​2024

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Decreased sialylation elicits complement-related microglia response and bipolar cell loss in the mouse retina

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3.09.2024 |  Glia

Cuevas-Rios G, Assale TA, Wissfeld J, Bungartz A, Hofmann J, Langmann T, Neumann H

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Abstract:

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1β were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3.

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Sialic acids on T cells are crucial for their maintenance and survival

 

14.6.2024 | Front Immunol

Schmidt  M, Linder AT, Korn M, Schellenberg N, Meyer SJ, Nimmerjahn F, Werner A, Abeln M, Gerardy-Schahn R, Münster-Kühnel AK, Nitschke L

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Abstract:

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.

 

>> Read publication <<

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IgG sialylation occurs in B cells pre antibody secretion

 

17.5.2024 | Front Immunol

Werner A, Hanić M, Zaitseva OO, Lauc G, Lux A, Nitschke L, Nimmerjahn F

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Abstract:

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

 

>> Read publication <<

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Therapeutic potential to target sialylation and SIGLECs in neurodegenerative and psychiatric diseases

 

11.3.2024 | Front Neurol

Wißfeld J, Abou Assale T, Cuevas-Rios G, Liao H, Neumann H

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Abstract:

Sialic acids, commonly found as the terminal carbohydrate on the glycocalyx of mammalian cells, are pivotal checkpoint inhibitors of the innate immune system, particularly within the central nervous system (CNS). Sialic acid-binding immunoglobulin-like lectins (SIGLECs) expressed on microglia are key players in maintaining microglial homeostasis by recognizing intact sialylation. The finely balanced sialic acid-SIGLEC system ensures the prevention of excessive and detrimental immune responses in the CNS. However, loss of sialylation and SIGLEC receptor dysfunctions contribute to several chronic CNS diseases. Genetic variants of SIGLEC3/CD33, SIGLEC11, and SIGLEC14 have been associated with neurodegenerative diseases such as Alzheimer's disease, while sialyltransferase ST8SIA2 and SIGLEC4/MAG have been linked to psychiatric diseases such as schizophrenia, bipolar disorders, and autism spectrum disorders. Consequently, immune-modulatory functions of polysialic acids and SIGLEC binding antibodies have been exploited experimentally in animal models of Alzheimer's disease and inflammation-induced CNS tissue damage, including retinal damage. While the potential of these therapeutic approaches is evident, only a few therapies to target either sialylation or SIGLEC receptors have been tested in patient clinical trials. Here, we provide an overview of the critical role played by the sialic acid-SIGLEC axis in shaping microglial activation and function within the context of neurodegeneration and synaptopathies and discuss the current landscape of therapies that target sialylation or SIGLECs.

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>> Read publication <<

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2023

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6'-sialyllactose ameliorates the ototoxic effects of the aminoglycoside antibiotic neomycin in susceptible mice

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7.12.2023 | Front Immunol

Abou Assale T, Kuenzel T, Schink T, Shahraz A, Neumann H, Klaus C 

 

Abstract:

Sialic acids are terminal sugars of the cellular glycocalyx and are highly abundant in the nervous tissue. Sialylation is sensed by the innate immune system and acts as an inhibitory immune checkpoint. Aminoglycoside antibiotics such as neomycin have been shown to activate tissue macrophages and induce ototoxicity. In this study, we investigated the systemic subcutaneous application of the human milk oligosaccharide 6'-sialyllactose (6SL) as a potential therapy for neomycin-induced ototoxicity in postnatal mice. Repeated systemic treatment of mice with 6SL ameliorated neomycin-induced hearing loss and attenuated neomycin-triggered macrophage activation in the cochlear spiral ganglion. In addition, 6SL reversed the neomycin-mediated increase in gene transcription of the pro-inflammatory cytokine interleukin-1β (Il-1b) and the apoptotic/inflammatory kinase Pik3cd in the inner ear. Interestingly, neomycin application also increased the transcription of desialylating enzyme neuraminidase 3 (Neu3) in the inner ear. In vitro, we confirmed that treatment with 6SL had anti-inflammatory, anti-phagocytic, and neuroprotective effects on cultured lipopolysaccharide-challenged human THP1-macrophages. Thus, our data demonstrated that treatment with 6SL has anti-inflammatory and protective effects against neomycin-mediated macrophage activation and ototoxicity.

