| 1. |
- Du, Jiqing, et al.
(författare)
-
Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1
- 2021
-
Ingår i: Nature Communications. - : Nature Research. - 2041-1723. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.
|
|
| 2. |
- Fauser, Joel, et al.
(författare)
-
Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD
- 2021
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.
|
|
| 3. |
- Gulen, Burak, et al.
(författare)
-
Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture
- 2020
-
Ingår i: Nature Chemistry. - : Nature Publishing Group. - 1755-4330 .- 1755-4349. ; 12:8, s. 732-739
-
Tidskriftsartikel (refereegranskat)abstract
- Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
|
|
| 4. |
- Hoepfner, Dorothea, et al.
(författare)
-
Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
- 2020
-
Ingår i: iScience. - Cambridge : Cell Press. - 2589-0042. ; 23:12
-
Tidskriftsartikel (refereegranskat)abstract
- AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.
|
|
| 5. |
- Kaspers, Marietta S., et al.
(författare)
-
Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b
- 2023
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.
|
|
| 6. |
- Behren, Sandra, et al.
(författare)
-
Bacteria Lectin Recognition Towards Fucose Binding Motifs Highlights the Impact of Presenting Mucin Core Glycopeptides
- 2024
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mucin glycoproteins are essential components of the mucosal protective barrier, which constantly senses and clears the host from pathogens. Throughout evolution, bacteria and virus have developed strategies to modulate and penetrate the mucosal barrier and cause virulence by interacting with the glycans of membrane-bound mucins at the epithelial cell-surface. These interactions may promote bacteria cell-adhesion, biofilm formation, protein toxin delivery, or cause an inflammatory environment. O-fucosylated glycan epitopes are commonly found on mucin glycoproteins, and are key ligands of many bacterial and viral lectins (glycan binding proteins). Herein we describe a chemoenzymatic synthesis strategy to efficiently prepare an extensive library of fucosylated mucin core tandem repeats glycopeptides to elucidate the fine fucose-binding specificities of the Pseudomonas aeruginosa lectin LecB and the Clostridium difficile toxin A. Therefore, glycan core structures were decorated with terminal Lewis and H-antigens, which play critical roles in infection biology. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of the glycan and peptide backbone presentation of these terminal antigens in binding interactions with the two bacterial lectins.
|
|
| 7. |
- Behren, Sandra, et al.
(författare)
-
Fucose binding motifs on mucin core glycopeptides impact bacterial lectin recognition
- 2023
-
Ingår i: Angewandte Chemie International Edition. - 1433-7851 .- 1521-3773. ; 62:32
-
Tidskriftsartikel (refereegranskat)abstract
- Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P. aeruginosa lectin LecB and the C. difficile toxin A.
|
|
| 8. |
- Gabba, Adele, et al.
(författare)
-
MUC1 glycopeptide vaccine modified with a GalNAc glycocluster targets the macrophage galactose c-type lectin on dendritic cells to elicit an improved humoral response
- 2023
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 145:24, s. 13027-13037
-
Tidskriftsartikel (refereegranskat)abstract
- Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.
|
|
| 9. |
|
|
| 10. |
- Germann, Matthias, et al.
(författare)
-
Optical frequency comb Fourier transform spectroscopy of formaldehyde in the 1250 to 1390 cm−1 range : experimental line list and improved MARVEL analysis
- 2024
-
Ingår i: Journal of Quantitative Spectroscopy and Radiative Transfer. - : Elsevier. - 0022-4073 .- 1879-1352. ; 312
-
Tidskriftsartikel (refereegranskat)abstract
- We use optical frequency comb Fourier transform spectroscopy to record high-resolution, low-pressure, room-temperature spectra of formaldehyde (H212C16O) in the range of 1250 to 1390 cm−1. Through line-by-line fitting, we retrieve line positions and intensities of 747 rovibrational transitions: 558 from the ν6 band, 129 from the ν4 band, and 14 from the ν3 band, as well as 46 from four different hot bands. We incorporate the accurate and precise line positions (0.4 MHz median uncertainty) into the MARVEL (measured active vibration-rotation energy levels) analysis of the H2CO spectrum. This increases the number of MARVEL-predicted energy levels by 82 and of rovibrational transitions by 5382, and substantially reduces uncertainties of MARVEL-derived H2CO energy levels over a large range: from pure rotational levels below 200 cm−1 up to multiply excited vibrational levels at 6000 cm−1. This work is an important step toward filling the gaps in formaldehyde data in the HITRAN database.
|
|
| 11. |
- Germann, Matthias, et al.
(författare)
-
Precision frequency comb spectroscopy in the 8 µm range
- 2023
-
Ingår i: CLEO 2023. - : Optical Society of America. - 9781957171258
-
Konferensbidrag (refereegranskat)abstract
- We use Fourier transform spectroscopy based on a compact difference frequency generation comb source emitting around 8 μm to record broadband high-resolution spectra of molecules relevant to astrophysics and environmental monitoring. From the spectra we obtain line lists with sub-MHz accuracy, an order of magnitude better than previously available, and use them to refine theoretical models of these molecules. Here we report results for formaldehyde, for which the 8 μm range is missing in HITRAN.
|
|
| 12. |
- Halim, Adnan, et al.
(författare)
-
Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC-MS/MS of Glycopeptides.
- 2014
-
Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 13:12, s. 6024-32
-
Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
|
|
| 13. |
- Malaker, Stacy A., et al.
