High Molecular Weight Protein Aggregates Research Articles

Effect of transglutaminase in conjunction with glucono‐δ‐lactone treatment on the antigenicity and conformational structure of β‐conglycinin

SummaryIn this study, β‐conglycinin was treated with transglutaminase (TGase) and glucono‐δ‐lactone (GDL) to meticulously examine the resulting changes in both antigenicity and structural attributes. The findings elucidated a significant reduction (P < 0.05) in the antigenicity of β‐conglycinin to 72.1% and 77.5% after the modified treatment employing TGase alone or in conjunction with GDL, respectively, in contrast to the untreated samples. Electrophoretic analysis showed a decrease in the density of 7S α, 7S α′, and 7S β, indicating the formation of high molecular weight protein aggregates because of the catalytic effect of TGase. Additionally, the simultaneous application of TGase and GDL induced modifications in the structural configuration of β‐conglycinin, as evidenced by a decrease in intrinsic fluorescence intensity, an augmentation in surface hydrophobicity, and a transition from β‐sheet to β‐turn and random coil structures. These observed alterations suggest that the decline in antigenicity is intricately linked to conformational changes, potentially influencing the exposure of antigenic epitopes. Overall, this study holds significant theoretical and practical implications for the development of hypoallergenic soy products and the enhancement of food safety.

Fundamentals of Diabetic Cataractogenesis and Promising Ways of its Pharmacological Correction

Cataracts in diabetes mellitus lead to decreased visual function and blindness. Cataract surgery for diabetes mellitus has limitations and complications. The search for effective means of conservative cataract therapy continues. The review presents the analysis of data from scientific sources, mainly for 2015–2020 using Internet resources (PubMed, Web of Science, Medline, eLibrary.Ru, Cyberleninka). In the work, diabetic cataractogenesis is presented as a sum of interrelated pathobiochemical processes. The main ones are the polyol pathway of glucose conversion, non-enzymatic glycation and oxidative modification of lens proteins, which are enhanced in diabetes mellitus. The lens has a high protein content. The formation of high molecular weight protein aggregates is of particular importance for the appearance of light scattering zones and a decrease in lens transparency. This review presents data on anti-cataract compounds that affect post-translational crystallin modification, prevent osmotic and oxidative stress in the lens, and exhibit antiglycation properties. This information shows that the search for means of pharmacological correction of cataractogenesis should be carried out among compounds with antioxidant and antiglycation activity.

Inherited cataracts: Genetic mechanisms and pathways new and old

Cataract(s) is the clinical equivalent of lens opacity and is caused by light scattering either by high molecular weight protein aggregates in lens cells or disruption of the lens microarchitecture itself. Genetic mutations underlying inherited cataract can provide insight into the biological processes and pathways critical for lens homeostasis and transparency, classically including the lens crystallins, connexins, membrane proteins or components, and intermediate filament proteins. More recently, cataract genes have been expanded to include newly identified biological processes such as chaperone or protein degradation components, transcription or growth factors, channels active in the lens circulation, and collagen and extracellular matrix components. Cataracts can be classified by age, and in general congenital cataracts are caused by severe mutations resulting in major damage to lens proteins, while age related cataracts are associated with variants that merely destabilize proteins thereby increasing susceptibility to environmental insults over time. Thus there might be separate pathways to opacity for congenital and age-related cataracts whereby congenital cataracts induce the unfolded protein response (UPR) and apoptosis to destroy the lens microarchitecture, while in age related cataract high molecular weight (HMW) aggregates formed by denatured crystallins bound by α-crystallin result in light scattering without severe damage to the lens microarchitecture.

