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Biomedical subjects

P Roepstorff

Publications and source records attributed to P Roepstorff.

At least 55 records · Page 3Linked to original sources
PubMed2000

The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

Adult↗
PubMed2000

The formation of heterodimers by vancomycin group antibiotics.

The formation of heterodimers in mixtures of glycopeptide antibiotics has been detected by electrospray ionization mass spectrometry (ESI-MS), and dimerization constants have been determined. By using NMR spectroscopy, it has been shown that these heterodimers indeed exist in aqueous solution. The dimerization constants obtained by NMR spectroscopy are in good agreement with those determined by ESI-MS. Structural information on the heterodimer interface of some of the heterodimers is obtained by using two-dimensional NMR techniques and reveals that these heterodimers are similar in structure to the homodimers.

Anti-Bacterial Agents↗
PubMed2000

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods.

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.

Alkylation↗
PubMed2000

Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards.

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance. A great advantages of the proposed method is the absence of molecular weight limitation for the protein quantitation and the possibility of quantitation without previous fractionation of proteins and peptides. Using this strategy, the peptide angiotensinogen and two proteins, RNase and its protein inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.

Animals↗
PubMed2000

MALDI-TOF mass spectrometry in protein chemistry.

Mass spectrometry has in the last decade been accepted as a key analytical technique in protein chemistry. It is now the preferred technique for identification of proteins separated by one- or two-dimensional polyacrylamide gel electrophoresis, i.e. in proteome analysis. It is the dominating technique for determination of posttranslational modifications in proteins. The two ionization techniques presently widely used in protein studies are matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight (TOF) mass analyzers and electrospray ionization (ESI) in combination with a variety of mass analyzers. In this chapter the principles and performance of MALDI-TOF mass spectrometry will be described as well as the application of this technique to a variety of applications.

Amino Acid Sequence↗
PubMed2000

Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis.

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.

Amino Acid Sequence↗
PubMed2000

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

Base Sequence↗
PubMed2000

The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity.

BACKGROUND: Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. OBJECTIVE: To study whether Fel d 1 is associated with enzyme activity. METHODS: Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. RESULTS: The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. CONCLUSION: These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.

Allergens↗
PubMed2000

Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.

Bacterial Proteins↗
PubMed2000

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.

High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Amino Acid Sequence↗
PubMed2000

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

Amino Acid Sequence↗
PubMed2000

Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts.

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

Amino Acid Sequence↗
PubMed2000

Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

Animals↗
PubMed2000

[Study of dimerization of polysynthetic derivatives of the antibiotic eremomycin by ESI MS and its role in elucidating antibacterial activity].

The dimerization constants for glycopeptide antibiotics vancomycin, ristocetin, and eremomycin and nine semisynthetic eremomycin derivatives were determined by the electrospray ionization mass spectrometry; the constants for natural antibiotics turned out to be close to those previously determined by NMR. No correlation between these dimerization constants and antibacterial activities of all the compounds toward the clinical strains of Gram-positive bacteria was found.

Anti-Bacterial Agents↗