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Although surface proteins of most enveloped viruses are glycosylated, among non-enveloped viruses only few express glycoproteins in their capsid as infective virions. Noroviruses belong to the latter group and are known to express one major capsid protein (VP1) that lacks genuine glycosylation. In the context of vaccine development based on VLPs and in searches for food additives offering potential prophylactic or therapeutic applications an increasing number of reports refers to the use of virus-like particles (VLPs) that were produced as secretory products in insect cells. We asked the question whether recombinant VLPs (GII.4 Sydney, 2012) produced via the baculovirus vector in H5 insect cells may be glycosylated in the protruding domains that are involved in receptor binding and immune reactivity. Mass spectrometric analysis of tryptic VP1 peptides prior to and after beta-elimination Michael addition in 70% ethylamine revealed Thr238, and Ser519 in the P1 domain, and Thr350, Thr369, Thr371, and Thr381 in the P2 domain as modified. Thr65, Ser67, and Thr350 were revealed by liquid chromatography-mass spectrometry to carry HexNAc or Hex-HexNAc modifications, respectively. Monosaccharide analysis by gas chromatography-mass spectrometry confirmed the presence of GlcNAc on VLP protein, whereas immunoassays with lectins and antibodies demonstrated O-linked GlcNAc on VP1 protein. Post-translational modifications of virus capsid proteins may contribute to a modulation of immunodominant surface epitopes and need to be considered in anti-norovirus vaccine design. Some modifications are located near amino acid side chains involved in the binding of blood group active sugar receptors.

Author summary Cutis laxa syndromes are pleiotropic disorders of the connective tissue, characterized by skin redundancy and variable systemic manifestations. Cutis laxa syndromes are caused by pathogenic variants in genes encoding structural and regulatory components of the extracellular matrix or in genes encoding components of cellular trafficking, metabolism, and mitochondrial function. Pathogenic variants in genes coding for vacuolar-ATPases, a multisubunit complex responsible for the acidification of multiple intracellular vesicles, cause type 2 cutis laxa syndromes, a group of cutis laxa subtypes further characterized by neurological, skeletal, and rarely cardiopulmonary manifestations. To investigate the pathomechanisms of vacuolar-ATPase dysfunction, we generated zebrafish models that lack a crucial subunit of the vacuolar-ATPases. The mutant zebrafish models show morphological and functional features reminiscent of the phenotypic manifestations in cutis laxa patients carrying pathogenic variants in ATP6V1E1. In-depth analysis at multiple -omic levels identified biological signatures that indicate impairment of signaling pathways, lipid metabolism, and mitochondrial respiration. We anticipate that these data will contribute to a better understanding of the pathogenesis of cutis laxa syndromes and other disorders involving defective v-ATPase function, which may eventually improve patient treatment and management. The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely

Norovirus infections belong to the most common causes of human gastroenteritis worldwide and epidemic outbreaks are responsible for hundreds of thousands of deaths annually. In humans, noroviruses are known to bind to gastrointestinal epithelia via recognition of blood-group active mucin-type O-glycans. Considering the involvement of l-alpha-fucose residues in these glycans, their high valency on epithelial surfaces far surpasses the low affinity, though specific interactions of monovalent milk oligosaccharides. Based on these findings, we attempted to identify polyfucoses (fucans) with the capacity to block binding of the currently most prevalent norovirus strain GII.4 (Sydney, 2012, JX459908) to human and animal gastrointestinal mucins. We provide evidence that inhibitory effects on capsid binding are exerted in a competitive manner by alpha-fucosyl residues on Fucus vesiculosus fucoidan, but also on the galacto-fucan from Undaria pinnatifida and their oligo-fucose processing products. Insight into novel structural aspects of fucoidan and derived oligosaccharides from low-mass Undaria pinnatifida were revealed by GCMS and MALDI mass spectrometry. In targeting noroviral spread attenuation, this study provides first steps towards a prophylactic food additive that is produced from algal species.

