Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Current Understanding of the Structure and Function of Fungal Immunomodulatory Proteins
    Liu, Yusi ; Bastiaan-Net, Shanna ; Wichers, Harry J. - \ 2020
    Frontiers in Nutrition 7 (2020). - ISSN 2296-861X
    FIPs - glycosylation - hemagglutination - immunomodulaion - immunomodulatory proteins - structure-function relationship

    Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi, which are extensively studied for their immunomodulatory activity. Currently, more than 38 types of FIPs have been described. Based on their conserved structure and protein identity, FIPs can be classified into five subgroups: Fve-type FIPs (Pfam PF09259), Cerato-type FIPs (Pfam PF07249), PCP-like FIPs, TFP-like FIPs, and unclassified FIPs. Among the five subgroups, Fve-type FIPs are the most studied for their hemagglutinating, immunomodulating, and anti-cancer properties. In general, these small proteins consist of 110–125 amino acids, with a molecular weight of ~13 kDa. The other four subgroups are relatively less studied, but also show a noticeable influence on immune cells. In this review, we summarized the protein modifications, 3-dimensional structures and bioactivities of all types of FIPs. Moreover, structure-function relationship of FIPs has been discussed, including relationship between carbohydrate binding module and hemagglutination, correlation of oligomerization and cytokine induction, relevance of glycosylation and lymphocyte activation. This summary and discussion may help gain comprehensive understanding of FIPs' working mechanisms and scope future studies.

    Serum Protein N-Glycans in Colostrum and Mature Milk of Chinese Mothers
    Elwakiel, Mohèb ; Bakx, Edwin J. ; Szeto, Ignatius M. ; Li, Yitong ; Hettinga, Kasper A. ; Schols, Henk A. - \ 2020
    Journal of Agricultural and Food Chemistry 68 (2020)25. - ISSN 0021-8561 - p. 6873 - 6883.
    glycoproteins - glycosylation - intestinal mucosal barrier - oligosaccharides

    To study the Chinese human milk N-glycome over lactation, N-glycans were released and separated from serum proteins, purified by solid-phase extraction, and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 66 different putative N-glycans were found in the colostrum (week 1) and mature milk (week 4) of seven Chinese mothers. A clear difference was observed between milk of five secretor and two nonsecretor mothers, based on the type and relative amounts of the individual N-glycans. The relative levels of the total neutral nonfucosylated and the fucosylated N-glycans in milk of five secretor mothers increased and decreased over lactation, respectively. This pattern could not be observed for the milk from the two nonsecretor mothers. Overall, this was the first study that provided detailed information on individual N-glycans in milk among mothers and over time as well as that fucosylation of N-glycans in milk was associated with the mother's secretor status.

    Modification of Prenylated Stilbenoids in Peanut (Arachis hypogaea) Seedlings by the Same Fungi That Elicited Them : The Fungus Strikes Back
    Aisyah, Siti ; Gruppen, Harry ; Slager, Mathijs ; Helmink, Bianca ; Vincken, Jean Paul - \ 2015
    Journal of Agricultural and Food Chemistry 63 (2015)42. - ISSN 0021-8561 - p. 9260 - 9268.
    Arachis hypogaea - Aspergillus oryzae - detoxification - fungal metabolism - glycosylation - groundnut - mass spectrometry - oxidative cleavage - peanut - Rhizopus oryzae

    Aspergillus oryzae and Rhizopus oryzae were compared for inducing the production of prenylated stilbenoids in peanut seedlings. The fungus was applied at two different time points: directly after soaking (day 1) or after 2 days of germination (day 3). Aspergillus- and Rhizopus-elicited peanut seedlings accumulated an array of prenylated stilbenoids, with overlap in compounds induced, but also with compounds specific to the fungal treatment. The differences were confirmed to be due to modification of prenylated stilbenoids by the fungus itself. Each fungus appeared to deploy different strategies for modification. The content of prenylated stilbenoids modified by fungi accounted for around 8% to 49% (w/w) of total stilbenoids. The contents of modified prenylated stilbenoids were higher when the fungus was applied on day 1 instead of day 3. Altogether, type of fungus and time point of inoculation appeared to be crucial parameters for optimizing accumulation of prenylated stilbenoids in peanut seedlings.

