serum samples collected from patients with ovarian cancer (n=24) and normal donors (n=16) and showed slight improvement in sensitivity (~12%) compared to the standard in-house KLK6 assay. We conclude that KLK6 is present in biological fluids either as free form, or bound to A1AT, and the bound form performs better than total KLK6 as a biomarker of ovarian carcinoma.

Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.

Differential Glycosylation Between Recombinant Factor VIII Produced in Baby Hamster Kidney and Chinese Hamster Ovary Cells Confers Differences in Immunogenicity in a Humanized Hemophilia Α Mouse Model

IntroductionThe formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations.MethodsFactor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS.Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT.FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay.ResultsLectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p<0.01), and greater levels of sialic acid capping (p<0.01) and fucosylated glycans (p<0.01). Mass spectra confirmed higher levels of sialic acid and identified two additional N-linked sites bearing high mannose glycans on CHO-rFVIII. In this mouse model we observed that BHK-rFVIII had a circulating half-life of 6.06 hours compared to the 10.01 hour half-life of CHO-rFVIII (p<0.0001).The immunogenicity of the BHK- and CHO-rFVIII products was next evaluated in vivo. In mice primed with a single 6 IU dose of BHK-rFVIII, we identified a higher proportion of FVIII-specific IFN-γ secreting splenocytes after seven days. Furthermore, long-term studies showed that 100% of mice subcutaneously exposed to BHK-rFVIII developed anti-FVIII IgG compared to the 47% that received CHO-rFVIII (p<0.01). Coincidently, when FVIII inhibitors were measured, we observed an incidence of 100% vs 37% (p<0.01), respectively. While the titres of FVIII-specific IgG were higher in mice exposed to BHK-rFVIII (p<0.01), there were no significant differences in the inhibitor concentrations. Similarly, we observed increased titres of FVIII-specific IgG in mice exposed intravenously (1 ug LPS with the first infusion) to BHK-rFVIII compared to CHO-rFVIII. However, there were no differences in the incidence of FVIII-specific IgG, nor in the incidence and concentration of inhibitors between the intravenously-infused mice.ConclusionsOur results demonstrate that BHK-rFVIII exhibits altered pharmacokinetic and immunogenic properties compared to CHO-rFVIII in humanized hemophilia A mice. The observed early increase in the proportion FVIII-specific IFN-γ producing cells in the spleen suggests an intrinsic immunogenic element of BHK-rFVIII. Similarly, the substantially increased immunogenicity of BHK-rFVIII in mice when treated subcutaneously complements the previously-reported clinical evidence. These differences may be attributed to the significant disparities in N-linked glycosylation, most notably high mannose and sialic acid containing glycans. Additional studies are underway to directly address the role of the these specific glycans and their potential impact on immunogenicity of rFVIII. DisclosuresLillicrap:Octapharma: Research Funding; Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding.

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate.

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics.