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Daniel Kolarich
Group Leader, Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems
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Vox sanguinis 2010 Oct;
Biochemical, molecular and preclinical characterization of a double-virus-reduced human butyrylcholinesterase preparation designed for clinical use.
Background and Objectives A human plasma-derived butyrylcholinesterase preparation manufactured on the industrial scale is described. Material and Methods The human butyrylcholinesterase (hBChE) product was extensively investigated for its purity u... expand abstractsing immunological and electrophoretic methods and characterized by thorough glycoproteomic approaches. A comprehensive preclinical testing programme addressing safety and pharmacokinetic parameters supplemented the biochemical characterization. Results The high-purity hBChE preparation is tetrameric and has high specific activity and molecular integrity of the protein backbone. Acute toxicity studies and in vivo thrombogenicity studies provided evidence of a sufficient safety margin for use in humans. Conclusion Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans. collapse abstract
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Omics : a journal of integrative biology 2010 Jul; 14(4)
The lectin riddle: glycoproteins fractionated from complex mixtures have similar glycomic profiles.
Abstract One common method used for analyzing the glycoproteome is chromatography using multiple lectins that display different affinities toward oligosaccharide structures. Much has been done to determine lectin affinity using standard glycoproteins... expand abstract with known glycosylation; however, a knowledge of the selectivity and specificity of lectins exposed to complex mixtures of proteins is required if they are to be used as a means of studying the glycoproteome. In the present study, three lectins (Concanavalin A, Jacalin, and Wheat Germ Agglutinin) were used to fractionate glycoproteins from two different complex environments: (1) cell membranes and (2) plasma. Reproducible enrichment of glycoproteins from these samples has been shown to result from the combined use of these lectins. However, the global glycan profiles of the released N- and O-linked oligosaccharides from the glycoproteins retained by the lectins, and from those glycoproteins that did not bind, using both these complex samples, were found to be very similar. That is, although the lectins selectively and reproducibly retained some glycoproteins, other proteins with the same attached oligosaccharide structures did not bind. Some small N- and O-glycan differences were observed in the bound fractions but there was little absolute specificity toward individual oligosaccharide structures known to have high affinity to these lectins. These data indicate that lectins are useful for fractionating glycoproteins from complex mixtures, but that the overall glycoproteome is not isolated by this approach. collapse abstract
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Analytical chemistry 2010 Apr; 82(9)
Challenges of determining O-glycopeptide heterogeneity: a fungal glucanase model system.
O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike doma... expand abstractin glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at individual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy. collapse abstract
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Journal of proteome research 2010 Feb; 9(2)
GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass ... expand abstractspectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked,
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Molecular and biochemical parasitology 2010 Apr; 171(1)
The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways.
Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro g... expand abstractroup, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole. collapse abstract
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Molecular & cellular proteomics : MCP 2010 Mar; 9(4)
Comparison of Methods for Profiling O-Glycosylation: HUMAN PROTEOME ORGANISATION HUMAN DISEASE GLYCOMICSPROTEOME INITIATIVE MULTI-INSTITUTIONAL STUDY OF IgA1.
The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly ... expand abstractendorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful. collapse abstract
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The FEBS journal 2009 Dec; 277(1)
Mucin-type O-glycosylation--putting the pieces together.
The O-glycosylation of Ser and Thr by N-acetylgalactosamine-linked (mucin-type) oligosaccharides is often overlooked in protein analysis. Three characteristics make O-linked glycosylation more difficult to analyse than N-linked glycosylation, namely:... expand abstract (a) no amino acid consensus sequence is known; (b) there is no universal enzyme for the release of O-glycans from the protein backbone; and (c) the density and number of occupied sites may be very high. For significant biological conclusions to be drawn, the complete picture of O-linked glycosylation on a protein needs to be determined. This review specifically addresses the analytical approaches that have been used, and the challenges remaining, in the characterization of both the composition and structure of mucin-type O-glycans, and the determination of the occupancy and heterogeneity at each amino acid attachment site. collapse abstract
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Molecular microbiology 2009 Mar; 72(2)
Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.
Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we repo... expand abstractrt that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs. collapse abstract
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Journal of proteome research 2009 Jan; 8(2)
Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture.
Past proteomic studies of membrane proteins have often been hampered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and thi... expand abstracts characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites. collapse abstract
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Molecular nutrition & food research 2008 Nov; 52(12)
Identification of four IgE-reactive proteins in raspberry (Rubus ideaeus L.).
IgE-reactive proteins in raspberry (Rubus ideaus L.) were identified using PCR, RT-PCR, 2-DE and MS/MS peptide sequencing. Specific polyclonal antibodies and patient sera were used in Western blotting to identify crossreactive epitopes. Initially, tw... expand abstracto potential allergens Rub i 1 and Rub i 3 were detected using PCR, showing high sequence identity to proteins in Rosaceous species like Mal d 1 and Mal d 3 from apple, Pru av 1 and Pru av 3 from cherry and Pru p 1 and Pru p 3 from peach. Furthermore, de novo identified peptides of a protein band at about 30 kDa reacting with most of the patient sera tested (> 80%) revealed a high sequence homology with class III chitinases. Raspberry chitinase, when subjected to glycoproteomic analysis, showed typical complex plant-type N-glycans with a core alpha1,3 fucose and a beta1,2 xylose at least at one position, indicating the presence of crossreacting carbohydrate determinants (CCDs). Finally, MS/MS analysis revealed an IgE-reactive raspberry cyclophilin, homologous to Bet v 7. Results obtained suggest that the consumption of raspberries might be responsible for adverse reactions in sensitised individuals. collapse abstract
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Analytical biochemistry 2009 Jan; 384(2)
Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method.
Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other tha... expand abstractn the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation. collapse abstract
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Proteomics 2008 Jun; 8(14)
Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides.
Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specif... expand abstractic quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods. collapse abstract
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Proteomics 2007 Dec; 8(2)
Glycoproteomic characterization of butyrylcholinesterase from human plasma.
Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspi... expand abstractrin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated. collapse abstract
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PLoS biology 2007 Jul; 5(8)
Nitroimidazole action in Entamoeba histolytica: a central role for thioredoxin reductase.
Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but li... expand abstractttle is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms. collapse abstract
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Proteomics 2007 Apr; 7(10)
A proteomic study of the major allergens from yellow jacket venoms.
The venoms of stinging insects belong to the most dangerous allergen sources and can cause fatal anaphylactic reactions. Reliable prediction of a patient's risk to anaphylactic reactions is vital, and diagnosis requires the knowledge of the relevant ... expand abstractallergens. Recently, a new hyaluronidase -like glycoprotein from Vespula vulgaris (Ves v 2b) was identified. This led us to investigate hyaluronidases and also other major allergens from V. germanica and four additional Vespula species. By MALDI-Q-TOF-MS, the new hyaluronidase-like protein was shown to be the major component of the 43-kDa band in all Vespula species studied. LC-ESI-Q-TOF-MS/MS sequencing of Ves g 2a and Ves g 2b facilitated the cloning of their cDNA. Ves v 2b and Ves g 2b turned out to be essentially identical on protein level. Whereas the less abundant "a" form displayed enzymatic activity, the new "b" homologue did not. This is probably caused by amino acid exchanges in the active site, and it raises questions about the physiological role of this protein. Sequence comparisons by MS/MS of antigen 5 and phospholipases from V. vulgaris, germanica, maculifrons, pensylvanica, flavopilosa and squamosa revealed the latter as a taxonomic outlier and led to the discovery of several not previously reported amino acid differences. collapse abstract
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Electrophoresis 2007 Jan; 28(3)
Mapping of Malus domestica allergens by 2-D electrophoresis and IgE-reactivity.
