Changes in the N-glycosylation of porcine immune globulin G during postnatal development.

N-glycosylation influences the effectiveness of immune globulin G (IgG) and thus the immunological downstream responses of immune cells. This impact arises from the presence of N-glycans within the Fc region, which not only alters the conformation of IgG but also influences its steric hindrance. Consequently, these modifications affect the interaction between IgG and its binding partners within the immune system. Moreover, this posttranslational modification vary according to the physiological condition of each individual. In this study, we examined the N-glycosylation of IgG in pigs from birth to five months of age. Our analysis identified a total of 48 distinct N-glycan structures. Remarkably, we observed defined changes in the composition of these N-glycans during postnatal development. The presence of agalactosylated and sialylated structures increases in relation to the number of N-glycans terminated by galactose residues during the first months of life. This shift may indicate a transition from passively transferred antibodies from the colostrum of the sow to the active production of endogenous IgG by the pig's own immune system.

Cancer-specific glycosylation of CD13 impacts its detection and activity in preclinical cancer tissues.

Harnessing the differences between cancer and non-cancer tissues presents new opportunities for selective targeting by anti-cancer drugs. CD13, a heavily glycosylated protein, is one example with significant unmet clinical potential in cancer drug discovery. Despite its high expression and activity in cancers, CD13 is also expressed in many normal tissues. Here, we report differential tissue glycosylation of CD13 across tissues and demonstrate for the first time that the nature and pattern of glycosylation of CD13 in preclinical cancer tissues are distinct compared to normal tissues. We identify cancer-specific O-glycosylation of CD13, which selectively blocks its detection in cancer models but not in normal tissues. In addition, the metabolism activity of cancer-expressed CD13 was observed to be critically dependent on its unique glycosylation. Thus, our data demonstrate the existence of discrete cancer-specific CD13 glycoforms and propose cancer-specific CD13 glycoforms as a clinically useful target for effective cancer-targeted therapy.

Knockout of the polysialyltransferases ST8SiaII and ST8SiaIV leads to a dilatation of rete testis during postnatal development.

Polysialic acid (polySia) is a carbohydrate polymer that modulates several cellular processes, such as migration, proliferation and differentiation processes. In the brain, its essential impact during postnatal development is well known. However, in most other polySia positive organs, only its localization has been described so far. For instance, in the murine epididymis, smooth muscle cells of the epididymal duct are polysialylated during the first 2 weeks of postnatal development. To understand the role of polySia during the development of the epididymis, the consequences of its loss were investigated in postnatal polySia knockout mice. As expected, no polysialylation was visible in the absence of the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, cGMP-dependent protein kinase I (PGK1), which is essentially involved in smooth muscle cell relaxation, was not detectable in peritubular smooth muscle cells when tissue sections of polySia knockout mice were analyzed by immunohistochemistry. In contrast to this signaling molecule, the structural proteins smooth muscle actin (SMA) and calponin were expressed. As shown before, in the duct system of the testis, even the expression of these structural proteins was impaired due to the loss of polySia. We now found that the rete testis, connecting the duct system of the testis and epididymis, was extensively dilated. The obtained data suggest that less differentiated smooth muscle cells of the testis and epididymis result in disturbed contractility and thus, fluid transport within the duct system visible in the enlarged rete testis.

Fc N-glycosylation of autoreactive Aß antibodies as a blood-based biomarker for Alzheimer's disease.

INTRODUCTION: Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aß42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aß42 . METHODS: nAbs-Aß42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aß42 were analyzed for their effect on Aß42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. RESULTS: Deglycosylation of nAbs-Aß42 had a major impact on Aß42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). DISCUSSION: N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aß antibodies. These data have consequences for the development of monocloncal Aß antibodies and may open new avenues for diagnostics.

Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Immunoglobulin Glycosylation - An Unexploited Potential for Immunomodulatory Strategies in Farm Animals.