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 >> Read publication <<

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​The monosialoganglioside GM1a protects against complement attack

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25.8.2023 | Cell Death Discov

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Wedekind H, Beimdiek J, Rossdam C, Kats E, Wittek V, Schumann L, Sörensen-Zender I, Fenske A, Weinhold B, Schmitt R, Tiede A, Büttner FFR, Münster-Kühnel A, Abeln M

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Abstract: 

The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, itsactivation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed thatloss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface,ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, wehere generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell deathin vitro. Glycolipid-analysis by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF)allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenouslyadministered GM1a integrated into the plasma membrane of trophoblasts, substantially increased binding of complement factor H(FH) and was sufficient to protect the cells from complement attack and cell death. GM1a treatment also rescued humanendothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantlyreduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). Thisstudy demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way forsialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.

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The inhibitory receptor Siglec-G controls the severity of chronic lymphocytic leukemia

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3.8.2023 | EMBO Rep

Röder B, Fahnenstiel H, Schäfer S, Budeus B, Dampmann M, Eichhorn M, Angermüller S, Brost C, Winkler TH, Seifert M, Nitschke L

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Abstract:

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

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 >> Read publication <<

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Polysialic acid promotes remyelination in cerebellar slice cultures by Siglec-E-dependent modulation of microglia polarization​

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10.7.2023 | ​Front Cell Neurosci

​Schröder L-J, Thiesler H, Gretenkort L, Möllenkamp T, Stangel M, Gudi V, Hildebrandt H

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Abstract: 

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. Spontaneous restoration of myelin after demyelination occurs, but its efficiency declines during disease progression. Efficient myelin repair requires fine-tuning of inflammatory responses by brain-resident microglia and infiltrating macrophages. Accordingly, promising therapeutic strategies aim at controlling inflammation to promote remyelination. Polysialic acid (polySia) is a polymeric glycan with variable chain lengths, presented as posttranslational modification on select protein carriers. PolySia emerges as a negative regulator of inflammatory microglia and macrophage activation and has been detected on oligodendrocyte precursors and reactive astrocytes in multiple sclerosis lesions. As shown recently, polySia-modified proteins can also be released by activated microglia, and the intrinsically released protein-bound and exogenously applied free polySia were equally able to attenuate proinflammatory microglia activation via the inhibitory immune receptor Siglec-E. Here, we explore polySia as a candidate substance for promoting myelin regeneration by immunomodulation. Lysophosphatidylcholininduced demyelination of organotypic cerebellar slice cultures was used as experimental model to analyze the impact of polySia with different degrees of polymerization (DPs) on remyelination and inflammation. In lysophosphatidylcholin-treated cerebellar slice cultures, polySia-positive cells were abundant during demyelination but largely reduced during remyelination. Based on determination of DP24 as the minimal polySia chain length required for inhibition of inflammatory BV-2 microglia activation, pools with short and long polySia chains (DP8-14 and DP24-30) were generated and applied to slice cultures during remyelination. Unlike DP8-14, treatment with DP24-30 significantly improved remyelination, increased arginase-1-positive microglia ratios, and reduced the production of nitric oxide in wildtype, but not Siglec-E-deficient slice cultures. In vitro differentiation of oligodendrocytes was not affected by DP24-30. Collectively, these results suggest a beneficial effect of exogenously applied polySia DP24-30 on remyelination by Siglec-E-dependent microglia regulation.

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​Mannosylated glycans impair normal T-cell development by reprogramming commitment and repertoire diversity

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21.6.2023 | Cell Mol Immunol​

Vicente MM, Alves I, Fernandes Â, Dias AM, Santos-Pereira B, Pérez-Anton E, Santos S, Yang T, Correia A, Münster-Kühnel A, Almeida ARM, Ravens S, Rabinovich GA, Vilanova M, Sousa AE, Pinho SS

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Abstract: 

T-cell development ensures the formation of diverse repertoires of T-cell receptors (TCRs) that recognize a variety of antigens. Glycosylation is a major posttranslational modification present in virtually all cells, including T-lymphocytes, that regulates activity/functions. Although these structures are known to be involved in TCR-selection in DP thymocytes, it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease. Here, we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes, as well as dynamic alterations. After restricting the N-glycosylation profile of thymocytes to high-mannose structures, using specific glycoengineered mice (Rag1CreMgat1fl/fl), we showed remarkable defects in key developmental checkpoints, including ß-selection, regulatory T-cell generation and γδT-cell development, associated with increased susceptibility to colon and kidney inflammation and infection. We further demonstrated that a single N-glycan antenna (modeled in Rag1CreMgat2fl/fl mice) is the sine-qua-non condition to ensure normal development. In conclusion, we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.

