Site-Specific O-glycosylation of SARS-CoV-2 Spike Protein and Its Impact on Immune and Autoimmune Responses.

The world-wide COVID-19 pandemic has promoted a series of alternative vaccination strategies aiming to elicit neutralizing adaptive immunity in the human host. However, restricted efficacies of these vaccines targeting epitopes on the spike (S) protein that is involved in primary viral entry were observed and putatively assigned to viral glycosylation as an effective escape mechanism. Besides the well-recognized N-glycan shield covering SARS-CoV-2 spike (S) proteins, immunization strategies may be hampered by heavy O-glycosylation and variable O-glycosites fluctuating depending on the organ sites of primary infection and those involved in immunization. A further complication associated with viral glycosylation arises from the development of autoimmune antibodies to self-carbohydrates, including O-linked blood group antigens, as structural parts of viral proteins. This outline already emphasizes the importance of viral glycosylation in general and, in particular, highlights the impact of the site-specific O-glycosylation of virions, since this modification is independent of sequons and varies strongly in dependence on cell-specific repertoires of peptidyl-N-acetylgalactosaminyltransferases with their varying site preferences and of glycan core-specific glycosyltransferases. This review summarizes the current knowledge on the viral O-glycosylation of the SARS-CoV-2 spike protein and its impact on virulence and immune modulation in the host.

Revised structure model of norovirus-binding fucoidan from Undaria pinnatifida: oligofucose chains branch off from a ß6-galactane.

Fucoidans are discussed as antiviral agents, and fucoidan from Undaria pinnatifida (UpF), in particular has gained interest as potential food additive in antinoroviral strategies. As the competitive blocking activity of antinoroviral agents increases with the valency of terminal nonreducing fucose on the competitor, an effective processing of fucoidans to inhibitory oligosaccharides will depend on basic structural features of the polysaccharide. We demonstrate increased antiviral binding activity of processed low-mass UpF generated by hydrothermal degradation contrasting with decreased efficacy of low-mass fucoidan from Fucus vesiculosus. As this finding is in conflict with current structural models of UpF, we undertook a re-investigation of the glycan backbone in UpF. Applying solvolytical desulfation combined with enzymatic cleavage of low-mass fucoidan by endo-ß6-galactanase and terminal labeling of oligosaccharides by deutero-reduction and bis-5-phenyl-3-methyl-1-pyrazolone (PMP) substitution, evidence from mass spectrometry and methylation linkage analysis of the oligosaccharides indicates that fucoses and galactoses in the glycan backbone are organized in homomeric blocks, where oligo-fucoses branch off from a galactane-type core: Fuc(1-3Fuc)n1-3[Gal(1-6Gal)n1-6]Gal(1-6Gal)n.

Recombinant norovirus capsid protein VP1 (GII.4) expressed in H5 insect cells exhibits post-translational modifications with potential impact on lectin activity and vaccine design.

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 virus-like particles (VLPs) and in searches for food additives offering potential prophylactic or therapeutic applications an increasing number of reports refers to the use of 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.

Sensitive asprosin detection in clinical samples reveals serum/saliva correlation and indicates cartilage as source for serum asprosin.

The C-terminal pro-fibrillin-1 propeptide asprosin is described as white adipose tissue derived hormone that stimulates rapid hepatic glucose release and activates hunger-promoting hypothalamic neurons. Numerous studies proposed correlations of asprosin levels with clinical parameters. However, the enormous variability of reported serum and plasma asprosin levels illustrates the need for sensitive and reliable detection methods in clinical samples. Here we report on newly developed biochemical methods for asprosin concentration and detection in several body fluids including serum, plasma, saliva, breast milk, and urine. Since we found that glycosylation impacts human asprosin detection we analyzed its glycosylation profile. Employing a new sandwich ELISA revealed that serum and saliva asprosin correlate strongly, depend on biological sex, and feeding status. To investigate the contribution of connective tissue-derived asprosin to serum levels we screened two cohorts with described cartilage turnover. Serum asprosin correlated with COMP, a marker for cartilage degradation upon running exercise and after total hip replacement surgery. This together with our finding that asprosin is produced by primary human chondrocytes and expressed in human cartilage suggests a contribution of cartilage to serum asprosin. Furthermore, we determined asprosin levels in breast milk, and urine, for the first time, and propose saliva asprosin as an accessible clinical marker for future studies.

Fucoidan and Derived Oligo-Fucoses: Structural Features with Relevance in Competitive Inhibition of Gastrointestinal Norovirus Binding.

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-α-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 α-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.

Loss of zebrafish atp6v1e1b, encoding a subunit of vacuolar ATPase, recapitulates human ARCL type 2C syndrome and identifies multiple pathobiological signatures.

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 unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo.

Novel Class of Human Milk Oligosaccharides Based on 6'-Galactosyllactose Containing N-Acetylglucosamine Branches Extended by Oligogalactoses.

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 (GlcNAc) 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 ß1-3,4-specific galactosidase supported the presence of terminal galactose-linked ß1-6 to one of the two GlcNAc branches. The mass spectrometry glycomic data have been deposited at the GlycoPOST archive with the data set identifier GPST000191 (Username: franz.hanisch@uni-koeln.de; Password: Soma1Dita2Carb. Watanabe, Y. GlycoPOST realizes FAIR principles for glycomics mass spectrometry data. Nucleic Acids Res.2021,49, D1523-D1528).

Echovirus-30 Infection Alters Host Proteins in Lipid Rafts at the Cerebrospinal Fluid Barrier In Vitro.

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.

Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors.

Norovirus infections cause severe gastroenteritis in millions of people every year. Infection requires the recognition of histo-blood group antigens (HBGAs), but such interactions can be inhibited by human milk oligosaccharides (HMOs), which act as structurally-similar decoys. HMO supplements could help to prevent norovirus infections, but the industrial production of complex HMOs is challenging. Here we describe a large-scale fermentation process that yields several kilograms of lacto-N-fucopentaose I (LNFP I). The product was synthesized in Escherichia coli BL21(DE3) cells expressing a recombinant N-acetylglucosaminyltransferase, ß(1,3)galactosyltransferase and α(1,2)fucosyltransferase. Subsequent in vitro enzymatic conversion produced HBGA types A1 and B1 for norovirus inhibition assays. These carbohydrates inhibited the binding of GII.17 virus-like particles (VLPs) to type A1 and B1 trisaccharides more efficiently than simpler fucosylated HMOs, which were in turn more effective than any non-fucosylated structures. However, we found that the simpler fucosylated HMOs were more effective than complex molecules such as LNFP I when inhibiting the binding of GII.17 and GII.4 VLPs to human gastric mucins and mucins from human amniotic fluid. Our results show that complex fucosylated HMOs can be produced by large-scale fermentation and that a combination of simple and complex fucosylated structures is more likely to prevent norovirus infections.

Oligosaccharides and Viral Infection: Human Milk Oligosaccharides versus Algal Fucan-Type Polysaccharides.

Norovirus infections belong to the most common causes of human gastroenteritis worldwide, and epidemic outbreaks are responsible for hundreds of thousands deaths annually. Strikingly, no antiviral treatment is available due to the difficulty in cultivating virions or in generating a vaccine, and due to the fact that their infection mechanisms are poorly understood. However, there is consent that noroviruses bind to histo-blood group antigens (HBGAs) on their way through the digestive tract. The HBGA profiles vary individually, making people more or less susceptible to different norovirus strains. In our current work, we tried to decipher the HBGA specificity of the most prevalent and clinically relevant norovirus GII.4 subfamily (Sydney 2012, JX459908) and its preferences for human milk oligosaccharides (HMOs) as potential anti-infectives. The structural evidence provided can explain at the molecular level why individuals with certain blood groups are at higher risk of infection, and how these infections may be prevented and treated by application of food additives. A central finding was that low-affinity binding of HMOs is surpassed by high-avidity binding of multivalent oligo- and polyfucoses as found in algal polysaccharides (fucoidans). Insight into structural details of fucoidans and their impact on noroviral-blocking efficiency is provided and discussed.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells.

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 KI 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.

Redox Proteomes in Human Physiology and Disease Mechanisms.

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.

Pandemic GII.4 Sydney and Epidemic GII.17 Kawasaki308 Noroviruses Display Distinct Specificities for Histo-Blood Group Antigens Leading to Different Transmission Vector Dynamics in Pacific Oysters.

Noroviruses are the major cause of foodborne outbreaks of acute gastroenteritis, which are often linked to raw oyster consumption. Previous studies have suggested histo-blood group antigens (HBGA)-like structures in the oyster tissues as ligands for norovirus binding and persistence. To better understand how oysters function as vectors for the most common human noroviruses, we first tested the ability of the norovirus strains GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 strains to interact with oyster tissues. Secondly, we explored how the HBGA preferences of these strains can affect their persistence in oyster tissues. We found limited HBGA expression in oyster tissues. HBGAs of A and H type 1 were present in the digestive tissues and palps of the Pacific oyster Crassostrea gigas, while the gills and mantle lacked any HBGA structures. By using Virus-like particles (VLPs), which are antigenically and morphologically similar to native virions, we were able to demonstrate that VLPs of GI.1 West Chester norovirus reacted with the digestive tissues and palps. Despite of the lack of HBGA expression in mantle, dominant GII.4 Sydney strain readily bound to all the oyster tissues, including the digestive tissues, gills, palps, and mantle. In contrast, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the investigated oyster tissues was observed. In synthetic HBGA and saliva-binding assays, GI.1 reacted with A type, H type, and Leb (Lewis b) HBGAs. GII.4 Sydney VLPs showed a broad binding pattern and interacted with various HBGA types. Compared to GI.1 and GII.4 VLPs, the GII.17 Kawasaki308 VLPs only weakly associated with long-chain saccharides containing A type, B type, H type, and Leb blood group epitopes. Our findings indicate that GI.1 and GII.4 noroviruses are likely to be concentrated in oysters, by binding to HBGA-like glycans, and therefore potentially leading to increased long term transmission. In regards to the GII.17 Kawasaki308 strain, we suggest that oysters can only function as short term transmission vector in periods of high environmental virus concentrations.

Functional Roles of O-Glycosylation.

O-Glycosylation in general has impact on a diversity of biological processes covering cellular aspects (targeted transport of glycoproteins), molecular aspects (protein conformation, resistance to proteolysis), and aspects involved in cellular communication (cell-cell and cell-matrix interaction). [...].

Avidity of α-fucose on human milk oligosaccharides and blood group-unrelated oligo/polyfucoses is essential for potent norovirus-binding targets.

There is agreement with respect to norovirus infection routes in humans regarding binding of the pathogen to gastrointestinal epithelia via recognition of blood group-active mucin-typeO-glycans as the initiating and essential event. Among food additives playing a potential role in applications to protect newborns, human milk oligosaccharides (HMOs) as competitors are of major importance. By focusing on fractions of high-molecular mass HMOs with high fucose contents, we attempted to identify the structural elements required for norovirus GII.4 (Sydney 2012, JX459908) capsid binding in neoglycolipid-based arrays. We provide evidence that HMO fractions with the strongest binding capacities contained hepta- to decasaccharides expressing branches with terminal blood group H1 or Lewis-b antigen. H2 antigen, as recognized by UEA-I lectin, is apparently not expressed in high-mass HMOs. Beyond affinity, sterical and valency effects contribute more to virus-like particle binding, as revealed for oligovalent fucose conjugates of α-cyclodextrin and oligofucoses from fucoidan. Accordingly, high-mass HMOs with oligovalent fucose can exhibit stronger binding capacities compared with monovalent fucose HMOs. The above features were revealed for the most clinically relevant and prevalent GII.4 strain and are distinct from other strains, like GII.10 (Vietnam 026, AF504671), which showed a preference for blood group Lewis-a positive glycans.

The Double Face of Mucin-Type O-Glycans in Lectin-Mediated Infection and Immunity.

Epithelial human blood group antigens (HBGAs) on O-glycans play roles in pathogen binding and the initiation of infection, while similar structures on secretory mucins exert protective functions. These double-faced features of O-glycans in infection and innate immunity are reviewed based on two instructive examples of bacterial and viral pathogens. Helicobacter pylori represents a class 1 carcinogen in the human stomach. By expressing blood group antigen-binding adhesin (BabA) and LabA adhesins that bind to Lewis-b and LacdiNAc, respectively, H. pylori colocalizes with the mucin MUC5AC in gastric surface epithelia, but not with MUC6, which is cosecreted with trefoil factor family 2 (TFF2) by deep gastric glands. Both components of the glandular secretome are concertedly up-regulated upon infection. While MUC6 expresses GlcNAc-capped glycans as natural antibiotics for H. pylori growth control, TFF2 may function as a probiotic lectin. In viral infection human noroviruses of the GII genogroup interact with HBGAs via their major capsid protein, VP1. HBGAs on human milk oligosaccharides (HMOs) may exert protective functions by binding to the P2 domain pocket on the capsid. We discuss structural details of the P2 carbohydrate-binding pocket in interaction with blood group H/Lewis-b HMOs and fucoidan-derived oligofucoses as effective interactors for the most prevalent norovirus strains, GII.4 and GII.17.

Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations.

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

Autosomal Tubulointerstitial Kidney Disease-MUC1 Type: Differential Proteomics Suggests that Mutated MUC1 (insC) Affects Vesicular Transport in Renal Epithelial Cells.

Autosomal dominant tubulointerstitial kidney disease associated to the MUC1 gene (ADTKD-MUC1; formerly MCKD1) belongs to a heterogeneous group of rare hereditary kidney diseases that is prototypically caused by frameshift mutations in the MUC1 repeat domain. The mutant MUC1 (insC) lacks the transmembrane domaine, exhibits aberant cellular topology, and hence might gain a function during the pathological process. To get insight into potential pathomechanisms we perform differential proteomics of extracellular vesicles shed by renal epithelia into the urine of patients. The study is based on three ADTKD patients and individual controls applying iTRAQ/LC-MS/MS. A total of 796 proteins were identified across all biological and technical replicates, and 298 proteins were quantified. A proportion of 47 proteins were fold-changed species. GO Term Enrichment analysis revealed proteins with significantly changed expression in ADTKD-associated extracellular vesicles as vesicular transport-associated proteins. Among these VTA1 is involved in the endosomal multivesicular body pathway associated with transport to lysosomes or export via exosomes. VTA1 is also claimed to play roles as a cofactor of the AAA ATPases VPS4A and VPS4B in the disassembly of ESCRT III. Protein interaction databases list VPS4B, CHMP2A, and IST1 as VTA1 binding partners. (Data are available via ProteomeXchange with identifier PXD008389.).

Human Milk Oligosaccharides as Promising Antivirals.

Human milk oligosaccharides (HMOs) are diverse unconjugated carbohydrates that are highly abundant in human breast milk. These glycans are investigated in the context of exhibiting multiple functions in infant growth and development. They seem to provide protection against infectious diseases, including a number of poorly manageable viral infections. Although the potential mechanism of the HMO antiviral protection is rather broad, much of the current experimental work has focused on studying of HMO antiadhesive properties. HMOs may mimic structures of viral receptors and block adherence to target cells, thus preventing infection. Still, the potential of HMOs as a source for new antiviral drugs is relatively unexploited. This can be partly attributed to the extreme complexity of the virus-carbohydrate interactions and technical difficulties in HMO isolation, characterization, and manufacturing procedures. Fortunately, we are currently entering a period of major technological advances that have enabled deeper insights into carbohydrate mediated viral entry, rational selection of HMOs as anti-entry inhibitors, and even evaluation of individual synthetic HMO structures. Here, we provide an up-to-date review on glycan binding studies for rotaviruses, noroviruses, influenza viruses, and human immunodeficiency viruses. We also discuss the preventive and therapeutic potential of HMOs as anti-entry inhibitors and address challenges on the route from fundamental studies to clinical trials.

Classical Galactosemia: Insight into Molecular Pathomechanisms by Differential Membrane Proteomics of Fibroblasts under Galactose Stress.

Classical galactosemia, a hereditary metabolic disease caused by the deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), results in an impaired galactose metabolism and serious long-term developmental affection of the CNS and ovaries, potentially related in part to endogenous galactose-induced protein dysglycosylation. In search for galactose-induced changes in membrane raft proteomes of GALT-deficient cells, we performed differential analyses of lipid rafts from patient-derived (Q) and sex- and age-matched control fibroblasts (H) in the presence or absence of the stressor. Label-based proteomics revealed of the total 454 (female) or 678 (male) proteins a proportion of ∼12% in at least one of four relevant ratios as fold-changed. GALT(-) cell-specific effects in the absence of stressor revealed cell-model-dependent affection of biological processes related to protein targeting to the plasma membrane (female) or to cellular migration (male). However, a series of common galactose-induced effects were observed, among them the strongly increased ER-stress marker GRP78 and calreticulin involved in N-glycoprotein quality control. The membrane-anchored N-glycoprotein receptor CD109 was concertedly decreased under galactose-stress together with cadherin-13, GLIPR1, glypican-1, and semaphorin-7A. A series of proteins showed opposite fold-changes in the two cell models, whereas others fluctuated in only one of the two models.