    Label-Free Glycoprofiling with Multiplex Surface Plasmon Resonance: A Tool To Quantify Sialylation of Erythropoietin
    Geuijen, K.P.M. ; Halims, L.A. ; Schellekens, H. ; Schasfoort, R.B. ; Wijffels, R.H. ; Eppink, M.H.M. - \ 2015
    Analytical Chemistry 87 (2015)16. - ISSN 0003-2700 - p. 8115 - 8122.
    liquid chromatography/mass spectrometry - recombinant-human-erythropoietin - lectin microarray - biological function - real-time - cho-cells - glycosylation - carbohydrate - glycoproteins - hemoglobin
    Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.
    Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans
    Westerhof, L.B. ; Wilbers, R.H.P. ; Raaij, D.R. van; Nguyen, D. ; Goverse, A. ; Henquet, M.G.L. ; Hokke, C.H. ; Bosch, D. ; Bakker, J. ; Schots, A. - \ 2014
    Plant Biotechnology Journal 12 (2014)9. - ISSN 1467-7644 - p. 1333 - 1342.
    monoclonal-antibodies - subcellular-localization - recombinant antibodies - hybrid immunoglobulin - terminal propeptide - insect cells - heavy-chain - glycosylation - protein - infliximab
    The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1¿ and IgG1¿ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-a, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1¿-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1¿- and IgG1¿-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1¿-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core a1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core a1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.
    Glycoform-selective prion formation in sporadic and familial forms of prion disease
    Xiao, X. ; Yuan, J. ; Haïk, S. ; Cali, I. ; Zhan, Y. ; Moudjou, M. ; Li, B. ; Laplanche, J.L. ; Laude, H. ; Langeveld, J.P.M. ; Gambetti, P. - \ 2013
    PLoS ONE 8 (2013)3. - ISSN 1932-6203 - 9 p.
    creutzfeldt-jakob-disease - prpsc formation - protein - scrapie - glycosylation - mutation - antibody - cells - susceptibility - conformers
    The four glycoforms of the cellular prion protein (PrP(C)) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrP(Sc)) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrP(Sc) in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJD(V180I)) or from Thr to Ala at residue 183 (fCJD(T183A)). Here we report that fCJD(V180I), but not fCJD(T183A), exhibits a proteinase K (PK)-resistant PrP (PrP(res)) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrP(res) species in both fCJD(V180I) and VPSPr is likewise attributable to the absence of PrP(res) glycosylated at the first N-linked glycosylation site at residue 181, as in fCJD(T183A). In contrast to fCJD(T183A), both VPSPr and fCJD(V180I) exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrP(V180I) with a typical glycoform profile from cultured cells generates detectable PrP(res) that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJD(V180I) share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrP(C) to PrP(Sc) is inhibited, probably by a dominant-negative effect, or by other co-factors.
    UHPLC/PDA–ESI/MS Analysis of the Main Berry and Leaf Flavonol Glycosides from Different Carpathian Hippophaë rhamnoides L. Varieties
    Pop, R.M. ; Socaciu, C. ; Pintea, A. ; Buzoianu, A.D. ; Sanders, M.G. ; Gruppen, H. ; Vincken, J.P. - \ 2013
    Phytochemical Analysis 24 (2013)5. - ISSN 0958-0344 - p. 484 - 492.