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Neuroimmunomodulatory properties of polysialic acid

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12.5.2023 | Glycoconj J 

​Gretenkort L, Thiesler H, Hildebrandt H

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Abstract: 

Polymeric sialic acid (polysialic acid, polySia) is a remarkable posttranslational modification of only few select proteins. The major, and most prominent polySia protein carrier is the neural cell adhesion molecule NCAM. Here, the key functions of polySia are to regulate interactions of NCAM and to balance cellular interactions in brain development and plasticity. During recent years, however, increasing evidence points towards a role of polySia in the modulation of immune responses. These immunomodulatory functions can be mediated by polySia on proteins other than NCAM, presented either on the cell surface or released into the extracellular space. This perspective review summarizes our current knowledge and addresses major open questions on polySia and polySia receptors in modulating innate immune responses in the brain.

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Proinflammatory macrophage activation by the polysialic acid-Siglec-16 axis is linked to increased survival of patients with glioblastoma

 

14.4.2023 | Clin Cancer Res​ 

Thiesler H, Gretenkort L, Hoffmeister L, Albers I, Ohlmeier L, Röckle I, Verhagen A, Banan R, Köpcke N, Krönke N, Feuerhake F, Behling F, Barrantes-Freer A, Mielke D, Rohde V, Hong B, Varki A, Schwabe K, Krauss JK, Stadelmann C, Hartmann C, Hildebrandt H

 

​Abstract: 

Purpose: Interactions with tumor-associated microglia and macrophages (TAM) are critical for glioblastoma progression. Polysialic acid (polySia) is a tumor-associated glycan, but its frequency of occurrence and its prognostic value in glioblastoma are disputed. Through interactions with the opposing immune receptors Siglec-11 and Siglec-16, polySia is implicated in the regulation of microglia and macrophage activity. However, due to a nonfunctional SIGLEC16P allele, SIGLEC16 penetrance is less than 40%. Here, we explored possible consequences of SIGLEC16 status and tumor cell-associated polySia on glioblastoma outcome.

Experimental design: FFPE specimens of two independent cohorts with 70 and 100 newly diagnosed glioblastoma patients were retrospectively analyzed for SIGLEC16 and polySia status in relation to overall survival. Inflammatory TAM activation was assessed in tumors, in heterotypic tumor spheroids consisting of polySia-positive glioblastoma cells and Siglec-16-positive or -negative macrophages, and by exposing Siglec-16-positive or -negative macrophages to glioblastoma cell-derived membrane fractions.

Results: Overall survival of SIGLEC16 carriers with polySia-positive tumors was increased. Consistent with proinflammatory Siglec-16 signaling, levels of TAM positive for the M2 marker CD163 were reduced, whereas the M1 marker CD74 and TNF expression were increased, and CD8+ T cells enhanced in SIGLEC16/polySia double-positive tumors. Correspondingly, TNF production was elevated in heterotypic spheroid cultures with Siglec-16-expressing macrophages. Furthermore, a higher, mainly M1-like cytokine release and activating immune signaling was observed in SIGLEC16-positive as compared to SIGLEC16-negative macrophages confronted with glioblastoma cell-derived membranes.

Conclusions: Collectively, these results strongly suggest that proinflammatory TAM activation causes the better outcome in glioblastoma patients with a functional polySia-Siglec-16 axis.

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Association of polysialic acid serum levels with schizophrenia spectrum and bipolar disorder-related structural brain changes and hospitalization

 

6.2.2023 | Sci Rep

Müller-Miny L, Thiel K, Meinert S, Hahn T, Kircher T, Nenadić I, Krug A, Hufschmidt F, Liao H, Neumann H, Dannlowski U, Lünemann JD

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Abstract:

Expression of polysialic acid (polySia) in the adult brain is enriched in areas of continuous neurogenesis and plasticity such as the hippocampus. Genome-wide association studies identified variants of glycosylation enzyme-encoding genes, required for the generation of polySia, to be associated with the development of schizophrenia and bipolar disorder. Here, we report that serum levels of polySia are increased in patients with schizophrenia spectrum disorder compared to patients with major depressive disorders or demographically matched healthy controls. Furthermore, elevated polySia serum levels are associated with structural hippocampal gray matter decline in schizophrenia spectrum and bipolar disorder. In patients with schizophrenia spectrum disorder, polySia serum levels correlate with the number, duration of disease-related hospitalizations, early retirement and medical leave as estimators of detrimental long-term disease trajectories. Our data show that polySia serum levels are linked to structural hippocampal brain changes in schizophrenia spectrum and bipolar disorders, and suggest a contribution of polySia to the pathophysiology of these diseases.