(författare)
-
The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins
- 2019
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:(15), s. 7278-7287
-
Tidskriftsartikel (refereegranskat)abstract
- Mucin-domain glycoproteins are found in nearly every tissue of the human body, and are important in biological processes ranging from embryogenesis to cancer. Because there are few tools to study mucin domains, their biological functions at the molecular scale remain unclear. Here, we help address a hurdle to the study of mucin-domain glycoproteins by characterizing a bacterial protease with selectivity for mucins. This mucinase selectively removes native mucins from cell surfaces and cuts them into fragments amenable to analysis.Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE’s unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.
|
|
| 14. |
- Pett, Christian, et al.
(författare)
-
Effective Assignment of α2,3/α2,6-Sialic Acid Isomers by LC-MS/MS-Based Glycoproteomics
- 2018
-
Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 57:30, s. 9320-9324
-
Tidskriftsartikel (refereegranskat)abstract
- Distinct structural changes of the α2,3/α2,6-sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high-throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large-scale glycoproteomics, thus allowing the site-specific structural characterization of sialic acid isomers.
|
|
| 15. |
- Wu, Xuanjun, et al.
(författare)
-
Protective Epitope Discovery and the Design of MUC1 Based Vaccine for Effective Tumor Protections in Immunotolerant Mice
- 2018
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:48, s. 16596-16609
-
Tidskriftsartikel (refereegranskat)abstract
- Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qβ carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qβ-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qβ-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.
|
|
| 16. |
- Wu, Xuanjun, et al.
(författare)
-
Synthesis and Immunological Evaluation of Disaccharide Bearing MUC-1 Glycopeptide Conjugates with Virus-like Particles
- 2019
-
Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 14:10, s. 2176-2184
-
Tidskriftsartikel (refereegranskat)abstract
- Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen–Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.
|
|
| 17. |
- Wu, Xuanjun, et al.
(författare)
-
Synthesis and immunological evaluation of the unnatural β-linked mucin-1 Thomsen-Friedenreich conjugate
- 2021
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 19:11, s. 2448-2455
-
Tidskriftsartikel (refereegranskat)abstract
- MUC1 glycopeptides are attractive antigens for anti-cancer vaccine development. One potential drawback in using the native MUC1 glycopeptide for vaccine design is the instability of theO-glycosyl linkage between the glycan and the peptide backbone to glycosidase. To overcome this challenge, a MUC1 glycopeptide mimic has been synthesized with the galactose-galactosamine disaccharide linked with threonine (Thomsen-Friedenreich or Tf antigen) through an unnatural β-glycosyl bond. The resulting MUC1-β-Tf had a much-enhanced stability toward a glycosidase capable of cleaving the glycan from the corresponding MUC1 glycopeptide with the natural α-Tf linkage. The MUC1-β-Tf was subsequently conjugated with a powerful carrier bacteriophage Qβ. The conjugate induced high levels of IgG antibodies in clinically relevant human MUC1 transgenic mice, which cross-recognized not only the natural MUC1-α-Tf glycopeptide but also MUC1 expressing tumor cells, supporting the notion that a simple switch of the stereochemistry of the glycan/peptide linkage can be a strategy for anti-cancer vaccine epitope design for glycopeptides.
|
|
| 18. |
- Yin, Zhaojun, et al.
(författare)
-
Antitumor Humoral and T Cell Responses by Mucin-1 Conjugates of Bacteriophage Qβ in Wild-type Mice
- 2018
-
Ingår i: ACS Chemical Biology. - : American Chemical Society. - 1554-8929 .- 1554-8937. ; 13:6, s. 1668-1676
-
Tidskriftsartikel (refereegranskat)abstract
- Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. In order to generate effective anti-MUC1 immune responses as potential anticancer vaccines, MUC1 peptides and glycopeptides have been covalently conjugated to bacteriophage Qβ. Immunization of mice with these constructs led to highly potent antibody responses with IgG titers over one million, which are among the highest anti-MUC1 IgG titers reported to date. Furthermore, the high IgG antibody levels persisted for more than six months. The constructs also elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The unique abilities of Qβ-MUC1 conjugates to powerfully induce both antibody and cytotoxic T cell immunity targeting tumor cells bode well for future translation of the constructs as anticancer vaccines.
|
|
| 19. |
- Yu, Jin, et al.
(författare)
-
Distinctive MS/MS Fragmentation Pathways of Glycopeptide-Generated Oxonium Ions Provide Evidence of the Glycan Structure.
- 2016
-
Ingår i: Chemistry (Weinheim an der Bergstrasse, Germany). - : Wiley. - 1521-3765. ; 22:3, s. 1114-1124
-
Tidskriftsartikel (refereegranskat)abstract
- Post-translational glycosylation of proteins play key roles in cellular processes and the site-specific characterisation of glycan structures is critical to understanding these events. Given the challenges regarding identification of glycan isomers, glycoproteomic studies generally rely on the assumption of conserved biosynthetic pathways. However, in a recent study, we found characteristically different HexNAc oxonium ion fragmentation patterns that depend on glycan structure. Such patterns could be used to distinguish between glycopeptide structural isomers. To acquire a mechanistic insight, deuterium-labelled glycopeptides were prepared and analysed. We found that the HexNAc-derived m/z 126 and 144 oxonium ions, differing in mass by H2 O, had completely different structures and that high-mannose N-glycopeptides generated abundant Hex-derived oxonium ions. We describe the oxonium ion decomposition mechanisms and the relative abundance of oxonium ions as a function of collision energy for a number of well-defined glycan structures, which provide important information for future glycoproteomic studies.
|
|