Human milk oligosaccharides (HMOs) have attracted much attention in recent years not only as a prebiotic factor but also in particular as an essential component of infant nutrition in relation to their impact on innate immunity. The backbone structures of complex HMOs generally contain single or repetitive lacto-N-biose (type 1) or lactosamine (type 2) units in either linear or branched chains extending from a lactose core. While all known branched structures originate from the 3,6-substitution of the lactosyl core galactose, we here describe a new class of HMOs that tentatively branch at the terminal galactose of 6'-galactosyllactose. Another novel feature of this class of HMOs was found in linear oligo-galactosyl chains linked to one of the N-acetylglucosamine (GIcNAc) branches. The novel structures exhibit general formulas with hexose versus hexosamine contents of 5/2 to 8/2 and can be designated as high-galactose (HG)-HMOs. In addition, up to three fucosyl residues are linked to the octa- to dodecasaccharides, which were detected in two human milk samples from the Lewis blood-group-defined donors. Structural analyses of methylated glycans and their alditols comprised matrix-assisted laser desorption ionization mass spectrometry, electrospray-(collision-induced dissociation) mass spectrometry and linkage analyses by gas chromatography-mass spectrometry of the derived partially methylated alditol acetates. Enzymatic degradation by the application of beta 1-3,4-specific galactosidase supported the presence of terminal galactose-linked beta 1-6 to one of the two GIcNAc branches. The mass spectrometry glycomic data have been deposited at the GlycoPOST archive with the data set identifier GPST000191 (Username: franz@uni-kowln.de; Password: Soma1Dita2Carb. Watanabe, Y. GlycoPOST realizes FAIR principles for glycomics mass spectrometry data.

Redox proteomics is a field of proteomics that is concerned with the characterization of the oxidation state of proteins to gain information about their modulated structure, function, activity, and involvement in different physiological pathways. Oxidative modifications of proteins have been shown to be implicated in normal physiological processes of cells as well as in pathomechanisms leading to the development of cancer, diabetes, neurodegenerative diseases, and some rare hereditary metabolic diseases, like classic galactosemia. Reactive oxygen species generate a variety of reversible and irreversible modifications in amino acid residue side chains and within the protein backbone. These oxidative post-translational modifications (Ox-PTMs) can participate in the activation of signal transduction pathways and mediate the toxicity of harmful oxidants. Thus the application of advanced redox proteomics technologies is important for gaining insights into molecular mechanisms of diseases. Mass-spectrometry-based proteomics is one of the most powerful methods that can be used to give detailed qualitative and quantitative information on protein modifications and allows us to characterize redox proteomes associated with diseases. This Review illustrates the role and biological consequences of Ox-PTMs under basal and oxidative stress conditions by focusing on protein carbonylation and S-glutathionylation, two abundant modifications with an impact on cellular pathways that have been intensively studied during the past decade.

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a K-I of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

Echovirus-30 (E-30) is a non-polio enterovirus responsible for meningitis outbreaks in children worldwide. To gain access to the central nervous system (CNS), E-30 first has to cross the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCSFB). E-30 may use lipid rafts of the host cells to interact with and to invade the BCSFB. To study enteroviral infection of the BCSFB, an established in vitro model based on human immortalized brain choroid plexus papilloma (HIBCPP) cells has been used. Here, we investigated the impact of E-30 infection on the protein content of the lipid rafts at the BCSFB in vitro. Mass spectrometry analysis following E-30 infection versus uninfected conditions revealed differential abundancy in proteins implicated in cellular adhesion, cytoskeleton remodeling, and endocytosis/vesicle budding. Further, we evaluated the blocking of endocytosis via clathrin/dynamin blocking and its consequences for E-30 induced barrier disruption. Interestingly, blocking of endocytosis had no impact on the capacity of E-30 to induce loss of barrier properties in HIBCPP cells. Altogether, these data highlight the impact of E-30 on HIBCPP cells microdomain as an important factor for host cell alteration.