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 >> Read publication <<

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2022

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Response to Oswald et al.: ST6Gal1 in plasma is dispensable for IgG sialylation

 

31.10.2022 | Glycobiology

Werner A, Nimmerjahn F

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Abstract:

The presence or absence of terminal sialic acid residues in the sugar moiety attached to the Fc-domain of IgG molecules modulates IgG activity and is associated with autoimmune or infection related inflammation. In a recent paper, Oswald and colleagues suggest that IgG sialylation may occur post IgG secretion from plasma cells, which would be a major issue for therapeutic antibodies injected into patients. In contrast, we argue that previous work rather demonstrates that IgG sialylation occurs within B cells and that the experimental system used by the authors is not suitable to address this critical question.

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 >> Read publication <<

 

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HINGEneering IgG for enhanced immune activation

 

15.7.2022 | Sci Immunol

Werner A, Nimmerjahn F

Abstract:

Two recent studies in Science Immunology by Orr et al. and Foss et al. demonstrate that the hinge region of immunoglobulin G (IgG) is a major regulator of IgG2 and IgG3 activity, respectively. Engineering of the IgG hinge may therefore be useful for optimizing the activity of IgG subclasses.

 

 >> Read publication <<

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Sialic acids on B cells are crucial for their survival and provide protection against apoptosis

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21.6.2022 | Proc Natl Acad Sci USA 

​Linder AT, Schmidt M, Hitschfel J, Abeln M, Schneider P, Gerardy-Schahn R, Münster-Kühnel AK, Nitschke L

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Abstract: 

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

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>> Read publication <<

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Low molecular weight polysialic acid binds to properdin and reduces the activity of the alternative complement pathway

 

6.4.2022 | Sci Rep 

Shahraz A, Lin Y, Mbroh J, Winkler J, Liao H, Lackmann M, Bungartz A, Zipfel PF, Skerka C, Neumann H

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Abstract: 

Sialic acids as the terminal caps of the cellular glycocalyx play an essential role in self-recognition and were shown to modulate complement processes via interaction between α2,3-linked sialic acids and complement factor H. Previously, it was suggested that low molecular weight α2,8-linked polysialic acid (polySia avDP20) interferes with complement activation, but the exact molecular mechanism is still unclear. Here, we show that soluble polySia avDP20 (molecular weight of ~ 6 kDa) reduced the binding of serum-derived alternative pathway complement activator properdin to the cell surface of lesioned Hepa-1c1c7 and PC-12 neuroblastoma cells. Furthermore, polySia avDP20 added to human serum blocked the alternative complement pathway triggered by plate-bound lipopolysaccharides. Interestingly, no inhibitory effect was observed with monosialic acid or oligosialic acid with a chain length of DP3 and DP5. In addition, polySia avDP20 directly bound properdin, but not complement factor H. These data show that soluble polySia avDP20 binds properdin and reduces the alternative complement pathway activity. Results strengthen the previously described concept of self-recognition of sialylation as check-point control of complement activation in innate immunity.

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News and Views on Polysialic Acid: From Tumor Progression and Brain Development to Psychiatric Disorders, Neurodegeneration, Myelin Repair and Immunomodulation

 

4.4.2022 | Front Cell Dev Biol 

Thiesler H, Küçükerden M, Gretenkort L, Röckle I, Hildebrandt H

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Abstract: 

