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Biomedical subjects

Peter Roepstorff

Publications and source records attributed to Peter Roepstorff.

At least 37 records · Page 2Linked to original sources
PubMed2005

Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom.

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.

Amino Acid Sequence↗
PubMed2005

A proteomic approach for investigation of photosynthetic apparatus in plants.

The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.

Chlorophyll↗
PubMed2005

Mass spectrometric analysis of protein interactions.

Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably.

Amides↗
PubMed2005

The extensible alloscutal cuticle of the tick, Ixodes ricinus.

The proteins in the distensible alloscutal cuticle of the blood-feeding tick, Ixodes ricinus, have been characterized by electrophoresis and chromatography, two of the proteins were purified and their total amino acid sequence determined. They show sequence similarity to cuticular proteins from the spider, Araneus diadematus, and the horseshoe crab, Limulus polyphemus, and to a lesser extent to insect cuticular proteins. They contain a conserved sequence region, which is closely related to the chitin-binding Rebers-Riddiford consensus sequence present in many insect cuticular proteins. Only a fraction of the alloscutal proteins can be readily dissolved, and the dissolved proteins are difficult to separate by electrophoresis and column chromatography. The insoluble fraction can only be dissolved after degradation to smaller peptides. The mixture of extractable proteins as well as hydrolysates of the insoluble fraction are fluorescent when exposed to ultraviolet light, and the fluorescence corresponds in excitation and emission maxima to the fluorescence of the rubber-like arthropodan protein, resilin, and to the amino acid dityrosine. Small amounts of dityrosine were obtained from ticks in the early phase of a blood meal when the cuticle weighs less than 4 mg; increasing amounts were obtained from animals in the initial period of feeding, during which the cuticular weight increases from 4 to 11 mg, whereas little increase in dityrosine content was observed during the final period of engorgement. Cuticle from fully distended ticks contains about 60-80 nmole dityrosine per tick, corresponding to 2-3 microg/mg cuticle. It is suggested that the major part of the cuticular proteins is made inextractable by cross-linking by dityrosine residues, and that dityrosine plays a role in stabilizing the cuticular structure during the extensive distension occurring during a blood meal. Small amounts of 3-monochlorotyrosine and 3,5-dichlorotyrosine were obtained from the distended tick cuticle, corresponding to chlorination of between 0.5% and 1.5% of the tyrosine residues. It is suggested that the chlorotyrosines are a side-product of oxidative processes in the cuticle.

Amino Acid Sequence↗
PubMed2005

Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism.

By comparison of proteome profiles of purified Chlamydia pneumoniae and whole lysates of C. pneumoniae infected HEp-2 cells, an N-terminal fragment of the previously uncharacterized chlamydial protein Cpn0796 was identified as a secreted protein. A 38 kDa cleavage product of Cpn0796 was present in infected cells, whereas only the 65 kDa full-length Cpn0796 could be detected in purified Chlamydia. Confocal immunofluorescence microscopy demonstrated that Cpn0796 was localized in the Chlamydia membrane in young inclusions. However, at 36 h post infection and later Cpn0796 was detected in the cytoplasm of C. pneumoniae infected HEp-2 and BHK cells. Furthermore, Cpn0796 was detected in the cytoplasm of infected cells in the lungs of C. pneumoniae infected C57Bl mice. When cleavage was inhibited, Cpn0796 was retained in the chlamydiae. We propose that Cpn0796 is an autotransporter the N-terminal of which is translocated to the host cell cytoplasm. This is the first example of secretion of a Chlamydia autotransporter passenger domain into the host cell cytoplasm. Cpn0796 is specific for C. pneumoniae, where five homologous proteins are encoded by clustered genes. None of these five proteins were found to be secreted.

Amino Acid Sequence↗
PubMed2005

Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

Amino Acid Sequence↗
PubMed2004

Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry.

Using amide hydrogen exchange combined with electrospray ionization mass spectrometry, we have in this study determined the number of amide hydrogens on several peptides that become solvent-inaccessible as a result of their high-affinity interaction with the urokinase-type plasminogen activator receptor (uPAR). These experiments reveal that at least six out of eight amide hydrogens in a synthetic nine-mer peptide antagonist (AE105) become sequestered upon engagement in uPAR binding. Various uPAR mutants with decreased affinity for this peptide antagonist gave similar results, thereby indicating that deletion of the favorable interactions involving the side chains of these residues in uPAR does not affect the number of hydrogen bonds established by the main chain of the peptide ligand. The isolated growth factor-like domain (GFD) of the cognate serine protease ligand for uPAR showed 11 protected amide hydrogens in the receptor complex. Interestingly, a naturally occurring O-linked fucose on Thr(18) confers protection of two additional amide hydrogens in GFD when it forms a complex with uPAR. Dissociation of the uPAR-peptide complexes is accompanied by a correlated exchange of nearly all amide hydrogens on the peptide ligand. This yields bimodal isotope patterns from which dissociation rate constants can be determined. In addition, the distinct bimodal isotope distributions also allow investigation of the exchange kinetics of receptor-bound peptides providing information about the local structural motions at the interface. These exchange experiments therefore provide both structural and kinetic information on the interaction between uPAR and these small peptide antagonists, which in model systems show promise as inhibitors of intravasation of human cancer cells.

