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Papers by bart devreese

Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS

European journal of biochemistry, 2001

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocr... more Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size‐exclusion, immunoaffinity, and reversed‐phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586–R602 (SR‐17) and is phosphorylated at one or two serine residues. Another novel peptide H603–Q636 (HQ‐34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin‐like peptide fragment (KR‐11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C‐terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.

Mikrochimica Acta, Dec 10, 2007

We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cyto... more We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4 0 -bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it most likely binds to HHC and results in a potential downshift of the voltammetric signals of the latter. Furthermore, the electrochemical determination of hydrogen peroxide at a RcCCP=HHC modified gold electrode is shown. The results demonstrate that HHC can substitute for cytochrome c 2 , the physiological electron donor. The buffer 2-[4-(2-hydroxyethyl)-piperazinyl]ethanesulfonic acid (HEPES) and tris(hydroxymethyl) methylamine (Tris) electrochemically are not as inert as previously believed. They can react with oxygen (radicals) during electrochemical measurements, and the products formed can give rise to additional redox peaks. We therefore also have conducted a voltammetric study on theses buffers.

Applied Microbiology and Biotechnology, Jun 2, 2012

This study represents two different large-scale proteomic experiments analyzing the antibiotic re... more This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of β-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Twodimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQ® differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in β-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 β-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the β-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in β-lactamase production.

Monitoring the zinc affinity of the metallo-�-lactamase CphA by automated nanoESI-MS

J Amer Soc Mass Spectrom, 2006

Analysis of the Importance of the Metallo-?-Lactamase Active Site Loop in Substrate Binding and Catalysis

Chem Biol, 2003

cDNA sequence of three isoforms of the hemocyanin from Helix vulgaris

Gene

Microbiology (Reading, England), 1999

The strongly hydrophobic bacteriocin amylovorin L471 from Lactobacillus amylovorus DCE 471 was is... more The strongly hydrophobic bacteriocin amylovorin L471 from Lactobacillus amylovorus DCE 471 was isolated and purified to homogeneity from complex culture broth by a novel, rapid and simple three-step protocol including (i) ammonium sulphate precipitation, (ii) chloroform/methanol extraction/precipitation and (iii) reversed-phase HPLC, the only chromatographic step involved. The molecular mass of the peptide was determined to be 4876.9 Da by electrospray mass spectrometric analysis. N-terminal amino acid sequencing identified 35 amino acid residues as being identical to the N-terminal sequence of lactobin A, a bacteriocin from another L. amylovorus strain. These non-identical strains produce bacteriocins that display small differences in molecular mass and inhibitory spectrum. The amino acid sequence of amylovorin L471 shared significant homology with lactacin X, one of the two bactericidal peptides produced by Lactobacillus johnsonii VPI11088. A purified amylovorin L471 preparation p...

Research in Microbiology, 2010

Shewanella oneidensis, a Gram-negative bacterium with unusual respiratory versatility, is found i... more Shewanella oneidensis, a Gram-negative bacterium with unusual respiratory versatility, is found in soil and sediment environments, and sporadically as an opportunistic pathogen in humans and aquatic animals. The ability to form biofilms is a critical factor in the environmental spread and survival of this bacterium. We subjected S. oneidensis MR-1 to random transposon insertion mutagenesis to identify genes contributing to the ability of the organism to form biofilms on polystyrene surfaces. Follow-up of the clone that was most heavily impaired in biofilm formation led to the identification of a novel 285 kDa multi-domain protein which we have termed biofilm-promoting factor A (BpfA). BpfA is secreted by a type I secretion system to the cell surface, where it is a requisite for biofilm development. The BpfA-dependent biofilm phenotype is positively modulated by sub to low millimolar amounts of calcium. Intriguingly, BpfA features structural motifs and sequence fingerprints that can be traced back to bacterial Bap-family and RTX family proteins, two protein families harboring putative and established calcium binding sites.

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2008

The fungal strain Aspergillus niger produces two superoxide dismutases, Cu/Zn-SOD and Mn-SOD. The... more The fungal strain Aspergillus niger produces two superoxide dismutases, Cu/Zn-SOD and Mn-SOD. The primary structure of the Cu/Zn-SOD has been determined by Edman degradation of peptide fragments derived from proteolytic digests. A single chain of the protein, consisting of 153 amino acid residues, reveals a very high degree of structural homology with the amino acid sequences of other Aspergillus Cu/Zn-SODs. The molecular mass of ANSOD, measured by MALDI-MS and ESI-MS, and calculated by its amino acid sequence, was determined to be 15821 Da. Only one Trp residue, at position 32, and one disulfide bridge were identified. However, neither a Tyr residue nor a carbohydrate chain occupying an N-linkage site (-Asn-Ile-Thr-) were found. Studies on the temperature and pH dependence of fluorescence, and on the temperature dependence of CD spectroscopic properties, confirmed that the enzyme is very stable, which can be explained by the stabilising effect of the disulfide bridge. The enzyme re...

Identification and characterization of novel chromogranin B‐derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS

European Journal of Biochemistry, 2001

Veterinary Research, 2014

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens t... more Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.