Polysialic acid (polySia) is a sugar homopolymer consisting of at least eight glycosidically linked sialic acid units. It is a posttranslational modification of a limited number of proteins with the neural cell adhesion molecule NCAM being the most prominent. As extensively reviewed before, polySia-NCAM is crucial for brain development and synaptic plasticity but also modulates tumor growth and malignancy. Functions of polySia have been attributed to its polyanionic character, its spatial expansion into the extracellular space, and its modulation of NCAM interactions. In this mini-review, we first summarize briefly, how the modulation of NCAM functions by polySia impacts tumor cell growth and leads to malformations during brain development of polySia-deficient mice, with a focus on how the latter may be linked to altered behaviors in the mouse model and to neurodevelopmental predispositions to psychiatric disorders. We then elaborate on the implications of polySia functions in hippocampal plasticity, learning and memory of mice in light of recently described polySia changes related to altered neurogenesis in the aging human brain and in neurodegenerative disease. Furthermore, we highlight recent progress that extends the range of polySia functions across diverse fields of neurobiology such as cortical interneuron development and connectivity, myelination and myelin repair, or the regulation of microglia activity. We discuss possible common and distinct mechanisms that may underlie these seemingly divergent roles of polySia, and provide prospects for new therapeutic approaches building on our improved understanding of polySia functions.

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>> Read publication <<

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Serum N-glycomics of a novel CDG-IIb patient reveals aberrant IgG glycosylation

 

27.2.2022 | Glycobiology 

Beimdiek J, Hennig R, Burock R, Puk O, Biskup S, Rapp E, Lesinski-Schiedat A, Buettner FFR, Das AM

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Abstract:

Rare genetic mutations of the MOGS gene affecting the function of the mannosyl-oligosaccharide glucosidase (glucosidase I) are the cause of the congenital disorder of glycosylation IIb (CDG-IIb). Glucosidase I specifically removes the distal α1,2-linked glucose from the protein bound precursor N-glycan Glc3Man9GlcNAc2 which is the initial step of N-glycan maturation. Here, we comparatively analyzed N-glycosylation of the whole serum proteome, serum-derived immunoglobulin G (IgG), transferrin (TF), and α-1-antitrypsin (AAT) of a female patient who is compound-heterozygous for two novel missense mutations in the MOGS gene, her heterozygous parents, and a sibling with wildtype genotype by multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) at unprecedented depth. Thereby, we detected the CDG-IIb-characteristic non-de-glucosylated N-glycans Glc3Man7-9GlcNAc2 as well as the free tetrasaccharide Glc3-Man in whole serum of the patient but not in the other family members. The N-glycan analysis of the serum proteome further revealed that relative intensities of IgG-specific complex type di-antennary N-glycans with core-fucosylation were considerably reduced in the patient's serum whereas TF- and AAT-characteristic sialylated di- and tri-antennary N-glycans were increased. This finding reflected the hypogammaglobulinemia diagnosed in the patient. We further detected aberrant oligo-mannose (Glc3Man7GlcNAc2) and hybrid type N-glycans on patient-derived IgGs and we attributed this defective glycosylation to be the reason for an increased IgG clearance. This mechanism can explain the hypogammaglobulinemia that is associated with CDG-IIb.

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2021

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Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis

 

5.12.2021 | Hepatology 

Taubert R, Engel B, Diestelhorst J, Hupa-Breier KL, Behrendt P, Baerlecken NT, Sühs KW, Janik MK, Zachou K, Sebode M, Schramm C, Londoño MC, Habes S; UK-AIH Consortium, Oo YH, Lalanne C, Pape S, Schubert M, Hust M, Dübel S, Thevis M, Jonigk D, Beimdiek J, Buettner FFR, Drenth JPH, Muratori L, Adams DH, Dyson JK, Renand A, Graupera I, Lohse AW, Dalekos GN, Milkiewicz P, Stangel M, Maasoumy B, Witte T, Wedemeyer H, Manns MP, Jaeckel E

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Abstract:

Background and aims: Detection of autoantibodies is a mainstay of diagnosing autoimmune hepatitis (AIH). However, conventional autoantibodies for the workup of AIH lack either sensitivity or specificity, leading to substantial diagnostic uncertainty. We aimed to identify more accurate serological markers of AIH with a protein macroarray.

