Evolution, Three-Dimensional Model and Localization of Truncated Hemoglobin PttTrHb of Hybrid Aspen

<div><p>Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (<i>Populus tremula</i> L. × <i>tremuloides</i> Michx.), the model system of tree biology. Additionally, we studied the <i>PttTrHb</i> expression in relation to non-symbiotic class1 Hb gene (<i>PttHb1</i>) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a <i>Bacillus subtilis</i> -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in <i>Bs</i>-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of <i>in vitro</i> -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.</p></div

Crystal structure of the human vascular adhesion protein-1: Unique structural features with functional implications

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role

Structure-based sequence alignment of selected group II TrHb sequences from bacteria and plant species.

<p>The sequence alignment was generated by superposing the structures and then aligning other bacterial and plant sequences. The α-helices of the X-ray structure of Bs-HbO (1UX8) are shown as grey background on the top of the alignment. The α-helices in vertebrate Hbs with 3-on-3 arrangement of α-helices are conventionally labelled as A, B, C, D, E, F, G and H, with topological sites numbered sequentially within each α-helix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088573#pone.0088573-Perutz1" target="_blank">[70]</a>. The same labelling of α-helices and topological sites are used here for the truncated Hbs. The conserved Gly motifs and F8His residues are in blue background with white font. The distal site residues B10, CD1, E11 and G8 are in pink background. All residues in the protein matrix tunnel cavity, originally in Mt-HbN (also added to the alignment), are in yellow background. The distal cavity residues discussed in the text are in pink background. The gate residue E7 is in cyan background.</p

Phylogenetic analysis of plant truncated hemoglobin (TrHb) proteins with significant bootstrap values.

<p>A: angiosperms, B: bacteria, Br: bryophytes, G: gymnosperms, gA: green alga.</p

Dual targeted poplar ferredoxin NADP+ oxidoreductase interacts with hemoglobin 1

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP+ oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol

Expression of <i>PttTrHb</i> and <i>PttHb1</i> in control line V617 and RNAi-silenced lines, H3, T2 and T3.

<p>Means ± SE (n = 5) calculated using <i>TUA</i> as reference gene.</p

Homology model of PttTrHb and heme pocket residues.

<p>A: Cartoon representation of the PttTrHb model in cyan colour. The heme is shown as sticks coloured as C, slate; N, blue; O, red and Fe, orange. B: Heme pocket residues shown in sticks. The proximal residues F8His and F7Arg are in C, orange. The carbon atom in heme is shown as slate. The cyanide ligand is with a yellow carbon. Same conventional vertebrate Hb labels are used for the ligand binding residues in cyan (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088573#pone-0088573-g001" target="_blank">Fig. 1</a>). Other residues (in white) are numbered by residue numbers. In the PttTrHb model, same residue numbering is used as 1UX8.</p

Immunolocalization of PttTrHb protein in hybrid aspen leaves and stem.

<p>A, Negative control obtained with leaf section incubated with primary antibody preabsorbed with the recombinant PttTrHb. B, Transversal section (TS) of young leaf from plant grown in normal conditions. C, TS of young leaf from 5 h SNP-treated plant. D, TS of mature leaf from plant grown in normal conditions. E, TS of mature leaf from 5 h SNP-treated plant. F, Negative control obtained with stem section incubated with primary antibody preabsorbed with the recombinant PttTrHb. G, TS of stem from plant grown in normal conditions. H and I, TS of stem from 5 h SNP-treated plant. c: cortex, ep: epidermis, p: phloem, pa: parenchyma, x: xylem. Bar  =  100 µm.</p

Immunolocalization of <i>PttTrHb</i> protein in hybrid aspen roots.

<p>Negative controls were obtained by root section incubated in A, without primary antibody or in B, with primary antibody preabsorbed with the recombinant PttTrHb. C to H: plant grown in normal conditions. C, Transversal section (TS) of lateral root. D, TS of adventitious root of plant rooted for 3 weeks. E, TS of adventitious root of plant rooted for 8 weeks. F, Longitudinal section of adventitious root of plant rooted for 8 weeks. G, TS of adventitious root tip of plant rooted for 8 weeks. H, TS of adventitious root of plant rooted for 8 weeks at the point of emergence of a lateral root (arrow). I, TS of adventitious root of plant rooted for 8 weeks after 5 h of SNP-treatment. c: cortex, e: endodermis, ep: epidermis, p: phloem, x: xylem. Bar  =  100 µm.</p

Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10–VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products