The function of antibodies, namely the identification and neutralization of pathogens, is mediated by their antigen binding site (Fab). In contrast, the subsequent signal transduction for activation of the immune system is mediated by the fragment crystallizable (Fc) region, which interacts with receptors or other components of the immune system, such as the complement system. This aspect of binding and interaction is more precise, readjusted by covalently attached glycan structures close to the hinge region of immunoglobulins (Ig). This fine-tuning of Ig and its actual state of knowledge is the topic of this review. It describes the function of glycosylation at Ig in general and the associated changes due to corresponding glycan structures. We discuss the functionality of IgG glycosylation during different physiological statuses, like aging, lactation and pathophysiological processes. Further, we point out what is known to date about Ig glycosylation in farm animals and how new achievements in vaccination may contribute to improved animal welfare.

The Loss of Polysialic Acid Impairs the Contractile Phenotype of Peritubular Smooth Muscle Cells in the Postnatal Testis.

In the testis, the germinal epithelium of seminiferous tubules is surrounded by contractile peritubular cells, which are involved in sperm transport. Interestingly, in postnatal testis, polysialic acid (polySia), which is also an essential player for the development of the brain, was observed around the tubules. Western blotting revealed a massive decrease of polySia from postnatal day 1 towards puberty, together with a fundamental reduction of the net-like intertubular polySia. Using polysialyltransferase knockout mice, we investigated the consequences of the loss of polySia in the postnatal testis. Compared to postnatal wild-type animals, polySia knockouts showed slightly reduced smooth muscle actin (SMA) immunostaining of peritubular smooth muscle cells (SMCs), while calponin, marking more differentiated SMCs, dramatically decreased. In contrast, testicular SMA and calponin immunostaining remained unchanged in vascular SMCs in all genotypes. In addition, the cGMP-dependent protein kinase PKG I, a key enzyme of SMC relaxation, was nearly undetectable in the peritubular SMCs. Cell proliferation in the peritubular layer increased significantly in the knockouts, as shown by proliferating cell nuclear anti (PCNA) staining. Taken together, in postnatal testis, the absence of polySia resulted in an impaired differentiation of peritubular, but not vascular, SMCs to a more synthetic phenotype. Thus, polySia might influence the maintenance of a differentiated phenotype of non-vascular SMCs.

The N-glycans of lactoferrin: more than just a sweet decoration.

Nearly all extracellular proteins undergo post-translational modification with sugar chains during their transit through the endoplasmic reticulum and the Golgi apparatus. These "sweet" modifications not only influence the activity of its carrier protein, but they themselves often have bioactivity, independent of the carrier function. Lactoferrin belongs to the group of glycoproteins and is modified with several different N-glycans. This minireview summarizes several studies dealing with the diverse glycosylation patterns of lactoferrin from different origins, and the potential impact of these post-translational modifications on the functionality of lactoferrin. A special emphasis is placed on the differences between human and bovine lactoferrin, because the latter form is often selected for the development of novel therapeutic approaches in humans. For this reason, the potential impact of the bovine-specific glycosylation patterns on the observed heterogeneous effects of lactoferrin in humans is discussed within this minireview.

Characterization of Sialic Acid-Binding Immunoglobulin-Type Lectins in Fish Reveals Teleost-Specific Structures and Expression Patterns.

The cellular glycocalyx of vertebrates is frequently decorated with sialic acid residues. These sialylated structures are recognized by sialic acid-binding immunoglobulin-type lectins (Siglecs) of immune cells, which modulate their responsiveness. Fifteen Siglecs are known to be expressed in humans, but only four Siglecs are regularly present in fish: Siglec1, CD22, myelin-associated glycoprotein (MAG), and Siglec15. While several studies have dealt with the physiological roles of these four Siglecs in mammals, little is known about Siglecs in fish. In the present manuscript, the expression landscapes of these Siglecs were determined in the two salmonid species Oncorhynchus mykiss and Coregonus maraena and in the percid fish Sander lucioperca. This gene-expression profiling revealed that the expression of MAG is not restricted to neuronal cells but is detectable in all analyzed blood cells, including erythrocytes. The teleostean MAG contains the inhibitory motif ITIM; therefore, an additional immunomodulatory function of MAG is likely to be present in fish. Besides MAG, Siglec1, CD22, and Siglec15 were also expressed in all analyzed blood cell populations. Interestingly, the expression profiles of genes encoding Siglecs and particular associated enzymes changed in a gene- and tissue-specific manner when Coregonus maraena was exposed to handling stress. Thus, the obtained data indicate once more that stress directly affects immune-associated processes.