The importance of apple allergens has been repeatedly emphasized, and their presence has been confirmed both in pollen and in fruits. In the present study, a combination of proteomic tools have been used to build a complete allergen map of apple. The... expand abstract water-soluble fraction of an apple extract was precipitated using a phenol-based procedure and separated by 2-DE. Initially four previously classified allergens, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, could be identified in Western blots with polyclonal rabbit antibodies directed to the four respective allergens, and subsequently matched to the bands recognized by several patient sera. Further, all four known apple allergens were localized on a 2-DE map and they were matched with spots recognized by sera of patients with different allergic patterns. Moreover, a new, putative allergen could be identified using MS. We evaluated the influence of post-translational modifications and the immunoreactivity under different analytical conditions. The comparison of different visualization methods for 2-DE gels and blots revealed that even very low concentrations of the intact epitopes are detectable by IgEs of patients, and therefore might be sufficient to trigger allergic symptoms in sensitized individuals. collapse abstract
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Glycoconjugate journal 2006 Oct; 23(7-8)
Comparative characterisation of recombinant invertebrate and vertebrate peptide O-Xylosyltransferases.
Chondroitin and heparan sulphates have key functions in animal development and their synthesis is initiated by the action of UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26). cDNAs encoding this enzyme have been pr... expand abstracteviously cloned from mammalian species; this in turn facilitated identification of corresponding Caenorhabditis elegans (sqv-6) and Drosophila melanogaster (oxt) genes. In the present study, we report the expression in Pichia pastoris and subsequent assay using either MALDI-TOF MS or RP-HPLC of recombinant forms of the Caenorhabditis xylosyltransferase SQV-6 and the human xylosyltransferase I, in addition to extending our previous studies on the xylosyltransferase from Drosophila. The enzyme activities were tested with a number of peptide substrates based on portions of the human bikunin, human perlecan and Drosophila syndecan core peptides. Whereas a variant of the latter, containing two Ser-Gly motifs was only modified on one of these motifs, the perlecan peptide with three Ser-Gly motifs could be multiply modified in vitro. Using this substrate, we could for the first time follow, by mass spectrometry, the xylosylation of a peptide with multiple xylosyltransferase acceptor motifs. collapse abstract
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Transfusion 2006 Oct; 46(11)
Biochemical, molecular characterization, and glycoproteomic analyses of alpha(1)-proteinase inhibitor products used for replacement therapy.
BACKGROUND: Isoelectric focusing (IEF) of alpha(1)-proteinase inhibitor (A1PI) shows that commercial products and plasma have different glycoisoform band patterns. Those in Aralast (Grifols Biologicals) reflect an anodal shift of glycoisoforms, which... expand abstract has caused concern. The protein, including glycoproteomic analyses, and structural features of A1PI products were investigated by state-of-the-art techniques. STUDY DESIGN AND METHODS: Batches from Aralast, Prolastin (Bayer), and Zemaira (Aventis Behring LLC) were analyzed by high-resolution IEF and high-performance size-exclusion chromatography (HP-SEC). Preparative separated isoforms from IEF were further purified by chromatography and subjected to mass spectrometry for sequence analyses, peptide mapping, and glycosylation analysis. Deamidation was quantified by enzymatic isoaspartate detection. Multiple sequence alignments and structural bioinformatics analyses were performed. RESULTS: In HP-SEC, Prolastin had the highest aggregate content at approximately 30 percent. Isoforms from all products purified by high-resolution IEF were sequenced with an amino acid coverage of more than 98 percent. Deamidation of Asn116 and Asn314 in A1PI was to found to some extent in all products and confirmed quantitatively by enzymatic analysis. There were no signs of methionine oxidation. Cys232 was found to be cysteinylated in A1PI in Prolastin and Aralast as in plasma, but not in Zemaira. All products showed truncation of the C-terminal lysine. Intact A1PI concentrates contained mainly diantennary, disialylated and smaller amounts of triantennary, trisialylated N-glycans. The percentage of fucosylation was similar in all products. Site-specific glycan analysis revealed bands M6 contained only diantennary glycans, whereas the more acidic bands M4 and M2 also carried triantennary structures. The most acidic isoforms, M2 in Prolastin and Zemaira and M0 in Aralast, additionally exhibited tetraantennary N-glycans. CONCLUSION: Protein chemical characterization of A1PI showed that all A1PI products to some extent differ from A1PI circulating in human plasma. Bioinformatic analysis indicated that removal of C-terminal Lys394 and cysteinylation of Cys232 are unlikely to affect structure and/or function of A1PI but cysteinylation may influence interaction between A1PI and its physiologic ligands. Aralast, Prolastin, and Zemaira contain the same set of N-glycans in the same ratios as those in normal human plasma A1PI. Tri- and tetraantennary structures are responsible for the partitioning into IEF isoforms, with the migration shift of Aralast not being due to any difference in the N-glycosylation, but to the partial loss of the C-terminal lysine. collapse abstract
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Clinical and vaccine immunology : CVI 2006 Sep; 13(10)
Identification and characterization of a major cell wall-associated iron-regulated envelope protein (Irep-28) in Mycobacterium tuberculosis.
