MUC1 Glycopeptide Vaccine Modified with a GalNAc Glycocluster Targets the Macrophage Galactose C-type Lectin on Dendritic Cells to Elicit an Improved Humoral Response.

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.

Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

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.

Fucose Binding Motifs on Mucin Core Glycopeptides Impact Bacterial Lectin Recognition.

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.

Erratum: Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation.

[This corrects the article DOI: 10.1016/j.isci.

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

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.

Synthesis and immunological evaluation of the unnatural β-linked mucin-1 Thomsen-Friedenreich conjugate.

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 the O-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.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

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.

Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation.

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.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

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.

Synthesis and Immunological Evaluation of Disaccharide Bearing MUC-1 Glycopeptide Conjugates with Virus-like Particles.

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 mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.

Mucin domains are densely -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.

Protective Epitope Discovery and Design of MUC1-based Vaccine for Effective Tumor Protections in Immunotolerant Mice.

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.

Antitumor Humoral and T Cell Responses by Mucin-1 Conjugates of Bacteriophage Qβ in Wild-type Mice.

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.

Effective Assignment of α2,3/α2,6-Sialic Acid Isomers by LC-MS/MS-Based Glycoproteomics.

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.

Induction of Antibodies Directed Against Branched Core O-Mannosyl Glycopeptides-Selectivity Complimentary to the ConA Lectin.

Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain development. Recent advances in glycoproteomics methodology and the "SimpleCell" strategy have enabled rapid identification of glycoproteins and specific glycosylation sites. Despite the enormous progress made, the biological impact of the mammalian O-mannosyl glycoproteome remains largely unknown to date.

Microarray Analysis of Antibodies Induced with Synthetic Antitumor Vaccines: Specificity against Diverse Mucin Core Structures.

Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases.

Distinctive MS/MS Fragmentation Pathways of Glycopeptide-Generated Oxonium Ions Provide Evidence of the Glycan Structure.

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.

Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides.

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.

A convergent strategy for the synthesis of type-1 elongated mucin cores 1-3 and the corresponding glycopeptides.

Mucins are a class of highly O-glycosylated proteins found on the surface of cells in epithelial tissues. O-Glycosylation is crucial for the functionality of mucins and changes therein can have severe consequences for an organism. With that in mind, the elucidation of interactions of carbohydrate binding proteins with mucins, whether in morbidly altered or unaltered conditions, continue to shed light on mechanisms involved in diseases like chronic inflammations and cancer.

A unified strategy for the synthesis of mucin cores 1-4 saccharides and the assembled multivalent glycopeptides.

By displaying different O-glycans in a multivalent mode, mucin and mucin-like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core-glycosylated amino acids was developed.