Simultaneous characterization of insulin HMWP and protamine sulphate in complex formulations through SEC-coupled mass spectrometry

High molecular weight protein aggregates present in a recombinant human insulin and analogs are conventionally quantified by SEC-HPLC and identified by SEC-MALS as oligomeric population which lacks precise identification of species. The limitation of these two techniques is vanquished though simultaneous separation and identification by SEC coupled with MS. The identification was established with organic solvent based isocratic elution followed by MS for parallel separation and identification of HMWP species. The developed SEC-MS method is validated to establish the method capability and variability. Further investigations under stress conditions of Insulin analogues revealed the capability of the method to separate higher order oligomeric (Trimeric, and Tetrameric) species alongside covalent dimeric species. Additionally, the method holds good in separating and sequencing protamine peptides used in suspension (Neutral Protamine Hagedorn) and biphasic/mixed (70/30) formulations of Human insulin using ETD-MSMS. The data presented here shows insight towards utilization of state-of-the-art SEC-MS technique in the biopharmaceutical field as a tool to establish the structural comparability of higher order species in biosimilars and to investigate the lot to lot batch variability for protamine sulphate in-terms of sequence confirmation.

Conformational changes of β-thalassemia major hemoglobin and oxidative status of plasma after in vitro exposure to extremely low-frequency electromagnetic fields: An artificial neural network analysis

ABSTRACT Electromagnetic fields (EMF) can generate reactive oxygen species and induce oxidative modifications. We investigated the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on oxidative status of plasma and erythrocytes in β-thalassemia major patients and design artificial neural networks (ANN) for evaluating the oxyHb concentration. Blood samples were obtained from age and sex-matched healthy donors (n = 12) and major β-thalassemia patients (n = 12) and subjected to 0.5 and 1 mT and 50 Hz of EMF. Plasma oxidative status was estimated after 1 and 2 h exposure to ELE-EMF. Structural changes of plasma proteins were investigated by Native PAGE and SDS-PAGE. Moreover; multilayer perceptron (MLP) method was applied for designing a feed forward ANN model to predict the impact of these oxidative and antioxidative parameters on oxyHb concentration. Two hour exposure to ELF-EMF induced significant oxidative changes on major β-thalassemia samplesElectrophoretic profiles showed two high molecular weight (HMW) protein aggregates in plasma samples from healthy donors and major β-thalassemia patients. According to our ANN design, the main predictors of oxyHb concentration were optical density of Hb at 542, 340, 569, 630, 577, and 420 nm and metHb and hemichrome (HC) concentration. Accuracy of the proposed ANN model was shown by predicted by observed chart (y = 1.3 + 0.96x, R2 = 0.942), sum of squares errors (SSR), and relative errors (RE). Our results showed the detailed effects of ELF-EMF on Hb structure and oxidative balance of plasma in major β-thalassemia patients and significance of ANN analysis during normal and pathologic conditions.

Polyphenol-rich aronia melanocarpa fruit juice antagonises behavioural changes and oxidative stress in rats exposed to disruption of circadian rhythms

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities.Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite.The extract from A. melanocarpa (0.5–50 μg/ml) added to Fg 10 min before peroxynitrite (100 μM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50 μg/ml).The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Physicochemical and functional properties of high pressure-treated isolated pea protein

Abstract Commercial isolated yellow field pea protein isolate (IPP) was subjected to 200, 400 and 600 MPa high hydrostatic pressure (HHP) treatments followed by determination of some physicochemical and functional properties. Native polyacrylamide gel electrophoresis confirmed HHP-induced formation of high molecular weight protein aggregates. Intrinsic fluorescence showed most intense at 600 MPa where fluorescence intensity was less than half of the control IPP. The 600 MPa-treated IPP also showed more unfolded structure with an extensive red shift (384 nm) in wavelength of maximum tryptophan fluorescence. However, solubility profile was very similar and was not significantly (p > 0.05) affected by HHP treatment. At pH 3.0, HHP-treated IPP formed significantly (p

Nile Red fluorescence spectrum decomposition enables rapid screening of large protein aggregates in complex biopharmaceutical formulations like influenza vaccines

The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV–Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components.

Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid

In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved. Biological significanceIn this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved.