Approach and results: During the search for more-precise autoantibodies to distinguish AIH from non-AIH liver diseases (non-AIH-LD), IgG antibodies with binding capacities to many human and foreign proteins were identified with a protein macroarray and confirmed with solid-phase ELISAs in AIH patients. Subsequently, polyreactive IgG (pIgG) was exemplarily quantified by reactivity against human huntingtin-interacting protein 1-related protein in bovine serum albumin blocked ELISA (HIP1R/BSA). The diagnostic fidelity of HIP1R/BSA binding pIgG to diagnose AIH was assessed in a retrospective training, a retrospective multicenter validation, and a prospective validation cohort in cryoconserved samples from 1,568 adults from 10 centers from eight countries. Reactivity against HIP1R/BSA had a 25% and 14% higher specificity to diagnose AIH than conventional antinuclear and antismooth muscle antibodies, a significantly higher sensitivity than liver kidney microsomal antibodies and antisoluble liver antigen/liver pancreas antigen, and a 12%-20% higher accuracy than conventional autoantibodies. Importantly, HIP1R/BSA reactivity was present in up to 88% of patients with seronegative AIH and in up to 71% of AIH patients with normal IgG levels. Under therapy, pIgG returns to background levels of non-AIH-LD.

Conclusions: pIgG could be used as a promising marker to improve the diagnostic workup of liver diseases with a higher specificity for AIH compared to conventional autoantibodies and a utility in autoantibody-negative AIH. Likewise, pIgG could be a major source of assay interference in untreated AIH.

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>> Read publication <<

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Sweet Rules: Linking Glycosylation to Antibody Function

 

23.10.2021 | Exp Suppl (Chapter)

Nimmerjahn F, Werner A

  

Abstract:

Antibodies produced upon infections with pathogenic microorganisms are essential for clearing primary infections and for providing the host with long-lasting immunity. Moreover, antibodies have become the most widely used platform for developing novel therapies against cancer and autoimmunity, requiring an in-depth understanding of how antibodies mediate their activity in vivo and which factors modulate pro- or anti-inflammatory antibody activities. Since the discovery that select residues present in the sugar domain attached to the immunoglobulin G (IgG) fragment crystallizable (Fc) region can modulate both, pro- and anti-inflammatory effector functions, a wealth of studies has focused on understanding how IgG glycosylation is regulated and how this knowledge can be used to optimize therapeutic antibody activity. With the introduction of glycoengineered afucosylated antibodies in cancer therapy and the initiation of clinical testing of highly sialylated anti-inflammatory antibodies the proof-of-concept that understanding antibody glycosylation can lead to clinical innovation has been provided. The focus of this review is to summarize recent insights into how antibody glycosylation is regulated in vivo and how select sugar residues impact IgG function.

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 >> Read publication <<

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Targeting sialylation to treat central nervous system diseases

 

1.10.2021 | Trends Pharmacol Sci 

Lünemann JD, von Gunten S, Neumann H

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Abstract:

Sialic acid-binding immunoglobulin-type lectins (SIGLECs) are membrane receptors that are preferentially expressed on immune cells and recognize sialylated proteins, lipids, and RNA. Sialic acids and signaling through SIGLECs are increasingly recognized for their essential roles in immune system homeostasis as well as nervous system development, plasticity, and repair. Dysregulated sialylation and SIGLEC dysfunctions contribute to several chronic diseases of the central nervous system (CNS) in which current therapeutic options are very limited. While only a few therapies targeting SIGLECs are currently being tested in clinical trials, the area emerged as one of the most dynamic and active fields in glycobiology and drug development. This review highlights recent insights into sialic acid and SIGLEC function in CNS pathologies and illustrates opportunities and challenges for the development of sialic acid-based and SIGLEC-targeted therapies for neurological diseases.

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>> Read publication <<

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Low molecular weight polysialic acid prevents lipopolysaccharide-induced inflammatory dopaminergic neurodegeneration in humanized SIGLEC11 transgenic mice

 

18.8.2021 | Glia 

Liao H, Winkler J, Wißfeld J, Shahraz A, Klaus C, Neumann H

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Abstract:

