Macrophage- and CD4+ T cell-derived SIV differ in glycosylation, infectivity and neutralization sensitivity.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

Transition transferases prime bacterial capsule polymerization.

Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.

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.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR-Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.

Reliable N-Glycan Analysis-Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation.

Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates.

Characterization of a gene cluster involved in Aspergillus fumigatus zwitterionic glycosphingolipid synthesis.

The human pathogenic fungus Aspergillus fumigatus synthesizes the zwitterionic glycolipid Manα1,3Manα1,6GlcNα1,2IPC, named Af3c. Similar glycosphingolipids having a glucosamine (GlcN) linked in α1,2 to inositolphosphoceramide (IPC) as core structure have only been described in a few pathogenic fungi. Here, we describe an A. fumigatus cluster of 5 genes (AFUA_8G02040 to AFUA_8G02090) encoding proteins required for the glycan part of the glycosphingolipid Af3c. Besides the already characterized UDP-GlcNAc:IPC α1,2-N-acetylglucosaminyltransferase (GntA), the cluster encodes a putative UDP-GlcNAc transporter (NstA), a GlcNAc de-N-acetylase (GdaA), and 2 mannosyltransferases (OchC and ClpC). The function of these proteins was inferred from analysis of the glycolipids extracted from A. fumigatus strains deficient in one of the genes. Moreover, successive introduction of the genes encoding GntA, GdaA, OchC, and ClpC in the yeast Saccharomyces cerevisiae enabled the reconstitution of the Af3c biosynthetic pathway. Absence of Af3c slightly reduced the virulence of A. fumigatus in a Galleria mellonella infection model.

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.

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides.

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.

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.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo.

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb-/- mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

C. elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats.

Among the different types of protein glycosylation, C-mannosylation of tryptophan residues stands out because of the unique linkage formed between sugar and protein. Instead of the typical O- or N-glycosidic linkage, a C-C bond is used for attachment of a single mannose. C-mannose is characteristically found in thrombospondin type 1 repeats and in the WSXWS motif of type I cytokine receptors. The genetic base of the enzymatic activity catalyzing C-mannosylation was not known. Here we demonstrate that Caenorhabditis elegans DPY-19 is a C-mannosyltransferase. DPY-19 exhibits topological and sequential homology to the N-glycan oligosaccharyltransferase, highlighting an evolutionary link between N- and C-glycosylation. We show that the C. elegans surface receptors MIG-21 and UNC-5 are acceptor substrates of DPY-19 and that C-mannosylation is essential for the secretion of soluble MIG-21. Thereby, our data provide an explanation for the previously described identical Q neuroblast migration phenotypes of dpy-19 and mig-21 mutants.

Systematic analysis of the human tumor cell binding to human vs. murine E- and P-selectin under static vs. dynamic conditions.

Endothelial E- and P-selectins promote metastasis formation by interacting with sialyl-Lewis X and A (sLeX/sLeA) on circulating tumor cells. This interaction precedes extravasation and can take place under dynamic and static conditions. Metastasis formation is often studied in xenograft models. However, it is unclear whether species differences exist in the ligand specificity of human (h) vs. murine (m) selectins and whether different ligands are functional under dynamic vs. static conditions. We systematically compared the h vs. m E- and P-selectin (ESel/PSel) binding of a range of human tumor cells under dynamic vs. static conditions. The tumor cells were categorized by their sLeA/X status (sLeA+/sLeX+, sLeA-/sLeX+ and sLeA-/sLeX-). The general biological nature of the tumor-selectin interaction was analyzed by applying several tumor cell treatments (anti-sLeA/X blockade, neuraminidase, pronase and inhibition of O/N-glycosylation). We observed remarkable differences in the static vs. dynamic interaction of tumor cells with h vs. m ESel/PSel depending on their sLeA/X status. The tumor cell treatments mostly affected either static or dynamic as well as either h- or m-selectin interaction. mESel showed a higher diversity of potential ligands than hESel. Inhibition of O-GalNAc-glycosylation also affected glycosphingolipid synthesis. Summarized, different ligands on human tumor cells are functional under static vs. dynamic conditions and for the interaction with human vs. murine ESel/PSel. Non-canonical selectin ligands lacking the sLeA/X glycan epitopes exist on human tumor cells. These findings have important implications for the current development of glycomimetic, antimetastatic drugs and encourage the development of immunodeficient mice with humanized selectins.

Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3.

Thrombospondin type 1 repeats (TSRs) occur in diverse proteins involved in adhesion and signaling. The two extracellular TSRs of the netrin receptor UNC5A contain WxxWxxWxxC motifs that can be C-mannosylated on all tryptophans. A single C-mannosyltransferase (dumpy-19, DPY-19), modifying the first two tryptophans, occurs in Caenorhabditis elegans, but four putative enzymes (DPY-19-like 1-4, DPY19L1-4) exist in mammals. Single and triple CRISPR-Cas9 knockouts of the three homologs that are expressed in Chinese hamster ovary cells (DPY19L1, DPY19L3, and DPY19L4) and complementation experiments with mouse homologs showed that DPY19L1 preferentially mannosylates the first two tryptophans and DPY19L3 prefers the third, whereas DPY19L4 has no function in TSR glycosylation. Mannosylation by DPY19L1 but not DPY19L3 is required for transport of UNC5A from the endoplasmic reticulum to the cell surface. In vertebrates, a new C-mannosyltransferase has apparently evolved to increase glycosylation of TSRs, potentially to increase the stability of the structurally essential tryptophan ladder or to provide additional adhesion functions.

Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Quantitative Secretomics Reveals Extrinsic Signals Involved in Human Pluripotent Stem Cell Cardiomyogenesis.

Human pluripotent stem cells can be differentiated in vitro into cardiomyocytes (CMs) but the molecular mechanisms behind this process are still not fully understood. In particular, the identification of morphogens remained elusive because the mass spectrometry-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. An albumin-free cardiomyogenic differentiation medium is established in this study and applied for secretomics at seven different time points during in vitro differentiation. By this analysis 4832 proteins are identified with 1802 being significantly altered during differentiation and 431 of these are annotated as secreted. Numerous extrinsic components of Wnt, TGFß, Activin A, Nodal, BMP, or FGF signaling pathways are quantitatively assessed during differentiation. Notably, the abundance of pathway agonists is generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. It is hypothesized that TGFß, Activin A, and Nodal signaling are turned down shortly upon the initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation, and interestingly, Activin A and TGFß signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.