    isotope dilution assay - sea buckthorn berries - mass-spectrometry - ms analysis - hplc - glycosylation - cultivars - stability - storage - juice
    Introduction - Sea buckthorn (Hippophaë rhamnoides L.) is known to be rich in many bioactive compounds (such as vitamins, phenolics, carotenoids) important for human health and nutrition. Among the phenolics, berries and leaves contain a wide range of flavonols that are good quality and authenticity biomarkers. Objective - To compare the composition of the main flavonols of Romanian sea buckthorn berry and leaf varieties and to identify the specific biomarkers that contribute to sample differentiation among varieties. Material and methods - Six varieties of cultivated sea buckthorn (ssp. Carpatica) berries and leaves were analysed by UHPLC/PDA–ESI/MS. Results - Berries and leaves contained mainly isorhamnetin (I) glycosides in different ratios. Whereas I-3-neohesperidoside, I-3-glucoside, I-3-rhamnosylglucoside, I-3-sophoroside-7-rhamnoside and free isorhamnetin were predominant for berries (out of 17 compounds identified), I-3-rhamnosylglucoside, I-3-neohesperidoside, I-3-glucoside, quercetin-3-pentoside, kaempferol-3-rutinoside, and quercetin-3-glucoside were predominant in leaves (out of 19 compounds identified). Berries contained, on average, 917¿mg/100¿g DW flavonol glycosides. Leaves had higher content of flavonol glycosides than berries, on average 1118¿mg/100¿g DW. The variation of the quantitative dataset analysed using principal component analysis accounted for 91% of the total variance in the case of berries and 73% in case of leaves, demonstrating a good discrimination among samples. Conclusion - Based on quantitative analysis, by principal component analysis, the flavonol derivatives can be considered as biomarkers to discriminate among varieties and to recognise specifically the berry versus leaf composition
    Effective Chikungunya Virus-like Particle Vaccine Produced in Insect Cells
    Metz, S.W.H. ; Gardner, J. ; Geertsema, C. ; Le, T.T. ; Goh, L. ; Vlak, J.M. ; Suhrbier, A. ; Pijlman, G.P. - \ 2013
    PLoS Neglected Tropical Diseases 7 (2013)3. - ISSN 1935-2727
    equine encephalitis-virus - envelope proteins - baculovirus vectors - inactivated vaccine - expression system - dna vaccines - immunogenicity - infection - opportunities - glycosylation
    The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV) causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.
    Identification of alg3 in the mushroom-forming fungus Schizophyllum commune and analysis of the ¿alg3 knockout mutant
    Berends, E. ; Lehle, L. ; Henquet, M. ; Hesselink, T. ; Wösten, H.A.B. ; Lugones, L.G. ; Bosch, H.J. - \ 2013
    Glycobiology 23 (2013)2. - ISSN 0959-6658 - p. 147 - 154.
    saccharomyces-cerevisiae - n-glycan - gene - glycoproteins - enzyme - mannosyltransferase - glucocerebrosidase - oligosaccharides - glycosylation - therapy
    Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Delta alg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.
    Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin
    Vries, R.P. de; Smit, C.H. ; Bruin, E. de; Rigter, A. ; Vries, E. de; Cornelissen, A.H.M. ; Eggink, D. ; Chung, N.P.Y. ; Moore, J.P. ; Sanders, R.W. ; Hokke, C.H. ; Koopmans, M.P.G. ; Rottier, P.J.M. ; Haan, C.A.M. de - \ 2012
    Journal of Virology 86 (2012)21. - ISSN 0022-538X - p. 11735 - 11744.
    influenza-virus hemagglutinin - receptor-binding - carbohydrate moiety - dc-sign - glycosylation - glycoprotein - proteins - antibody - recognition - specificity
    Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH53) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2 moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.
    Characterization of the single-chain Fv-Fc antibody MBP10 produced in Arabidopsis alg3 mutant seeds
    Henquet, M.G.L. ; Eigenhuijsen, J. ; Hesselink, T. ; Spiegel, H. ; Schreuder, M.E.L. ; Duijn, E. van; Cordewener, J.H.G. ; Depicker, A. ; Krol, A.R. van der; Bosch, H.J. - \ 2011
    Transgenic Research 20 (2011)5. - ISSN 0962-8819 - p. 1033 - 1042.