Betaglycan (TßRIII) is a Key Factor in TGF-ß2 Signaling in Prepubertal Rat Sertoli Cells.

Transforming growth factor-ßs (TGF-ßs) signal after binding to the TGF-ß receptors TßRI and TßRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-ß2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-ß1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-ß1 as well as TGF-ß2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-ß signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-ß2 but not TGF-ß1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-ß1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-ß2 increased it. Silencing of BG as well as TIMP3 reduced TGF-ß2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-ß2 signaling. In contrast, this effect was not observed with TIMP3/TGF-ß1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-ß2 signaling in Sertoli cells.

The Bovine Antimicrobial Peptide Lactoferricin Interacts with Polysialic Acid without Loss of Its Antimicrobial Activity against Escherichia coli.

The lactoferrin-derived peptide lactoferricin (LFcin) belongs to the family of antimicrobial peptides, and its bovine form has already been successfully applied to counteract enterohemorrhagic Escherichia coli (EHEC) infection. Recently, it was described that LFcin interacts with the sugar polymer polysialic acid (polySia) and that the binding of lactoferrin to polySia is mediated by LFcin, included in the N-terminal domain of lactoferrin. For this reason, the impact of polySia on the antimicrobial activity of bovine LFcin was investigated. Initially, the interaction of LFcin was characterized in more detail by native agarose gel electrophoresis, demonstrating that a chain length of 10 sialic acid residues was necessary to bind LFcin, whereas approximately twice-as-long chains were needed to detect binding of lactoferrin. Remarkably, the binding of polySia showed, independently of the chain length, no impact on the antimicrobial effects of LFcin. Thus, LFcin binds polySia without loss of its protective activity as an antimicrobial peptide.

Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model.

In order to combat invading pathogens neutrophils can release neutrophil extracellular traps (NETs). However, since NETs can also damage endogenous cells, several control mechanisms for the formation of NETs must work effectively. For instance, neutrophil activation is silenced within blood circulation by the binding of sialylated glycoconjugates to sialic acid binding immunoglobulin-like lectins (Siglecs) on neutrophils. As neutrophils are recruited within the female reproductive tract, after mating, a comparable mechanism may also take place within the bovine cervix to prevent an exaggerated NET formation and thus, infertility. We examined, if the highly glycosylated mucins, which are the major functional fraction of biomolecules in mucus, represent a potential regulator of NET formation. The qPCR data revealed that in polymorphonuclear neutrophils (PMNs) inhibitory Siglecs are the most frequently expressed Siglecs and might be a potential target of sialylated glycans to modulate the activation of PMNs. Remarkably, the addition of bovine cervical mucins significantly inhibited the formation of NET, which had been induced in response to lipopolysaccharides (LPS) or a combination of phorbol myristate acetate (PMA) and ionomycin. The inhibitory effects were independent of the stage of estrous cycle (estrus, luteal, and follicular mucins). PMNs retained their segmented nuclei and membrane perforation was prevented. However, the inhibitory effects were diminished, when sialic acids were released under acidic conditions. Comparable results were achieved, when sialic acids were targeted by neuraminidase digestion, indicating a sialic acid dependent inhibition of NET release. Thus, bovine cervical mucins have an anti-inflammatory capability to modulate NET formation and might be further immunomodulatory biomolecules that support fertility.

Glycans as Modulators for the Formation and Functional Properties of Neutrophil Extracellular Traps: Used by the Forces of Good and Evil.

