Biology subjects

Rapp, E.

Publications and source records attributed to Rapp, E..

10 recordsLinked to original sources

bioRxiv2025

POMT-dependent O-mannosylation regulates integrin β1 maturation via N-glycan processing

Withdrawal StatementThe authors have withdrawn this manuscript because following a careful internal review conducted in the course of manuscript revision, the authors identified that parts of the presented data could not be reliably traced back to the corresponding original datasets in their current form. While the general biological interpretation is not necessarily invalidated by this finding, the integrity of the present data representation no longer meets the standard required for publication. For this reason, the authors have decided to withdraw this preprint and will refrain from pursuing publication until the underlying data situation has been fully clarified and, if possible, reconstructed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The authors regret any inconvenience caused to the scientific community.

cell biology↗

bioRxiv2024

Immunoglobulin A Carries Sulfated N-glycans Primarily at the Tailpiece Site -- An Oxonium-Ion-Guided Approach for Site-Specific N-glycan Identification

Sulfated N-glycans from human immunoglobulin A (IgA) were recently discovered via glycomic approaches. However, their site-specific description is still pending. Certain N-glycan structures at specific N-glycosylation sites in IgA are crucial for microbial neutralization and effector functions. For instance, sialylated N-glycans on the C-terminal tailpiece mediate anti-viral activity by interfering with sialic-acid-binding viruses. Sulfated N-glycan epitopes can be ligands for viral proteins and thus play a role in the immune response. In this study, we performed a site-specific screening for sulfated N-glycans in two commercially available human serum IgA samples employing an in-depth N-glycoproteomic approach, previously developed by us. We found evidence of complex-type and hybrid-type N-glycans containing sulfated N-acetylhexosamine (sulfated HexNAc) attached to the N-glycosylation sites in the tailpiece and the CH2 domain of both IgA subclasses. A detailed comparison of the N-glycosylation profiles of human serum IgA samples from two suppliers showed such N-glycans with sulfated HexNAc consistently in higher abundance in the tailpiece region. Surprisingly, also complex-type N-glycan compositions bearing O-acetylated sialic acid were identified in the tailpiece. These findings have not been described before for a site-specific glycopeptide analysis. Overall, our work provides a methodology for performing a dedicated site-specific search for sulfated and O-acetylated N-glycans that can be easily transferred, e.g. to human IgA derived from mucosal tissues, milk, or saliva. Our future aim is to include sulfated N-glycans into longitudinal studies of IgA N-glycosylation and to investigate their role as a biomarker and a treatment option. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/597690v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@3d0214org.highwire.dtl.DTLVardef@1537f95org.highwire.dtl.DTLVardef@dd11eeorg.highwire.dtl.DTLVardef@1c43744_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

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, the impact of these differences on viral spread and neutralization sensitivity remains largely unknown. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis revealed 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 differed 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 SIVs 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.

microbiology↗

bioRxiv2023

New avenues for human blood plasma biomarker discovery via improved in-depth analysis of the low-abundant N-glycoproteome

To understand implications of protein glycosylation for clinical diagnostic and biopharmaceuticals, innovative glycoproteomic technologies are required. Recently, significant advances were made, particularly toward structure-focused N-glyco-proteomic analyses. The mass spectrometric analysis of intact N-glycopeptides using stepped collision fragmentation along with glycan oxonium ion profiling now enables to reliably discriminate between different N-glycan structures. Still, there are weaknesses that current N-glycoproteomic approaches must overcome: 1) handling of incorrect identifications, 2) identification of rare and modified N-glycans, and 3) insufficient glycoproteomic coverage, especially in complex samples. To address these shortcomings, we have developed an innovative N-glycoproteomic workflow that aims at providing comprehensive site-specific and structural N-glycoproteomic data on human blood plasma glycoproteins. The workflow features protein depletion plus various fractionation strategies and the use of high-resolution mass spectrometry with stepped collision fragmentation. Furthermore, by including a decision tree procedure established for data validation, we could significantly improve the description of the N-glycan micro-heterogeneity. Our data analysis workflow allows the reliable differentiation of ambiguous N-glycan structures like antenna-versus core-fucosylation plus the modified and rare N-glycans such as sulfated and glucuronidated ones. With this workflow, we were able to advance in the analysis of human blood plasma glycoproteins to concentrations as low as 10 pg/mL. A total of 1,929 N-glycopeptides and 942 N-glycosites derived from 805 human middle-to low-abundant glycoproteins were identified. Overall, the presented workflow holds great potential to improve our understanding of protein glycosylation and to foster the discovery of blood plasma biomarkers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/562384v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1548a6eorg.highwire.dtl.DTLVardef@14c74dcorg.highwire.dtl.DTLVardef@8c2377org.highwire.dtl.DTLVardef@6c6845_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Synthetic glycans that control gut microbiome structure mitigate colitis in mice

Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. We describe the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures resulted in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increased survival, reduced weight loss, and improved clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome.

microbiology↗

bioRxiv2021

Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human {beta}-1,2 N-acetlyglucosamintransferases I and II (MGAT1{triangleup}TM and MGAT2{triangleup}TM) and the {beta}-1,4-galactosyltransferase (GalT{triangleup}TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

synthetic biology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

Estimation of Kinematics from Inertial Measurement Units Using a Combined Deep Learning and Optimization Framework

The difficulty of estimating joint kinematics remains a critical barrier toward widespread use of inertial measurement units in biomechanics. Traditional sensor-fusion filters are largely reliant on magnetometer readings, which may be disturbed in uncontrolled environments. Careful sensor-to-segment alignment and calibration strategies are also necessary, which may burden users and lead to further error in uncontrolled settings. We introduce a new framework that combines deep learning and top-down optimization to accurately predict lower extremity joint angles directly from inertial data, without relying on magnetometer readings. We trained deep neural networks on a large set of synthetic inertial data derived from a clinical marker-based motion-tracking database of hundreds of subjects. We used data augmentation techniques and an automated calibration approach to reduce error due to variability in sensor placement and limb alignment. On left-out subjects, lower extremity kinematics could be predicted with a mean ({+/-} STD) root mean squared error of less than 1.27 {degrees} ({+/-} 0.38 {degrees}) in flexion/extension, less than 2.52 {degrees} ({+/-} 0.98 {degrees}) in ad/abduction, and less than 3.34 {degrees} ({+/-} 1.02 {degrees}) internal/external rotation, across walking and running trials. Errors decreased exponentially with the amount of training data, confirming the need for large datasets when training deep neural networks. While this framework remains to be validated with true inertial measurement unit (IMU) data, the results presented here are a promising advance toward convenient estimation of gait kinematics in natural environments. Progress in this direction could enable large-scale studies and offer an unprecedented view into disease progression, patient recovery, and sports biomechanics.

bioengineering↗

bioRxiv2020

Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies known as -dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on -dystroglycan, which leads to a reduction in cell adhesion to the extracellular matrix. Mutations in protein O-mannose {beta}1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, are mainly associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, direct molecular mechanisms are largely unknown. We used POMGNT1 knock-out HEK293T cells and fibroblasts from a MEB patient to gain a deeper insight into the molecular changes in POMGNT1 deficiency. A combination of biochemical and molecular biological techniques with proteomics, glycoproteomics and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. We found that in POMGNT1 deficient cells ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable to the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or -dystroglycanopathy in general, and suggests a previously underestimated impact of changes in O-mannosylation on N-glycosylation.

biochemistry↗

bioRxiv2020

The medaka alg2 mutant is a model for hypo-N-glycosylation-associated retinitis pigmentosa

Patients suffering from Congenital Disorders of Glycosylation (CDG) carry mutations in components of the evolutionarily highly conserved protein-glycosylation-machinery. Employing targeted genome editing, we modeled alleles in medaka fish based on a mutation described in an ALG2-index patient. The multisystemic phenotypes in our alg2 model closely resembled the patients syndromes. Molecularly, the mutation results in a reduction of the abundance of N-glycans without altering the profile of glycan structures in fish as well as in patient fibroblasts. This hypo-N-glycosylation impacted on protein abundance in two directions. We discovered a putative compensatory upregulation of the basic glycosylation and glycoprotein processing machinery highlighting the regulatory topology of the network. Conversely, proteins of the retinal phototransduction machinery were massively downregulated in the alg2 model. Those relate to the specific loss of rod photoreceptors that fail to be maintained in the alg2 mutants, a condition known as retinitis pigmentosa. Transient supply of human or medaka alg2 mRNA efficiently rescued the phenotypic spectrum and restored viability demonstrating that our alg2 model delivers key traits for the potential treatment of the disorder.

developmental biology↗