    n-acetylglucosaminyltransferase-i - human beta-1,4-galactosyltransferase - monoclonal-antibody - mass-spectrometry - transgenic plants - linked glycans - glycosylation - cells - protein
    ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man5GlcNAc2 mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man5GlcNAc2 compared to Man8GlcNAc2 and Man7GlcNAc2 isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins
    Influence of Roasting on the Antioxidant Activity and HMF Formation of a Cocoa Bean Model Systems
    Oliviero, T. ; Capuano, E. ; Cämmerer, B. ; Fogliano, V. - \ 2009
    Journal of Agricultural and Food Chemistry 57 (2009)1. - ISSN 0021-8561 - p. 147 - 152.
    maillard reaction-products - coffee - glycosylation - melanoidins - components - capacity - glycine - lactose - xylose - color
    During the roasting of cocoa beans chemical reactions lead to the formation of Maillard reaction (MR) products and to the degradation of catechin-containing compounds, which are very abundant in these seeds. To study the modifications occurring during thermal treatment of fat and antioxidant rich foods, such as cocoa, a dry model system was set up and roasted at 180 °C for different times. The role played in the formation of MR products and in the antioxidant activity of the system by proteins, catechin, and cocoa butter was investigated by varying the model system formulation. Results showed that the antioxidant activity decreased during roasting, paralleling catechin concentration, thus suggesting that this compound is mainly responsible for the antioxidant activity of roasted cocoa beans. Model system browning was significantly higher in the presence of catechin, which contributed to the formation of water-insoluble melanoidins, which are mainly responsible for browning. HMF concentration was higher in casein-containing systems, and its formation was strongly inhibited in the presence of catechin. No effects related to the degree of lipid oxidation could be observed. Data from model systems obtained by replacing fat with water showed a much lower rate of MR development and catechin degradation but the same inhibitory effect of catechin on HMF formation.
    LEW3, encoding a putative a-1,2-mannosyltransferase (ALG11) in N-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in Arabidopsis thaliana
    Zhang, M. ; Henquet, M.G.L. ; Chen, Z. ; Zhang, H. ; Zhang, Y. ; Ren, X. ; Krol, A.R. van der; Gonneau, M. ; Bosch, H.J. ; Gong, Z. - \ 2009
    The Plant Journal 60 (2009)6. - ISSN 0960-7412 - p. 983 - 999.
    unfolded protein response - abscisic-acid - endoplasmic-reticulum - insensitive mutants - sugar responses - guard-cells - gene - glycosylation - tolerance - glycans
    N-linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with assembly of a dolichol-linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an a-1,2-mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol-linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N-glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low-level accumulation of Man3GlcNAc2 and Man4GlcNAc2 glycans, structures that are seldom detected in wild-type plants. In addition, the lew3 mutant has low levels of normal high-mannose-type glycans, but increased levels of complex-type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N-glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild-type. These results demonstrate that protein N-glycosylation plays crucial roles in plant development and the response to abiotic stresses
    Methionine Sulfoxides on PrPSc: A Prion-Specific Covalent Signature
    Canello, T. ; Engelstein, R. ; Moshel, O. ; Xanthopoulos, K. ; Langeveld, J.P.M. ; Sklaviadis, T. ; Gasset, M. ; Gabizon, R. - \ 2008
    Biochemistry 47 (2008)34. - ISSN 0006-2960 - p. 8866 - 8873.
    in-vitro - oxidative stress - protein - scrapie - disease - glycosylation - purification - proteasome - conversion - complexes
    Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrPSc, an aberrant form of the membrane protein PrPC. In the absence of an established form-specific covalent difference, the infectious properties of PrPSc were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27¿30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of ¿PrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrPSc are present as methionine sulfoxides this modification could not be found on brain PrPC as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrPSc differs both within and between species, adding another diversity factor to the structure of PrPSc molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrPSc and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.
