Axonal Odorant Receptors Mediate Axon Targeting

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine- binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons.Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting

Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

umor cells), and its tumoricidal activity has been well established.-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules.We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity

Differential effects of heparin and glucose on structural conformation of human alpha1 antitrypsin: evidence for a heparin-induced cleaved form of the inhibitor

alpha1 Antitrypsin (alpha1AT) is the archetypal member of the serpin superfamily. Current knowledge of its inhibitory mechanism does not provide for any heparin-induced enhancement of serine proteinase inhibition. Since previous results have shown that an apparently altered alpha1AT form may be purified from the plasma of insulin-dependent diabetics by means of heparin-affinity chromatography, in the present work the possibility was tested that heparin at various concentrations modifies the structural conformation and function of human alpha1AT in the absence and presence of glucose, used at concentrations of 15 mM to mimick mild hyperglycemic conditions. Heparin was observed to bind strongly to alpha1AT, causing maximal enhancement of tryptophan fluorescence emission at 50 microg/ml, mostly in the presence of glucose. Circular dichroism spectra revealed that heparin with glucose caused the most relaxed, ordered structure of the inhibitor with increased heat stability. Modification in conformation was accompanied by loss of inhibitory activity, as demonstrated by the inability of alpha1AT to block bovine trypsin in the specific assay and by alterations of its immunological properties. However, despite inactivation, in the presence of heparin-both with and without glucose-alpha1AT was still able to bind trypsin, as revealed by inhibitor-to-proteinase complexes visible in both SDS- and nondenaturing electrophoreses. These complexes showed the same feature regardless of trypsin concentration and differed from those formed at a molar excess of the inhibitor in the absence of heparin, since they underwent rapid, intense fragmentation accompanied by complete loss of the secondary structure of the inhibitor. Even in the absence of trypsin, cleavage of alpha1AT was also observed to occur at both Val321- and Glu344- in the primary sequence of the inhibitor in the presence of 50 microg/ml heparin, with and without glucose. These results suggest that heparin binding to alpha1AT causes profound structural changes in the molecule, involving both the expulsion of the reactive site out of the molecule plane and a relaxed, heat-stable form of the inhibitor, rendered a substrate for the proteinase. Although glucose apparently does not affect alpha1AT functioning, it does enhance the effects of heparin on the alpha1AT structure. The possibility is discussed that, while heparin and glucose binding occurs at different sites on alpha1AT, glucose favors heparin binding by inducing a partially relaxed form in the inhibitor. Differences in structure and charge between the two substances account for both different individual effects on alpha1AT and the predominance of the effects of heparin

Surface topography of histidine residues of tetanus toxin probed by immobilized-metal-ion affinity chromatography.

Tetanus toxin contains 14 histidine residues: six of them are localized in the light chain (L), one is present in the N-terminal half of the heavy chain (HN) and the remaining seven histidines are localized in the C-terminal half of the heavy chain (Hc). Using immobilized-metal-ion affinity chromatography with Chelating Superose-Zn(II), we show that histidines of Hc are exposed to the protein surface and are responsible for the binding of tetanus toxin and of Hc to the immobilized metal. The histidines of the L chain are not available for co-ordination of matrix-bound Zn2+; however, two of them and three of the histidines of fragment Hc are accessible to diethyl pyrocarbonate. Chromatography on Superose-Zn(II) is also shown to be a simple and efficient method for the rapid isolation of tetanus toxin and of its Hc fragment, which can be extended to the botulinum neurotoxins

The role of tryptophan in protein fibrillogenesis: relevance of Trp7 and Trp14 to the amyloidogenic properties of myoglobin

In order to understand the role of tryptophan in the mechanisms of fibrils formation, the ability of a series of analogs of the residue 7-18 span of myoglobin to form amyloid-like fibrils was investigated. Alternatively one or both tryptophans were substituted with alanine and leucine, to determine the contribution of hydrophobicity and aromaticity. The scale of aggregation propensity of the peptides determined indicates that tryptophan is crucial for the amyloidogenic process. Since the rare tryptophan residue is generally engaged in structural roles in proteins, or when exposed serves as binding sites, we surmise that its exposure in the amyloidogenic fragments allows for intermolecular clustering with residues from other molecules leading to the formation of amyloid aggregates

Partly folded states of members of the lysozyme/lactalbumin superfamily: A comparative study by circular dichroism spectroscopy and limited proteolysis

The partly folded states of protein members of the lysozyme (LYS)/α-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123- to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far- and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the β-subdomain (approximately chain region 34–57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured α-domain and a largely disrupted β-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule