Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis.

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.

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

Rare genetic mutations of the mannosyl-oligosaccharide glucosidase (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 2 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.

Plasma markers of COVID-19 severity: a pilot study.

BACKGROUND: SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect this wide spectrum of disease presentations. METHODS: Our pilot cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins (APPs) by nephelometry, and full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). RESULTS: When compared to controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, α1-acid glycoprotein (AGP), α1-antitrypsin (AAT), ceruloplasmin (CP), haptoglobin (HP), and high-sensitivity C-reactive protein (hs-CRP). The concentrations of α1-antichymotrypsin (ACT), α2-macroglobulin (A2MG) and serum amyloid A (SAA) proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose- and sialylated di-antennary glycans and decreased non-sialylated di-antennary glycan A2G2 in COVID-19 patients compared to controls. CONCLUSIONS: COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight complexity of inflammatory process and grant further investigations.

Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation.

Polysialic acid (polySia) emerges as a novel regulator of microglia activity. We recently identified polysialylated proteins in the Golgi compartment of murine microglia that are released in response to inflammatory stimulation. Since exogenously added polySia is able to attenuate the inflammatory response, we proposed that the release of polysialylated proteins constitutes a mechanism for negative feedback regulation of microglia activation. Here, we demonstrate that translocation of polySia from the Golgi to the cell surface can be induced by calcium depletion of the Golgi compartment and that polysialylated proteins are continuously released for at least 24 h after the onset of inflammatory stimulation. The latter was unexpected, because polySia signals detected by immunocytochemistry are rapidly depleted. However, it indicates that the amount of released polySia is much higher than anticipated based on immunostaining. This may be crucial for microglial responses during traumatic brain injury (TBI), as we detected polySia signals in activated microglia around a stab wound in the adult mouse brain. In BV2 microglia, the putative polySia receptor Siglec-E is internalized during lipopolysaccharide (LPS)-induced activation and in response to polySia exposure, indicating interaction. Correspondingly, CRISPR/Cas9-mediated Siglec-E knockout prevents inhibition of pro inflammatory activation by exogenously added polySia and leads to a strong increase of the LPS response. A comparable increase of LPS-induced activation has been observed in microglia with abolished polySia synthesis. Together, these results indicate that the release of the microglia-intrinsic polySia pool, as implicated in TBI, inhibits the inflammatory response by acting as a trans-activating ligand of Siglec-E.

The monosialoganglioside GM1a protects against complement attack.

The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss 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, we here generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell death in 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. Exogenously administered 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 human endothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantly reduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). This study demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.