    Predicting sub-Golgi localization of type II membrane proteins
    Dijk, A.D.J. van; Bosch, H.J. ; Braak, C.J.F. ter; Krol, A.R. van der; Ham, R.C.H.J. van - \ 2008
    Bioinformatics 24 (2008)16. - ISSN 1367-4803 - p. 1779 - 1786.
    subcellular-localization - transmembrane domains - lipid-bilayers - pathway - system - model - glycosyltransferases - glycosylation - retention - selection
    Motivation: Recent research underlines the importance of finegrained knowledge on protein localization. In particular, subcompartmental localization in the Golgi apparatus is important, for example, for the order of reactions performed in glycosylation pathways or the sorting functions of SNAREs, but is currently poorly understood. Results: We assemble a dataset of type II transmembrane proteins with experimentally determined sub-Golgi localizations and use this information to develop a predictor based on the transmembrane domain of these proteins, making use of a dedicated proteinstructure based kernel in an SVM. Various applications demonstrate the power of our approach. In particular, comparison with a large set of glycan structures illustrates the applicability of our predictions on a `glycomic¿ scale and demonstrates a significant correlation between sub-Golgi localization and the ordering of different steps in glycan biosynthesis
    Aggregation of -Lactoglobulin Regulated by Glucosylation
    Broersen, K. ; Elshof, E. ; Groot, J. de; Voragen, A.G.J. ; Hamer, R.J. ; Jongh, H.H.J. de - \ 2007
    Journal of Agricultural and Food Chemistry 55 (2007)6. - ISSN 0021-8561 - p. 2431 - 2437.
    heat-induced aggregation - amyloid formation - fibril formation - protein - glycoprotein - stability - hydrophobicity - glycosylation - calreticulin - denaturation
    A large number of proteins are glycosylated, either in vivo or as a result of industrial processing. Even though the effect of glycosylation on the aggregation of proteins has been studied extensively in the past, some reports show that the aggregation process is accelerated, whereas others found that the process is inhibited by glycosylation. This paper investigates the reasons behind these controversial results as well as the potential mechanism of the effect of glucosylation on aggregation using bovine -lactoglobulin as a model. Glucosylation was found to inhibit denaturant-induced aggregation, whereas heat-induced aggregation was accelerated. It was also found that the kinetic partitioning from an unfolded state was driven toward refolding for glucosylated protein, whereas aggregation was the preferred route for the nonglucosylated protein. Keywords: Aggregation; glucosylation; -lactoglobulin; hydrophobicity; electrostatic repulsion; unfolding/refolding
    Absence of N-linked glycans from the F2 subunit of the major baculovirus envelope fusion protein F enhances fusogenicity
    Long, G. ; Pan, X. ; Vlak, J.M. - \ 2007
    Journal of General Virology 88 (2007)2. - ISSN 0022-1317 - p. 441 - 449.
    newcastle-disease-virus - membrane-fusion - oligosaccharide chains - influenza-virus - intracellular-transport - endoplasmic-reticulum - cell-surface - glycosylation - hemagglutinin - neuraminidase
    The F protein is the major glycoprotein present in the envelopes of budded virus (BV) of members of the family Baculoviridae. The F protein mediates low-pH-activated fusion with insect cell membranes. Baculovirus F proteins are synthesized as a precursor (F0) and cleaved post-translationally into two disulfide-bonded subunits, F1 (C-terminal, large subunit) and F2 (N-terminal, small subunit). Recently, N-linked glycosylation of the F1 and F2 subunits of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was demonstrated [Long, G., Westenberg, M., Wang, H., Vlak, J. M. & Hu, Z. (2006). J Gen Virol 87, 839¿846]. Sequence analysis frequently predicts that one or more N-linked glycosylation sites are present in the F2 subunit of baculovirus F proteins. N-glycans on envelope fusion proteins are usually required for proper conformational integrity and biological function, such as infectivity. This study examined the importance of N-linked glycosylation of the F2 subunit of HearNPV by site-directed mutagenesis. The only putative N-linked glycosylation site in F2 was eliminated by mutating asparagine (N104) to glutamine (Q), resulting in the mutant HearNPVfN104Q. When inserted into an f-null HearNPV and a gp64-null bacmid of Autographa californica multiple nucleopolyhedrovirus, infectious BV could be retrieved that contained unglycosylated F2. The virulence of HearNPVfN104Q was enhanced, as BV was produced earlier after infection and yielded larger plaques than f-null HearNPV repaired with the wild-type f gene. HearNPVfN104Q BV also induced much more efficient low-pH-activated syncytium formation. These results indicate that N-linked glycosylation of the HearNPV baculovirus F2 subunit is not essential for viral infectivity and suggest that it is involved in BV production and fusogenicity
    Post-translational modifications of recombinant B-cinerea EPG 6
    Xie, M. ; Krooshof, G.H. ; Benen, J.A.E. ; Atwood, J.A. ; King, D. ; Bergmann, C. ; Orlando, R. - \ 2005
    Rapid Communications in Mass Spectrometry 19 (2005)22. - ISSN 0951-4198 - p. 3389 - 3397.