Parkinson's disease is one of the most common neurodegenerative diseases in the elderly population, with a pathophysiology linked to neuroinflammation, complement activation, and oxidative damage. Soluble polysialic acid with an average degree of polymerization 20 (polySia avDP20) prevents inflammation and oxidative burst in human macrophages via sialic acid-binding immunoglobulin like lectin-11 (SIGLEC11) receptor and interferes with alternative complement activation. Here, we confirmed the anti-inflammatory capacity of polySia avDP20 on cultured murine embryonic stem cell-derived microglia and analyzed the effect of polySia avDP20 in a lipopolysaccharide-triggered animal model of Parkinson's disease. We demonstrated a neuroprotective effect of intraperitoneally applied polySia avDP20 in humanized SIGLEC11 transgenic mice after repeated systemic challenge with lipopolysaccharide. Pathway enrichment analysis of the brain transcriptome on day 19 after disease initiation showed that intraperitoneal application of 10 μg/g body weight polySia avDP20 prevented excessive inflammation. In line with these data, polySia avDP20 attenuated the lipopolysaccharide-triggered increase in mRNA levels of immune-related genes (Il1b, Cd14, Myd88, Fcer1g, Itgam, C4, Cybb, Iba1 and Cd68) and cell death-related genes (Casp8, Ripk1 and Ripk3) in the brains of SIGLEC11 transgenic mice on day 19, but not on day 5. Moreover, immunohistochemistry demonstrated that polySia avDP20 reduced the lipopolysaccharide-induced increase in immunoreactivity of IBA1 and CD68 in the substantia nigra pars reticulata in SIGLEC11 transgenic and wild type mice on day 19. Furthermore, treatment with polySia avDP20 prevented the loss of dopaminergic neurons in the substantia nigra pars compacta induced by lipopolysaccharide challenge in both SIGLEC11 transgenic and wild type mice on day 19. Thus, our data demonstrate that polySia avDP20 ameliorates inflammatory dopaminergic neurodegeneration and therefore is a promising drug candidate to prevent Parkinson's disease-related inflammation and neurodegeneration.

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Uridine diphosphate-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase deletion in mice leads to lethal intracerebral hemorrhage during embryonic development

 

5.7.2021 | Glycobiology 

Wedekind H, Kats E, Weiss A-C, Thiesler H, Klaus C, Kispert A, Horstkorte R, Neumann H, Weinhold B, Münster-Kühnel A, Abeln M​

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Abstract: 

Among the enzymes of the biosynthesis of sialoglycoconjugates, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), catalyzing the first essential step of the sialic acid (Sia) de novo biosynthesis, and CMP-Sia synthase (CMAS), activating Sia to CMP-Sia, are particularly important. The knockout of either of these enzymes in mice is embryonically lethal. While the lethality of Cmas-/- mice has been attributed to a maternal complement attack against asialo fetal placental cells, the cause of lethality in Gne-deficient embryos has remained elusive. Here, we advanced the significance of sialylation for embryonic development through detailed histological analyses of Gne-/- embryos and placentae. We found that Gne-/- embryonic and extraembryonic tissues are hyposialylated, rather than completely deficient of sialoglycans which holds true for Cmas-/- embryos. Residual sialylation of Gne-/- cells can be explained by scavenging free Sia from sialylated maternal serum glycoconjugates via the lysosomal salvage pathway. The placental architecture of Gne-/- mice was unaffected, but severe hemorrhages in the neuroepithelium with extensive bleeding into the cephalic ventricles were present at E12.5 in the mutants. At E13.5, the vast majority of Gne-/- embryos were asystolic. This phenotype persisted when Gne-/- mice were backcrossed to a complement component 3-deficient background, confirming distinct pathomechanisms of Cmas-/- and Gne-/- mice. We conclude that the low level of sialylation observed in Gne-/- mice is sufficient, both for immune homeostasis at the fetal-maternal interface and for embryonic development until E12.5. However, formation of the neural microvasculature is the first critical process depending on a higher degree of sialylation during development of the embryo proper.

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Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

 

29.6.2021 | Sci Rep 

Wißfeld J, Mathews M, Mossad O, Picardi P, Cinti A, Redaelli L, Pradier L, Brüstle O, Neumann H

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Abstract:

CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer's disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2 (D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.

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The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid

 

28.4.2021 | Glycobiology 

Albers M, Schröter L, Belousov S, Hartmann M, Grove M, Abeln M, Mühlenhoff M

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Abstract: 

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid up-take system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

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Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype

 

4.2.2021 | Glia 

Wißfeld J, Nozaki I, Mathews M, Raschka T, Ebeling C, Hornung V, Brüstle O, Neumann H.

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Abstract:

Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33ΔE2 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-β1-42 and bacterial particles were increased after knockout of CD33 or CD33ΔE2 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-β1-42 or bacterial particles was increased after CD33 knockout but not in CD33ΔE2 -expressing microglia. In summary, deletion of CD33 or expression of CD33ΔE2 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid β1-42 , but potentially detrimental oxidative burst and inflammation, which was absent in CD33ΔE2 -expressing microglia.

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