A very common mechanism to trap pathogens is the release of DNA. Like flies in a spider's web, pathogens are enclosed in a sticky chromatin meshwork. Interestingly, plants already use this mechanism to catch bacteria. In mammals, especially neutrophils release their DNA to prevent an invasion of bacteria. These neutrophil extracellular traps (NETs) are equipped with antimicrobial molecules, including, for instance, histones, antimicrobial peptides, lactoferrin, and neutrophil elastase. Thus, in a defined area, pathogens and toxic molecules are directly adjacent. However, several of these antimicrobial substances are also cytotoxic for endogenous cells. It is, therefore, not surprising that distinct control mechanisms exist to prevent an exaggerated NETosis. Nevertheless, despite these endogenous control instruments, an extraordinary NET release is characteristic for several pathologies. Consequently, NETs are a novel target for developing therapeutic strategies. In this review, we summarize the roles of glycans in the biology of NETs; on the one hand, we focus on the glycan-dependent strategies of endogenous cells to control NET formation or to inactivate its cytotoxic effects, and, on the other hand, the "sweet" tricks of pathogens to inhibit the release of NETs or to prevent NET-mediated killing mechanisms are examined. Understanding both, the forces of good and evil, allows the development of novel glycan-based approaches to combat the harmful side of NETs during distinct pathologies.

Nanoparticles Equipped with α2,8-Linked Sialic Acid Chains Inhibit the Release of Neutrophil Extracellular Traps.

Neutrophils can combat the invasion of pathogens by the formation of neutrophil extracellular traps (NETs). The NET mechanism is not only an effective tool for combating pathogens, but is also associated with diseases. Therefore, NETs are a potential target for combating pathologies, such as cystic fibrosis and thrombosis. We investigated the potential of nanoparticles, which were modified with α2,8-linked sialic acid chains, to modulate NET release during phorbol myristate acetate stimulation. Interestingly, when these nanoparticles were applied, the formation of reactive oxygen species was partly inhibited and the release of NET was counteracted. However, although the release of NET fibers was prevented, the nuclei still lost their characteristic segmented structure and became swollen, indicating that only the release, and not complete activation was suppressed. Intriguingly, coincubation of α2,8-sialylated particles with free sialic acid chains prevented the outlined inhibitory effects. Thus, the sialic acid chains must be attached to a linker molecule to generate an active bioconjugate that is able to inhibit the release of NET.

Polysialic Acid Modulates the Binding of External Lactoferrin in Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are formed by neutrophils during inflammation. Among other things, these DNA constructs consist of antimicrobial proteins such as lactoferrin and histones. With these properties, NETs capture and destroy invading microorganisms. The carbohydrate polysialic acid (polySia) interacts with both lactoferrin and histones. Previous experiments demonstrated that, in humans, lactoferrin inhibits the release of NET and that this effect is supported by polySia. In this study, we examined the interplay of lactoferrin and polySia in already-formed NETs from bovine neutrophils. The binding of polySia was considered to occur at the lactoferricin (LFcin)-containing domain of lactoferrin. The interaction with the peptide LFcin was studied in more detail using groups of defined polySia chain lengths, which suggested a chain-length-dependent interaction mechanism with LFcin. The LFcin domain of lactoferrin was found to interact with DNA. Therefore, the possibility that polySia influences the integration of lactoferrin into the DNA-structures of NETs was tested by isolating bovine neutrophils and inducing NETosis. Experiments with NET fibers saturated with lactoferrin demonstrated that polySia initiates the incorporation of external lactoferrin in already-loaded NETs. Thus, polySia may modulate the constituents of NET.

Polysialic acid interacts with lactoferrin and supports its activity to inhibit the release of neutrophil extracellular traps.

Polysialic acid (polySia) is a linear carbohydrate polymer consisting of N-acetylneuraminic acid residues and is involved in several physiological processes. In the present study, we identified the multifunctional protein lactoferrin as a novel interaction partner for polySia. Lactoferrin co-precipitated when polySia was isolated from human blood, milk, and semen samples. The interaction between polySia and lactoferrin was verified using a native gel electrophoresis application, demonstrating that such interaction depends on the degree of polymerization. The interaction between the molecules could be inhibited by an antibody against lactoferricin (LFcin), which suggests that the LFcin domain of lactoferrin represents the potential binding area for sialic acid polymers. Because lactoferrin inhibits the formation of neutrophil extracellular traps (NETs), the potential impact of polySia on this function of lactoferrin was tested. Intriguingly, we observed that polySia increases the efficiency of lactoferrin to prevent the release of NET fibers. PolySia alone shows no activity. Therefore, together with lactoferrin, polySia may represent a natural regulatory system of NET release.

