Composition and in situ structure of the cell envelope and surface layer.

Archaea share genomic similarities with Eukarya and cellular architectural similarities with Bacteria, though archaeal and bacterial surface layers (S-layers) differ. Using cellular cryo-electron tomography, we visualized the S-layer lattice surrounding , a methanogenic archaeon. Though more compact than known structures, 's S-layer is a flexible hexagonal lattice of dome-shaped tiles, uniformly spaced from both the overlying cell sheath and the underlying cell membrane.

-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2).

The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the -glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid-capped -glycans. VEGFR2 is a highly glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer.

Altered Domain Structure of the Prion Protein Caused by Cu Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR.

The cellular isoform of the prion protein (PrP) serves as precursor to the infectious isoform (PrP), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu ions. PrP consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define.

Oligomannose Glycopeptide Conjugates Elicit Antibodies Targeting the Glycan Core Rather than Its Extremities.

Up to ∼20% of HIV-infected individuals eventually develop broadly neutralizing antibodies (bnAbs), and many of these antibodies (∼40%) target a region of dense high-mannose glycosylation on gp120 of the HIV envelope protein, known as the "high-mannose patch" (HMP). Thus, there have been numerous attempts to develop glycoconjugate vaccine immunogens that structurally mimic the HMP and might elicit bnAbs targeting this conserved neutralization epitope. Herein, we report on the immunogenicity of glycopeptides, designed by selection, that bind tightly to anti-HMP antibody 2G12.

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific -Glycosylation Profiles.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational -glycosylation on the CH2 domain, and the remodeling of the -linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody -glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody -glycosylation.

-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by tachyzoites.

is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by -mannose and -fucose in spp.

Glycomic and Proteomic Changes in Aging Brain Nigrostriatal Pathway.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy.

Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-containing Adhesins of the TRAP Family.

In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites.

Microfluidic Capillary Electrophoresis-Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides.

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers.

Asparagine-Linked Glycans of Contain a Single Long Arm, Are Barely Processed in the Endoplasmic Reticulum (ER) or Golgi, and Show a Strong Bias for Sites with Threonine.

causes severe diarrhea in infants in developing countries and in immunosuppressed persons, including those with AIDS. We are interested in the Asn-linked glycans (-glycans) of , because (1) the -glycan precursor is predicted to contain five mannose and two glucose residues on a single long arm nine mannose and three glucose residues on the three-armed structure common in host -glycans, (2) is a rare eukaryote that lacks the machinery for -glycan-dependent quality control of protein folding in the lumen of the Endoplasmic Reticulum (ER), and (3) ER and Golgi mannosidases, as well as glycosyltransferases that build complex -glycans, are absent from the predicted proteome. The -glycans reported here, which were determined using a combination of collision-induced dissociation and electronic excitation dissociation, contain a single, unprocessed mannose arm ± terminal glucose on the trimannosyl chitobiose core.

Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability.

Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

Human leukocyte antigen-antigen D related (HLA-DR) molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1593 non-redundant HLA-DR-presented peptides, derived from 870 source proteins.

Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1.

Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material.

Workflow for combined proteomics and glycomics profiling from histological tissues.

Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order the environment through which cells receive signals and communicate. Proteomic and glycomic molecular signatures from tissue surfaces can add diagnostic power to the immunohistochemistry workflows. Acquired in a spatially resolved manner, such proteomic and glycomic information can help characterize disease processes and be easily applied in a clinical setting.

Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.

A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step.

Identification of the major expressed S-layer and cell surface-layer-related proteins in the model methanogenic archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A.

Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics.

Epsilon-sarcoglycan mutations found in combination with other dystonia gene mutations.

Myoclonus-dystonia is a movement disorder associated with mutations in the epsilon-sarcoglycan gene (SGCE) in most families and in the DRD2 and DYT1 genes in two single families. In both of the latter families, we also found a mutation of SGCE. The molecular mechanisms through which the detected mutations may contribute to myoclonus-dystonia remain to be determined.