FTIR studies of the similarities between pathology induced protein aggregation in vivo and chemically induced protein aggregation ex vivo

Fourier transform infrared (FTIR) spectroscopy has been well documented to discriminate between protein secondary structures, at the micron scale. This capability has enabled in situ localization of β-sheet aggregate accumulation within the central nervous system during pathological protein misfolding associated with Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, and Parkinson’s Disease. In addition to the above diseases, similar spectral alterations occurring over the range ∼1625–1630cm−1 have been reported in other biological systems, including inclusion body formation within bacteria and during the formation of high molecular weight protein aggregates via protein oxidation and denaturation. Thus, the characteristic spectral alterations to the amide-I band observed during protein misfolding in neurological disorders are likely not specific to these diseases, but rather, reflect an aggregated protein end point, which can result from a range of biochemical events. For example, a common pathogenic component of many neurological conditions is oxidative stress, protein oxidation and altered ion homeostasis, which have the potential to denature proteins and promote the formation of high molecular weight aggregates.Oxidative stress is a generic feature of neurodegenerative diseases and also occurs during neurodegenerative conditions, such as stroke, multiple sclerosis, epilepsy and cerebral malaria. These neuropathological disorders do not have an established protein misfolding pathology, in contrast to Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, which do have an established protein misfolding pathology. Interestingly, recent studies using FTIR have confirmed the presence of protein aggregates within the central nervous system during stroke, cerebral malaria, epilepsy, multiple sclerosis. Such reports suggest FTIR spectroscopy may be a highly valuable research tool to study protein aggregation as a marker of oxidative stress and neurodegeneration in many diseases, not just those with a characteristic pathology for protein misfolding. This manuscript extends the recent literature and reports further characterization of the alterations to the amide I band that result from ubiquitous ex vivo protein aggregation in cerebral tissue. The new data presented highlights that the spectroscopic alterations to the amide I band often reported for amyloid-β plaques in Alzheimer’s disease, are spectroscopically very similar to spectroscopic alterations observed during ischemia induced neurodegeneration (stroke) and ex vivo induced protein aggregation. As such, this study further validates FTIR as a useful platform to study protein aggregation in neurological disorders, including those not characterized by protein misfolding pathology.

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan.

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes.

Aronia melanocarpa as a protector against nitration of fibrinogen

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities.Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite.The extract from A. melanocarpa (0.5–50μg/ml) added to Fg 10min before peroxynitrite (100μM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50μg/ml).The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild‐type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

Biochemical and proteomic characterization of alkaptonuric chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc.

Fluorescent substances and high molecular weight protein aggregates formed in rat heart mitochondria upon doxorubicin-induced lipid peroxidation.

A rat heart mitochondrial suspension was incubated with doxorubicin, FeCl3 and NADH. Fluorescent substances and high molecular weight protein aggregates were observed in the mitochondrial membranes upon the formation of thiobarbituric acid-reactive substances. Since both fluorescent substances and high molecular weight protein aggregates are retained in mitochondrial membranes, they can be of use in the clarification of the site of doxorubicin-induced lipid peroxidation.

High molecular weight protein aggregates formed in the liver of the rat following large doses of paracetamol.

Paracetamol (200 and 500 mg kg-1) was given intraperitoneally to rats pretreated with 3-methylcholanthrene for 3 days. Glutamic oxalacetic acid transaminase (GOT) activity in plasma increased in rats receiving 500 mg kg-1 paracetamol. Plasma GOT activity was low at the dose of 200 mg kg-1, but the same dose to diethyl maleate pretreated rats increased the GOT activity. High mol. wt protein aggregates were found to be formed in liver homogenates and microsomes of rats which showed high plasma GOT activity, accompanied by depletion of hepatic glutathione. The formation of protein aggregates in the liver of rats following large doses of paracetamol suggests a contribution of lipid peroxidation to paracetamol-induced hepatotoxicity.

Control of Polymorphism and Morphology of Calcium Carbonate Crystals by a Matrix Protein Aggregate in Fish Otoliths

We show that the high-molecular weight (HMW) protein aggregate of fish otolith matrix is involved in the regulation of crystal polymorphs during otolith biomineralization. Teleost fish otoliths are...

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.

Lysyl-tRNA Synthetase Is a Target for Mutant SOD1 Toxicity in Mitochondria

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.

Biomarkers for ragwort poisoning in horses: identification of protein targets

BackgroundIngestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods.ResultsOne dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin.ConclusionThese findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.