IgG Fc N-glycosylation: Alterations in neurologic diseases and potential therapeutic target?

Immunoglobulin G (IgG) is the most abundant antibody subclass of the human circulatory system and has important functions in the adaptive immune response. On the one hand, recognition and neutralization of antigens is mediated by the fab fragment, and on the other hand, processes such as phagocytosis, complement activation and inflammatory reactions are triggered by the Fc fragment. Here, the composition of conserved N-glycans attached to asparagine 297 of the IgG CH2 domain is a major critical factor that particularly modulates the effector functions of IgG. Additional attachments of fucoses, galactoses, N-acetylglucosamines, and sialic acids have been identified as factors that influence the affinity to a wide range of complement proteins and receptors and, thus, secondarily induce the secretion of pro- and anti-inflammatory cytokines. Consequently, alterations in the IgG Fc N-glycosylation pattern can provoke disruptions in the immunological state and are accompanied by various diseases, although the involvement of changed IgG glycosylation in disease outbreaks remains unknown. In addition to many autoimmune diseases, which have already been extensively reviewed, there are a number of further disorders related to altered IgG glycosylation patterns. In the present review, we focus on neurologic diseases, as in the last few years, an increasing number of studies have been published in this field. Due to the absence of reliable early biomarkers as well as therapeutic options in many cases, such analyses are of great interest and reveal possible future approaches.

Polysialic acid is released by human umbilical vein endothelial cells (HUVEC) in vitro.

BACKGROUND: Sialic acids represent common terminal residues on numerous mammalian glycoconjugates, thereby influencing e.g. lumen formation in developing blood vessels. Interestingly, besides monosialylated also polysialylated glycoconjugates are produced by endothelial cells. Polysialic acid (polySia) is formed in several organs during embryonal and postnatal development influencing, for instance, cell migration processes. Furthermore, the function of cytokines like basic fibroblast growth factor (bFGF) is modulated by polySia. RESULTS: In this study, we demonstrated that human umbilical vein endothelial cells (HUVEC) also secrete polysialylated glycoconjugates. Furthermore, an interaction between polySia and vascular endothelial growth factor (VEGF) was observed. VEGF modulates like bFGF the migration of HUVEC. Since both growth factors interact with polySia, we examined, if polySia modulates the migration of HUVEC. To this end scratch assays were performed showing that the migration of HUVEC is stimulated, when polySia was degraded. CONCLUSIONS: Since polySia can interact with bFGF as well as VEGF and the degradation of polySia resulted in an increased cell migration capacity in the applied scratch assay, we propose that polySia may trap these growth factors influencing their biological activity. Thus, polySia might also contribute to the fine regulation of physiological processes in endothelial cells.

Polysialic Acid in Human Plasma Can Compensate the Cytotoxicity of Histones.

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

Siglecs: A journey through the evolution of sialic acid-binding immunoglobulin-type lectins.

Siglecs (sialic acid-binding immunoglobulin-type lectins) are a family of immune regulatory receptors predominantly found on the cells of the hematopoietic system. A V-set Ig-like domain mediates the recognition of different sialylated glycoconjugates, which can lead to the activation or inhibition of the immune response, depending on the involved Siglecs. Siglecs are categorized into two subgroups: one including all CD33-related Siglecs and the other consisting of Siglec-1 (Sialoadhesin), Siglec-2 (CD22), Siglec-4 (myelin-associated glycoprotein, MAG) and Siglec-15. In contrast to the members of the CD33-related Siglecs, which share ∼50-99% sequence identity, Siglecs of the other subgroup show quite low homology (approximately 25-30% sequence identity). Based on the published sequences and functions of Siglecs, we performed phylogenetic analyses and sequence alignments to reveal the conservation of Siglecs throughout evolution. Therefore, we focused on the presence of Siglecs in different classes of vertebrates (fishes, amphibians, birds, reptiles and mammals), offering a bridge between the presence of different Siglecs and the biological situations of the selected animals.