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S Dewilde

Publications and source records attributed to S Dewilde.

16 recordsLinked to original sources
PubMed2001

Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family.

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O(2) supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe(2+)) form, indicating a His-Fe(2+)-His binding scheme. O(2) or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O(2) and CO binding is suggested to be slow in vivo. Because of this competition, the observed O(2) affinity of recombinant human neuroglobin is average (1 torr at 37 degrees C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 degrees C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E'(o) = -129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.

Animals↗
PubMed2001

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.

Amino Acid Sequence↗
PubMed2001

The primary structure of globin and linker chains from the chlorocruorin of the polychaete Sabella spallanzanii.

Annelid hemoglobins are organized in a very complex supramolecular network of interacting polypeptides, the structure of which is still not wholly resolved. We have separated by two-dimensional electrophoresis the 4-MDa chlorocruorin of Sabella spallanzanii and identified its components by amino-terminal sequencing. This work reveals a high rate of heterogeneity of constituent chains in a single animal as well as in the Sabella population. Using a cDNA library prepared from the hematopoietic tissue of this worm, we have isolated and fully sequenced most globin and linker cDNAs. The primary structure features of these polypeptides have been characterized by comparison with model globin and linker sequences.

Amino Acid Sequence↗
PubMed2001

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans.

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans was investigated by applying cladistic and distance-based approaches to reconstruct the phylogenetic relationships of this group of respiratory pigments. We performed this study using the aligned sequences of globin and linker chains that are the constituents of these complex molecules. Three novel globin and two novel linker chains of Sabella spallanzanii described in an accompanying paper (Pallavicini, A., Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384--26390) were also included. Our results allowed us to test previous hypotheses on the evolutionary pathways of these proteins and to formulate a new most parsimonious model of molecular evolution. According to this novel model, the genes coding for the polypeptides forming these composite molecules were already present in the common ancestor of annelids, vestimentiferans, and pogonophorans.

Amino Acid Sequence↗
PubMed2001

Protein and gene structure of a chlorocruorin chain of Eudistylia vancouverii.

The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.

Amino Acid Sequence↗
PubMed2000

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

Amino Acid Sequence↗
PubMed2000

Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale.

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.

2,3-Diphosphoglycerate↗
PubMed2000

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.

Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

Amino Acid Motifs↗
PubMed2000

A hemoglobin with an optical function.

Hemoglobins are best known as oxygen transport proteins. Here we describe a hemoglobin from the parasitic nematode Mermis nigrescens (Mn-GLB-E) that has an optical, light shadowing function. The protein accumulates to high concentration as intracellular crystals in the ocellus of mature phototactic adult females while also being expressed at low concentration in other tissues. It differs in sequence and expression pattern from Mn-GLB-B, a second Mermis globin. It retains the structure and oxygen-binding and light-absorbing properties typical of nematode hemoglobins. As such, recruitment to a shadowing role in the eye appears to have occurred by changes in expression without modification of biochemistry. Both globins are coded by genes interrupted by two introns at the conserved positions B12.2 and G7.0, which is in agreement with the 3exon/2intron pattern model of globin gene evolution.

Amino Acid Sequence↗
PubMed1999

Daphnia pulex didomain hemoglobin: structure and evolution of polymeric hemoglobins and their coding genes.

The high-molecular-weight extracellular hemoglobin of Daphnia pulex is composed of at least three different didomain globin chains. The primary structure of one of these chains was determined at the protein and cDNA levels. Each globin domain of the polypeptide chain displays the standard structural characteristics. The first domain is preceded by a 30-residue extension containing an 18-residue unprecedented threonine-rich segment and a 12-residue preA segment which is homologous to the preA segments of other nonvertebrate globin chains. Both domains are linked together by a preA' segment, which is homologous to other preA segments and lacks the threonine-rich segment. Dimerization of the globin chains by the formation of a disulphide bridge linking the unique cysteines near the amino-termini results in a covalent, vertebrate-like tetradomain structure. The flexible amino-terminal extension most likely facilitates dimerization. The gene coding for this globin chain is interrupted by six small introns. Each domain displays two intradomain introns at the conserved positions B12.2 and G7.0. A precoding intron occurs at position preA(-27.0) and a bridge intron at occurs preA'(-13.2). We propose a crossover event as the most likely mechanism for duplication. Arthropod globin trees reflect the added effects of gene diversification, gene duplication, and species evolution. The position of monodomain intracellular globins in the tree suggests that they resemble the ancestral globin more than the derived didomain extracellular globins do.

Amino Acid Sequence↗
PubMed1998

Structural, functional, and genetic characterization of Gastrophilus hemoglobin.

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.

Amino Acid Sequence↗
PubMed1998

Characterization of the myoglobin and its coding gene of the mollusc Biomphalaria glabrata.

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature. The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508). The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

Amino Acid Sequence↗
PubMed1997

Trematode myoglobins, functional molecules with a distal tyrosine.

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Amino Acid Sequence↗
PubMed1996

Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata.

The globin of the nerve cord of the polychaete annelid Aphrodite aculeata was isolated and purified to homogeneity. The native molecule has a pI of 6.3 and acts as a dimer of two identical Mr 15, 644.5 polypeptide chains as determined by electrospray mass spectrometry. It has an average affinity for oxygen (P50 = 1.24 torr) resulting from fast association (kon = 170 X 10(6) M-1 . s-1) and dissociation rates (koff = 360 s-1). The partial primary structure of this nerve globin was determined at the protein level and completed and confirmed by translation of the cDNA sequence. The globin chain has 150 amino acid residues and a calculated Mr of 15, 602.69 strongly suggesting that the amino terminus is acetylated. The absence of a leader sequence and the lack of Cys at the positions NA2 and H9 needed for the formation of the high Mr complexes found in extracellular annelid globins classify the Aphrodite globin with the cellular globin species. The Aphrodite nerve globin is unlikely to represent a separate globin family, as cDNA derived primers detect globin messenger RNA in muscle, gut, and pharynx tissue as well. The gene encoding this globin species is interrupted by a single intron, inserted at position G7.0. Comparison to other globin gene structures strongly suggest that introns can be lost independently, rather than simultaneously as a result of a single conversion event as suggested previously (Lewin, R. (1984) Science 226, 328).

Amino Acid Sequence↗
PubMed1995

A fairly conserved epitope on the hemagglutinin of influenza A (H3N2) virus with variable accessibility to neutralizing antibody.

A monoclonal antibody LMBH5 was derived from mice which had been immunized with A/Victoria/3/75 (H3N2)-type recombinant, secreted hemagglutinin (HA), and were subsequently challenged with a potentially lethal dose of X31 [A/Aichi/2/68 (H3N2) x A/PR/8/34 (H1N1)] virus. LMBH5 reacted strongly with the native and low-pH-induced conformations of the HA of A/Aichi (X31 strain) and A/Victoria (X47 strain), but very weakly with the native structure of the HA of A/Philippines/2/82 (X79 strain) and not at all with the HA of A/Guizhou/54/89 H3 (NIB25 strain). However, the acid-induced conformations of the latter two viruses were recognized by LMBH5. The antibody prevented infection of MDCK cells with X31 and X47, whereas X79 virus was partially neutralized by LMBH5. X31 monoclonal escape variants had single amino acid substitutions (Ser 227-->Pro) near the interface. The data obtained suggest that the neutralizing LMBH5 reacts with a fairly conserved epitope of influenza A (H3N2) virus, which as a result of antigenic drift becomes inaccessible in the native state of the HA.

Animals↗