Iron limitation and the expression of mycobactin and carboxymycobactin by Mycobacterium tuberculosis are known. Here, we report how iron regulated the coordinate expression of these two siderophores and a 28-kDa cell wall-associated iron-regulated pr... expand abstractotein (Irep-28). Irep-28 is identified as the DNA-binding HU homologue HupB protein (hupB [Rv2986c]). Antibodies to this protein were detected in sera from tuberculosis patients. The location of the protein in the cell wall makes it a potential drug target. collapse abstract
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Proteomics 2006 May; 6(11)
Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms.
Human alpha1-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces... expand abstract of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha1,6 type. collapse abstract
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Phytochemistry 2006 Mar; 67(7)
Molecular cloning and characterization of a plant alpha1,34-fucosidase based on sequence tags from almond fucosidase I.
Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps in... expand abstractcluding dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice. collapse abstract
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Bioorganic & medicinal chemistry 2006 May; 14(10)
Spin trapping of C- and O-centered radicals with methyl-, ethyl-, pentyl-, and phenyl-substituted EMPO derivatives.
In order to develop spin traps with an optimal ratio between hydrophilic and lipophilic properties, low toxicity, and high stability of spin adducts (especially with superoxide radicals), several EMPO-derived spin traps have recently been synthesized... expand abstract forming more stable superoxide adducts (t(1/2) > 20 min) than DMPO or DEPMPO. In this study, ESR-, 1H-, and 13C-NMR data of several phenyl- or n-pentyl-substituted EMPO derivatives are presented with full signal assignment. Methyl groups at position 3 or 4 stabilized the superoxide adducts considerably. Spin adducts from other oxygen- and carbon-centered radicals (e.g., derived from methanol or linoleic acid hydroperoxide) are also described. collapse abstract
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Planta 2006 May; 224(1)
Sialic acid concentrations in plants are in the range of inadvertent contamination.
The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-... expand abstract4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-D: -manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants. collapse abstract
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Biochimica et biophysica acta 2006 Mar; 1760(4)
Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry.
The lecithinase homolog (Hev b 4) from Hevea brasiliensis (Q6T4P0_HEVBR) is an important natural rubber latex allergen. Hev b 4 is a highly glycosylated protein and its carbohydrate moiety has been implicated in the binding of IgE from natural rubber... expand abstract latex allergic patients. The cDNA for Hev b 4 has recently been cloned and sequenced. Here, we have analyzed the post-translational modifications of natural Hev b 4 by liquid chromatography/electrospray ionization-mass spectrometry of tryptic peptides. Seven of the eight potential glycosylation sites were found to be occupied. One site, however, was only partially glycosylated. Asn224 was substituted by complex type N-glycans with fucose and xylose, whereas all other sites carried either oligomannose glycans or a mixture of oligomannose and complex N-glycans. Glycosylation site Asn308, the most C-terminal one of the eight sites, was only found in the non-glycosylated form. The complex type N-glycans apparently form the molecular basis for the immune reaction with patients' sera. A large fraction of Hev b 4 molecules contains two or more complex N-glycans and thus a physiological reaction against these polyvalent allergens on the basis of the carbohydrate is in theory possible. Aside from allowing glycosylation analysis, the mass spectrometric data defined the N-terminal cleavage site of Hev b 4. This study once more demonstrates the outstanding analytical potential of electrospray ionization-mass spectrometry coupled with liquid chromatographic separation. collapse abstract
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The FEBS journal 2005 Sep; 272(20)
The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris.
Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carb... expand abstractohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms. collapse abstract
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