    carotovora ssp. carotovora - botrytis-cinerea - mass-spectrometry - endopolygalacturonase genes - polygalacturonase - glycosylation - infection - identification - expression - virulence
    The fungus Botrytis cinerea is a ubiquitous plant pathogen that infects more than 200 different plant species and causes substantial economic losses in a wide range of agricultural crops and harvested products. Endopolygalacturonases (EPGs) are among the first array of cell-wall-degrading enzymes secreted by fungi during infection. Up to 13 EPG glycoforms have been described for B. cinerea. The presence of multiple N-linked glycosylation modifications in BcPG1-6 is predicted by their deduced amino acid sequences. In this work, the glycosylation sites and the attached oligosaccharide structures on BcPG6 were analyzed. The molecular mass of the intact glycoprotein was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis. BcPG6 contains seven potential N-linked glycosylation sites. Occupancy of these glycosylation sites and the attached carbohydrate structures were analyzed by tryptic digestion followed by liquid chromatography/mass spectrometry (LC/MS) using a stepped orifice voltage approach. Five out of seven potential N-linked sites present in BcPG6 were determined to be occupied by high-mannose-type oligosaccharides. Four of them were readily determined to be at Asn58 (T3 peptide), Asn198 (T7 peptide), Asn237 (T9 peptide) and Asn256 (T11 peptide), respectively. Another was located on the T8 peptide, which contained two potential N-linked sites, Asn224 and Asn227 (SNNN224VTN227ITFK). LC/MS/MS of a sample treated with N-glycanase placed the glycan in this peptide at Asn224 rather than at Asn227. The potential glycosylation site on Asn146 (T6 peptide) was not glycosylated. In addition, two disulfide bonds were observed, linking the Cys residues within the T13 and T16 peptides
    Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics
    Teeffelen, A.M.M. Van; Broersen, K. ; Jongh, H.H.J. de - \ 2005
    Protein Science 14 (2005)8. - ISSN 0961-8368 - p. 2187 - 2194.
    guanidine-hydrochloride - amino-acids - m-values - stability - thermostability - glycosylation - denaturation - spectroscopy - surface - identification
    Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded -lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of -amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by ~3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5°C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications
    Influence of Growth Conditions and Developmental Stage on N-Glycan Heterogeneity of Transgenic Immunoglobulin G and Endogenous Proteins in Tobacco Leaves
    Elbers, I.J.W. ; Stoopen, G.M. ; Bakker, H. ; Stevens, L.H. ; Bardor, M. ; Molthoff, J.W. ; Jordi, W.J.R.M. ; Bosch, H.J. ; Lommen, A. - \ 2001
    Plant Physiology 126 (2001). - ISSN 0032-0889 - p. 1314 - 1322.
    rheumatoid-arthritis - leaf senescence - plant-cells - glycosylation - antibodies - igg - oligosaccharides - biosynthesis - glycoproteins - expression
    Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15°C and 25°C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of high- mannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminal N-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibody N-glycan lacking both xylose and fucose is interesting
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