Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment.

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp ß-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp ß-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.

Distinguishing N-acetylneuraminic acid linkage isomers on glycopeptides by ion mobility-mass spectrometry.

Differentiating the structure of isobaric glycopeptides represents a major challenge for mass spectrometry-based characterisation techniques. Here we show that the regiochemistry of the most common N-acetylneuraminic acid linkages of N-glycans can be identified in a site-specific manner from individual glycopeptides using ion mobility-mass spectrometry analysis of diagnostic fragment ions.

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer.

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with ß1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides.

Mass spectrometry has made possible the field of proteomics and become an invaluable tool for identifying and quantifying post-translational modifications of proteins from complex mixtures. Because PTMs are recognized as key factors of biological activity, reliable PTM characterization is essential to understanding the relationship between protein isoform and activity. Protein glycosylations, in particular, can be especially difficult to characterize due to the range of different oligosaccharide entities that may be attached at any particular site. In this month' Special Feature Dr Daniel Kolarich of the Dept. of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces and his collaborators point out that quantitative label-free glycoproteomics can yield information about glycoprotein microand macroheterogeniety if the tools are sufficiently accurate. To understand and characterize the performance capability of MS tools they synthesized a panel of peptides and their glycopeptide derivatives as references and used these to investigate the qualitative and quantitative results from various ionization techniques and mass analyzers.

Disrupted intracellular redox balance of the diplomonad fish parasite Spironucleus vortens by 5-nitroimidazoles and garlic-derived compounds.

The 5-nitroimidazole, metronidazole, has traditionally been employed in veterinary medicine to treat a range of infections including the diplomonad fish parasite Spironucleus. This study aims to determine the mode of action of metronidazole on Spironucleus vortens, including the specific mechanism of activation of the pro-drug and subsequent cellular targets of the drug metabolites. Due to the ban on use of metronidazole in the treatment of production animals in Europe and USA, garlic-derived compounds were also investigated as natural alternatives to metronidazole chemotherapy. Scanning electron microscopy (SEM) provided an overview of gross cellular damage caused by metronidazole and garlic derivatives. Proteomic analyses by 2D gel electrophoresis identified the proteins involved in specific covalent adduct formation with nitroimidazoles. Furthermore, thioredoxin reductase (TrxR) activity and non-protein thiol concentration were assayed in extracts of S. vortens before and after treatment with nitroimidazoles and garlic-derivatives. Metronidazole and garlic-derived compounds caused severe damage of trophozoites indicated by membrane blebbing and lysed cell debris. Analysis of the S. vortens proteome identified several proteins capable of specific nitroimidazole binding, including; uridine phosphorylase, enolase, protein disulphide isomerase, aminoacyl-histidine dipeptidase and malic enzyme. Of the compounds tested, metronidazole and the garlic-derived compound ajoene were the most effective at inhibiting TrxR activity and depleting non-protein thiols. These data suggest TrxR-mediated activation of nitroimidazoles, leading to depletion of non-protein thiols. Redox imbalance due to antioxidant failure is implicated as the mode of action of nitroimidazoles and garlic-derived compounds, ultimately leading to cell death. Possible synergy between garlic derivatives and metronidazole should be further investigated in vitro in order to determine their theoretical implications.

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 using 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.

Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N-glycans as cross-reactive allergens in honeybee and yellow jacket venom.

BACKGROUND: IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy. OBJECTIVE: To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot. METHODS: Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N-glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N-glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated. RESULTS: Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3. CONCLUSIONS: The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization.

Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy.

BACKGROUND: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, chi(2) test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy.

Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions.

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

N-Glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1.

A method has been developed which allows the analysis of glycoproteins separated by SDS-PAGE. The procedure, though applicable to N-glycosylated glycoproteins of any origin, is particularly devised for glycoproteins potentially containing fucose in alpha1,3-linkage to the reducing GlcNAc as may be found in plants and invertebrates, e.g., insects and parasitic helminths. Starting with an established procedure for mass spectrometric peptide mapping, the analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry involved the use of peptide:N-glycosidase A, a triphasic microcolumn for sample cleanup, and a new matrix mixture consisting of 2,5-dihyhydroxybenzoic acid, 1-hydroxyisoquinoline, and arabinosazone. The method was tested on proteins with N-glycans of known structure, i.e., as horseradish peroxidase, zucchini ascorbate oxidase, soybean agglutinin, honeybee venom hyaluronidase, bovine ribonuclease B, and bovine fetuin. An electrophoretic band corresponding to 4 microg of glycoprotein was generally sufficient to allow detection of the major N-glycan species. As an additional benefit, a peptide mass map is generated which serves to identify the analyzed protein. The method was applied to glycoprotein allergens whose glycan structures were unknown. Ara h 1 and Ole e 1, major allergens from peanut and olive pollen, respectively, contained mainly xylosylated N-glycans with the composition Man(3(-4))XylGlcNAc(2) in the case of Ara h 1 and GlcNAc(1-2)Man(3)XylGlcNAc(2) in the case of Ole e 1 where also some GlcNAc(0-2)Man(3)XylFucGlcNAc(2) was found.