Lost and Found: The Family of NF-κB Inhibitors Is Larger than Assumed in Salmonid Fish.

NF-κB signalling is largely controlled by the family of 'inhibitors of NF-κB' (IκB). The relevant databases indicate that the genome of rainbow trout contains multiple gene copies coding for iκbα (nfkbia), iκbε (nfkbie), iκbδ (nkfbid), iκbζ (nfkbiz), and bcl3, but it lacks iκbß (nfkbib) and iκbη (ankrd42). Strikingly, three nfkbia paralogs are apparently present in salmonid fish, two of which share a high sequence identity, while the third putative nfkbia gene is significantly less like its two paralogs. This particular nfkbia gene product, iκbα, clusters with the human IκBß in a phylogenetic analysis, while the other two iκbα proteins from trout associate with their human IκBα counterpart. The transcript concentrations were significantly higher for the structurally more closely related nfkbia paralogs than for the structurally less similar paralog, suggesting that iκbß probably has not been lost from the salmonid genomes but has been incorrectly designated as iκbα. In the present study, two gene variants coding for iκbα (nfkbia) and iκbε (nfkbie) were prominently expressed in the immune tissues and, particularly, in a cell fraction enriched with granulocytes, monocytes/macrophages, and dendritic cells from the head kidney of rainbow trout. Stimulation of salmonid CHSE-214 cells with zymosan significantly upregulated the iκbα-encoding gene while elevating the copy numbers of the inflammatory markers interleukin-1-beta and interleukin-8. Overexpression of iκbα and iκbε in CHSE-214 cells dose-dependently quenched both the basal and stimulated activity of an NF-κB promoter suggesting their involvement in immune-regulatory processes. This study provides the first functional data on iκbε-versus the well-researched iκbα factor-in a non-mammalian model species.

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.

Engineering of complex protein sialylation in plants.

Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

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.

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.

Milk Polysialic Acid Levels Rapidly Decrease in Line with the N-Acetylneuraminic Acid Concentrations during Early Lactation in Dairy Cows.

Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health.

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 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.

Individual Impact of Distinct Polysialic Acid Chain Lengths on the Cytotoxicity of Histone H1, H2A, H2B, H3 and H4.

Neutrophils are able to neutralize pathogens by phagocytosis, by the release of antimicrobial components, as well as by the formation of neutrophil extracellular traps (NETs). The latter possibility is a DNA-meshwork mainly consisting of highly concentrated extracellular histones, which are not only toxic for pathogens, but also for endogenous cells triggering several diseases. To reduce the negative outcomes initiated by extracellular histones, different approaches like antibodies against histones, proteases, and the polysaccharide polysialic acid (polySia) were discussed. We examined whether each of the individual histones is a binding partner of polySia, and analyzed their respective cytotoxicity in the presence of this linear homopolymer. Interestingly, all of the histones (H1, H2A, H2B, H3, and H4) seem to interact with α2,8-linked sialic acids. However, we observed strong differences regarding the required chain length of polySia to bind histone H1, H2A, H2B, H3, and H4. Moreover, distinct degrees of polymerization were necessary to act as a cytoprotective agent in the presence of the individual histones. In sum, the outlined results described polySia-based strategies to bind and/or to reduce the cytotoxicity of individual histones using distinct polySia chain length settings.

Artificial Polysialic Acid Chains as Sialidase-Resistant Molecular-Anchors to Accumulate Particles on Neutrophil Extracellular Traps.

Neutrophils are involved in numerous immunological events. One mechanism of neutrophils to combat pathogens is the formation of neutrophil extracellular traps (NETs). Thereby, neutrophils use DNA fibers to form a meshwork of DNA and histones as well as several antimicrobial components to trap and kill invaders. However, the formation of NETs can lead to pathological conditions triggering among other things (e.g., sepsis or acute lung failure), which is mainly a consequence of the cytotoxic characteristics of accumulated extracellular histones. Interestingly, the carbohydrate polysialic acid represents a naturally occurring antagonist of the cytotoxic properties of extracellular histones. Inspired by polysialylated vesicles, we developed polysialylated nanoparticles. Since sialidases are frequently present in areas of NET formation, we protected the sensitive non-reducing end of these homopolymers. To this end, the terminal sialic acid residue of the non-reducing end was oxidized and directly coupled to nanoparticles. The covalently linked sialidase-resistant polysialic acid chains are still able to neutralize histone-mediated cytotoxicity and to initiate binding of these polysialylated particles to NET filaments. Furthermore, polysialylated fluorescent microspheres can be used as a bioanalytical tool to stain NET fibers. Thus, polySia chains might not only be a useful agent to reduce histone-mediated cytotoxicity but also an anchor to accumulate nanoparticles loaded with active substances in areas of NET formation.

In vitro generation of polysialylated cervical mucins by bacterial polysialyltransferases to counteract cytotoxicity of extracellular histones.

Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone-mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell-based experiments demonstrated the activity of elongated mucins against histone-mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.