Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor

Autocatalytic formation of a covalent link between tryptophan 41 and the heme in ascorbate peroxidase.

Electronic spectroscopy, HPLC analyses, and mass spectrometry (MALDI-TOF and MS/MS) have been used to show that a covalent link from the heme to the distal Trp41 can occur on exposure of ascorbate peroxidase (APX) to H2O2 under noncatalytic conditions. Parallel analyses with the W41A variant and with APX reconstituted with deuteroheme clearly indicate that the covalent link does not form in the absence of either Trp41 or the heme vinyl groups. The presence of substrate also precludes formation of the link. Formation of a protein radical at Trp41 is implicated, in a reaction mechanism that is analogous to that proposed [Ghiladi, R. A., et al. (2005) Biochemistry 44, 15093-15105] for formation of a covalent Trp-Tyr-Met link in the closely related catalase peroxidase (KatG) enzymes. Collectively, the data suggest that radical formation at the distal tryptophan position is not an exclusive feature of the KatG enzymes and may be used more widely across other members of the class I heme peroxidase family

Peroxide-dependent formation of a covalent link between Trp51 and the heme in cytochrome c peroxidase.

Ascorbate peroxidase (APX), cytochrome c peroxidase (CcP), and the catalase-peroxidases (KatG) share very similar active site structures and are distinguished from other peroxidases by the presence of a distal tryptophan residue. In KatG, this distal tryptophan forms a covalent link to an adjacent tyrosine residue, which in turn links to a methionine residue. We have previously shown [ Pipirou, Z. et al. ( 2007 ) Biochemistry 46 , 2174 - 2180 ] that reaction of APX with peroxide leads, over long time scales, to formation of a covalent link with the distal tryptophan (Trp41) in a mechanism that proceeds through initial formation of a compound I species bearing a porphyrin pi-cation radical followed by radical formation on Trp41, as implicated in the KatG enzymes. Formation of such a covalent link in CcP has never been reported, and we proposed that this could be because compound I in CcP uses Trp191 instead of a porphyrin pi-cation radical. To test this, we have examined the reactivity of the W191F variant of CcP with H(2)O(2), in which formation of a porphyrin pi-cation radical occurs. We show, using electronic spectroscopy, HPLC, and mass spectroscopy, that in W191F partial formation of a covalent link from Trp51 to the heme is observed, as in APX. Radical formation on Trp51, as seen for KatG and APX, is implicated; this is supported by QM/MM calculations. Collectively, the data show that all three members of the class I heme peroxidases can support radical formation on the distal tryptophan and that the reactivity of this radical can be controlled either by the protein structure or by the nature of the compound I intermediate

Evidence for heme oxygenase activity in a heme peroxidase.

The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the alpha-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism

Covalent Attachment of Heme to the Protein Moiety in an Insect E75 Nitric Oxide Sensor

We have recombinantly expressed and purified the ligand binding domains (LBDs) of four insect nuclear receptors of the E75 family. The Drosophila melanogaster and Bombyx mori nuclear receptors were purified as ferric hemoproteins with Soret maxima at 424 nm, whereas their ferrous form had a Soret maximum at 425 nm that responds to ·NO and CO binding. In contrast, the purified LBD of Oncopeltus fasciatus displayed a Soret maximum at 415 nm for the ferric protein that shifted to 425 nm in its ferrous state. Binding of ·NO to the heme moiety of D. melanogaster and B. mori E75 LBD resulted in the appearance of a peak at 385 nm, whereas this peak appeared at 416 nm in the case of the O. fasciatus hemoprotein, resembling the behaviour displayed by its human homolog Rev-erbβ. HPLC analysis revealed that, unlike the D. melanogaster and B. mori counterparts, the heme group of O. fasciatus is covalently attached to the protein through the side-chains of two amino acids. The large sequence homology with O. fasciatus E75 led us to clone and express the LBD of Blattella germanica, which established that its spectral properties closely resemble those of O. fasciatus and that it also has the heme group covalently bound to the protein. Hence, ·NO/CO regulation of the transcriptional activity of these nuclear receptors might be differently controlled among various insect species. In addition, covalent heme binding provides strong evidence that at least some of these nuclear receptors function as diatomic gas sensors rather than heme sensors. Finally, our findings expand the classes of hemoproteins in which the heme group is normally covalently attached to the polypeptide chain