Sebastian P. Galuska

Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam−/− mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam−/− brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam−/− background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam−/− brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Impact of the Polysialyltransferases ST8SiaII and ST8SiaIV on Polysialic Acid Synthesis during Postnatal Mouse Brain Development

Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by ∼70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.

Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate ∼90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.

Enzyme-dependent Variations in the Polysialylation of the Neural Cell Adhesion Molecule (NCAM) in Vivo

Polysialic acid (polySia), an α2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.

Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1

The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.

High negative charge-to-size ratio in polyphosphates and heparin regulates factor VII-activating protease

Factor VII‐activating protease (FSAP) circulates as an inactive zymogen in the plasma. FSAP also regulates fibrinolysis by activating pro‐urokinase or cellular activation via cleavage of platelet‐derived growth factor BB (PDGF‐BB). As the Marburg I polymorphism of FSAP, with reduced enzymatic activity, is a risk factor for atherosclerosis and liver fibrosis, the regulation of FSAP activity is of major importance. FSAP is activated by an auto‐catalytic mechanism, which is amplified by heparin. To further investigate the structural requirements of polyanions for controlling FSAP activity, we performed binding, activation and inhibition studies using heparin and derivatives with altered size and charge, as well as other glycosaminoglycans. Heparin was effective in binding to and activating FSAP in a size‐ and charge density‐dependent manner. Polyphosphate was more potent than heparin with regard to its interactions with FSAP. Heparin was also an effective co‐factor for inhibition of FSAP by plasminogen activator inhibitor 1 (PAI‐1) and antithrombin, whereas polyphosphate served as co‐factor for the inhibition of FSAP by PAI‐1 only. For FSAP‐mediated inhibition of PDGF‐BB‐induced vascular smooth muscle cell proliferation, heparin as well as a polyphosphate served as efficient co‐factors. Native mast cell‐derived heparin exhibited identical properties to those of unfractionated heparin. Despite the strong effects of synthetic polyphosphate, the platelet‐derived material was a weak activator of FSAP. Hence, negatively charged polymers with a high charge‐to‐size ratio are responsible for the activation of FSAP, and also act as co‐factors for its inhibition by serine protease inhibitors.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing “neutrophil extracellular traps”, which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

Background: Polysialylated glycoproteins play an import role during numerous biological processes. Results: Polysialylated ST8SiaII and NCAM are components of mammalian semen and are partially associated with spermatozoa. Conclusion: Polysialic acid represents a further glyco-motif in mammalian ejaculates, which is known to influence the immune system. Significance: Administration of polysialic acid during insemination might be useful to increase the number of spermatozoa escaping the female immune system. Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.

Polysialylation of the Synaptic Cell Adhesion Molecule 1 (SynCAM 1) Depends Exclusively on the Polysialyltransferase ST8SiaII in Vivo

Background: Polysialic acid is a developmentally regulated posttranslational modification. Results: Loss of the polysialyltransferase ST8SiaII but not ST8SiaIV abolished polysialylation of SynCAM 1 in the mouse brain. Conclusion: Polysialylation of SynCAM 1 is mediated by ST8SiaII throughout postnatal mouse brain development. Significance: Studying the molecular requirements for protein polysialylation is crucial for understanding how this process is regulated. Polysialic acid is a unique carbohydrate polymer specifically attached to a limited number of glycoproteins. Among them is synaptic cell adhesion molecule 1 (SynCAM 1), a member of the immunoglobulin (Ig) superfamily composed of three extracellular Ig-like domains. Polysialylation of SynCAM 1 is cell type-specific and was exclusively found in NG2 cells, a class of multifunctional progenitor cells that form specialized synapses with neurons. Here, we studied the molecular requirements for SynCAM 1 polysialylation. Analysis of mice lacking one of the two polysialyltransferases, ST8SiaII or ST8SiaIV, revealed that polysialylation of SynCAM 1 is exclusively mediated by ST8SiaII throughout postnatal brain development. Alternative splicing of the three variable exons 8a, 8b, and 8c can theoretically give rise to eight transmembrane isoforms of SynCAM 1. We detected seven transcript variants in the developing mouse brain, including three variants containing exon 8c, which was so far regarded as a cryptic exon in mice. Polysialylation of SynCAM 1 was restricted to four isoforms in perinatal brain. However, cell culture experiments demonstrated that all transmembrane isoforms of SynCAM 1 can be polysialylated by ST8SiaII. Moreover, analysis of domain deletion constructs revealed that Ig1, which harbors the polysialylation site, is not sufficient as an acceptor for ST8SiaII. The minimal polypeptide required for polysialylation contained Ig1 and Ig2, suggesting an important role for Ig2 as a docking site for ST8SiaII.