A kinetic approach to the dependence of dissimilatory metal reduction byShewanella oneidensisMR-1 on the outer membrane cytochromescOmcA and OmcB

The FEBS Journal, 2007

The Gram-negative bacterium Shewanella oneidensis MR-1 shows a remarkably versatile anaerobic res... more The Gram-negative bacterium Shewanella oneidensis MR-1 shows a remarkably versatile anaerobic respiratory metabolism. One of its hallmarks is its ability to grow and survive through the reduction of metallic compounds. Among other proteins, outer membrane decaheme cytochromes c OmcA and OmcB have been identified as key players in metal reduction. In fact, both of these cytochromes have been proposed to be terminal Fe(III) and Mn(IV) reductases, although their role in the reduction of other metals is less well understood. To obtain more insight into this, we constructed and analyzed omcA, omcB and omcA/omcB insertion mutants of S. oneidensis MR-1. Anaerobic growth on Fe(III), V(V), Se(VI) and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III) reduction, a redundant function in V(V) reduction, and no apparent involvement in Se(VI) and U(VI) reduction. Growth of the omcB(-) mutant on Fe(III) was more affected than growth of the omcA(-) mutant, suggesting OmcB to be the principal Fe(III) reductase. This result was corroborated through the examination of whole cell kinetics of OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is approximately 11.5 and approximately 6.3 times faster than OmcA at saturating and low nonsaturating concentrations of Fe(III)-nitrilotriacetic acid, respectively, whereas the omcA(-) omcB(-) double mutant was devoid of Fe(III)-nitrilotriacetic acid reduction activity. These experiments reveal, for the first time, that OmcA and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis MR-1. Kinetic inhibition experiments further revealed vanadate (V(2)O(5)) to be a competitive and mixed-type inhibitor of OmcA and OmcB, respectively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Neither sodium selenate nor uranyl acetate were found to inhibit OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken together with our growth experiments, this suggests that proteins other than OmcA and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.

European Journal of Biochemistry, 1997

In functional units d and g from the P,-haemocyanin of the gastropod Helix pornatia, aminoacid an... more In functional units d and g from the P,-haemocyanin of the gastropod Helix pornatia, aminoacid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d m i s 6 2 ; H. pomatiu g m H i s 6 8 ) . This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units. The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatiu d and on peptides obtained by proteolysis of carboxymethylated H. pomutiu d with trypsin and pronase. Upon sequence analysis the cysteine-histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2-thiolhistidine. A peptide with a cysteine-histidine thioether bridge was isolated from pepsinolysates of functional units c, e, ,f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. ofiicinalis and 0. vulgaris. A cysteine-histidine thioether bridge, which was reported previously for tyrosinase of Neumsporu crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.

PROTEOMICS, 2003

Shewanella oneidensis MR‐1 is a gram‐negative facultative aerobic bacterium living at oxic‐anoxic... more Shewanella oneidensis MR‐1 is a gram‐negative facultative aerobic bacterium living at oxic‐anoxic interfaces in nature. The plasticity of terminal electron‐acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear. In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide. By analysis of two‐dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins. The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano‐high‐performance liquid chromatography coupled to an electrospray ionization‐quadrupole‐time of flight instrument, a recently introduced matrix‐assisted laser desorption/ionization (MALDI) tandem‐time of flight mass spectrometer. The instrument allows the acquisition of high quality spectra, in both the mass spectromet...

Microbial Biotechnology, 2007

SummaryMany studies have reported microorganisms as efficient biocatalysts for colour removal of ... more SummaryMany studies have reported microorganisms as efficient biocatalysts for colour removal of dye‐containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR‐1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox‐active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. T...

Journal of Proteome Research, 2007

The vast majority of proteins functions in complex with one or more of the same or other proteins... more The vast majority of proteins functions in complex with one or more of the same or other proteins, indicating that protein-protein interactions play crucial roles in biology. Here, we present a β-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fused to two non-functional but complementing β-galactosidase truncations (∆R and ∆ω). The level of complemented β-galactosidase activity, driven by the protein-protein recognition between both nonβ-galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Our approach was validated by reconfirming some well-established Escherichia coli cytoplasmic and membranous interactions, including well-chosen mutants, and providing the first in vivo evidence for the transient periplasmic interaction between Rhodobacter capsulatus cytochrome c 2 and cytochrome c peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analyses of interactions are not limited to particular cellular compartments, ii) the potential of using the system in mutation-driven structure-function studies, and iii) the possibility of its application to transiently interacting proteins. These benefits demonstrate the relevance of the method as a powerful new tool in the broad spectrum of interaction assessment methods.

Mass spectrometric identification ofin vivo carbamylation of the amino terminus ofEctothiorhodospira mobilis high-potential iron-sulfur protein, isozyme 1

Journal of Mass Spectrometry, 2002

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 ... more The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.

Structure of Hemocyanin Subunit CaeSS2 of the Crustacean Mediterranean Crab Carcinus aestuarii

Journal of Biochemistry, 2005

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exh... more Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.

Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin

Glycoconjugate Journal, 2011

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin ... more The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.

Sequence, overproduction and purification of the family 11 endo-β-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38

Gene, 1999

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening a... more The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.