Amides↗
PubMed2004

Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry.

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Acetonitriles↗
PubMed2004

Molecular characterization of major cat allergen Fel d 1: expression of heterodimer by use of a baculovirus expression system.

Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization.

Allergens↗
PubMed2004

The molecular basis for oat intolerance in patients with celiac disease.

BACKGROUND: Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. METHODS AND FINDINGS: We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine-->glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. CONCLUSIONS: We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.

Atrophy↗
PubMed2004

Screening for enzyme inhibitors by surface plasmon resonance combined with mass spectrometry.

We have developed a novel strategy to identify enzyme inhibitors that interact directly with their enzyme targets. In the approach, an enzyme is immobilized on a sensor chip, and it is determined whether the immobilized enzyme is still active by incubation with model substrates and mass spectrometric analysis of the products. Putative inhibitors or mixtures containing putative inhibitors are then injected over the sensor chip for binding analysis with surface plasmon resonance. It is then tested whether the bound compounds inhibit the enzymatic activity by subsequent incubation with the model substrate and mass spectrometric analysis. If the bound compound inhibits the enzyme, the inhibitor is eluted from the enzyme and characterized by mass spectrometry. To test the strategy, it has been applied to the well-characterized interaction between trypsin and pure bovine pancreas trypsin inhibitor. Furthermore, fractions of plant extracts were screened for binding to and inhibition of carboxypeptidase B.

Aprotinin↗
PubMed2004

Absolute quantitation of proteins by a combination of acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry.

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.

Amino Acids↗
PubMed2004

Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae.

In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and carboxypeptidase Y is slightly delayed in Acb1p-depleted cells, whereas the maturation of alkaline phosphatase and Gas1p is unaffected. The fact that Gas1p maturation is unaffected by Acb1p depletion, despite the lowered ceramide content in these cells, indicates that ceramide synthesis in yeast could be compartmentalized. We suggest that the reduced ceramide synthesis in Acb1p-depleted cells leads to severely altered vacuole morphology, perturbed vacuole assembly and strong inhibition of homotypic vacuole fusion.

Carrier Proteins↗
PubMed2004

Identification of protein-protein interfaces implicated in CD80-CD28 costimulatory signaling.

The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.

Animals↗
PubMed2004

Differentiation of isomeric oligosaccharide structures by ESI tandem MS and GC-MS.

A mixture of arabinoxylan oligosaccharides from wheat seedling was permethylated and analyzed by electrospray ion trap MS and GC-MS. The presence of isomeric structures differing in degree of branching and position of the branched residue along the xylose backbone was demonstrated for oligosaccharides with four and five monosaccharide residues. No isomeric structures were found for oligosaccharides with three monosaccharide residues. Linkage analysis by GC-MS reveals that xylose residues were substituted with single arabinoxyl residues at C-3.

Carbohydrate Sequence↗
PubMed2004

Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III.

A common strategy in proteomics to improve the number and quality of peptides detected by mass spectrometry (MS) is to desalt and concentrate proteolytic digests using reversed phase (RP) chromatography prior to analysis. However, this does not allow for detection of small or hydrophilic peptides, or peptides altered in hydrophilicity such as phosphopeptides. We used microcolumns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analyzed. MS revealed that some phosphopeptides did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve terminals. The detection of this peptide was greatly enhanced by graphite micropurification. Sequencing by tandem MS confirmed the presence of phosphate at both Ser-774 and Ser-778, while a singly phosphorylated peptide was predominantly phosphorylated only on Ser-774. The method further revealed a singly and doubly phosphorylated peptide in dynamin III, analogous to the dynamin I sequence. A pair of dynamin III phosphorylation sites were found at Ser-759 and Ser-763 by tandem MS. The results directly define the in vivo phosphorylation sites in dynamins I and III for the first time. The findings indicate a large improvement in the detection of small amounts of phosphopeptides by MS and the approach has major implications for both small- and large-scale projects in phosphoproteomics.

Animals↗
PubMed2004

Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides.

Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.

Amino Acid Sequence↗