Aphrodisin, an aphrodisiac lipocalin secreted in hamster vaginal secretions

Vertebrates communicate through pheromones, which favor biological regulations within each species. Aphrodisin, a protein belonging to the lipocalin superfamily, found in hamster vaginal secretions, is detected by the male accessory olfactory system and induces or facilitates its copulatory behavior. Although much is known about aphrodisin structure, the question of whether aphrodisin bears itself the pheromonal function or is simply a carrier for hydrophobic small pheromones has not been definitely solved. Arguments based on use of recombinant aphrodisin deprived of any natural ligand and its capability to convey hamster pheromonal compounds will be discussed, together with progresses concerning putative natural ligand(s).     

Crystal structure of aphrodisin, a sex pheromone from female hamster

We have solved the crystal structure of aphrodisin, a pheromonal protein inducing a copulatory behaviour in male hamster, using MAD methods with selenium, at 1.63 A resolution. The monomeric protein belongs to the lipocalin family, and possesses a disulfide bridge in a loop between strands 2 and 3. This disulfide bridge is characteristic of a family of lipocalins mainly identified in rodents, and is analogous to the fifth disulfide bridge of the long neurotoxins, such as alpha cobratoxin. An elongated electron density was found inside the buried cavity, which might represent a serendipitous ligand of unknown origin. The analysis of the water accessible surfaces of the side-chains bordering the cavity indicates that Phe76 may be the door for the natural ligand to access the cavity. This residue defines the entry of the cavity as belonging to the consensus for lipocalins. The face bearing Phe76 might also serve for the interaction with the receptor.     

Pheromone signalling in the mouse: role of urinary proteins and vomeronasal organ.

The lipocalin protein family is characterized in structure by a conserved hydrophobic pocket which can bind small volatile odorants. The Major Urinary Proteins (MUPs) are a class of lipocalins found in the urine of adult male mice which concentrate in the urine odorants which confer a characteristic odor. The behavioural as well as the endocrine effects of mouse urine and MUPs are briefly reviewed, suggesting a complex role is pheromonal communication. Some recent data on the molecular receptors of the vomeronasal organ further suggest a complex interaction with the MUP system.     

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.     

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

Lipocalins are extracellular proteins (17-25 kDa) that bind and transport small lipophilic molecules. The three-dimensional structure of the first lipocalin from a metatherian has been determined at different values of pH both with and without bound ligands. Trichosurin, a protein from the milk whey of the common brushtail possum, Trichosurus vulpecula, has been recombinantly expressed in Escherichia coli, refolded from inclusion bodies, purified and crystallized at two different pH values. The three-dimensional structure of trichosurin was solved by X-ray crystallography in two different crystal forms to 1.9 A (1 A=0.1 nm) and 2.6 A resolution, from crystals grown at low and high pH values respectively. Trichosurin has the typical lipocalin fold, an eight-stranded anti-parallel beta-barrel but dimerizes in an orientation that has not been seen previously. The putative binding pocket in the centre of the beta-barrel is well-defined in both high and low pH structures and is occupied by water molecules along with isopropanol molecules from the crystallization medium. Trichosurin was also co-crystallized with a number of small molecule ligands and structures were determined with 2-naphthol and 4-ethylphenol bound in the centre of the beta-barrel. The binding of phenolic compounds by trichosurin provides clues to the function of this important marsupial milk protein, which is highly conserved across metatherians.     

Le rôle des protéines liant les odeurs (OBP) dans la transduction olfactive

This paper reviews biochemical and functional properties of a family of proteins involved in the transduction process of chemical signals. Odorant-binding proteins (OBPs) are small soluble proteins highly concentrated in the chemosensory organs of Insects and Vertebrates. They are preferentially expressed in the nasal mucus of Vertebrates and in the sensillar lymph of Insects. They have been found to bind reversibly small hydrophobic molecules detected via the olfactory system. The vertebrate OBPs bind non-specific odorants with low affinities. They belong to the lipocalin family as well as other proteins involved in chemical communication and associated to different organs and functions. Nevertheless, no specific ligand for OBPs has been yet identified in Vertebrates. However, the large microdiversity of OBPs in the same animal suggests that OBPs could be involved in the discrimination of odors. Chemical communication in Noctuid moths was used as a model to study the molecular mechanisms of odor recognition. The pheromonal system is extremely sensitive and specific since the male is able to detect only few molecules of the pheromone and to recognize specific blends of the same molecules. The chemical signals were identified for a large number of Lepidopteran species and the associated behaviours they elicit were fully characterized. The Lepidopteran OBPs are divided into two sub-classes according to their ligands: pheromone-binding proteins (PBP) are expressed in sensilla trichodea responding to pheromonal compounds while general odorant binding proteins (GOBP) are associated with sensilla basiconica tuned to the detection of general odors, such as plant volatiles. The PBPs selectively bind components of the female sex-pheromone with measurable affinities. The ligand binding site was localized in the 40–60 aminoacids region. Substitutions in the binding site of different proteins are correlated with the fixation of different ligands, leading to the hypothesis that the primary structure encodes the ligand specificity. Other proteins expressed in chemosensory organs of other orders of Insects were cloned or purified. In absence of functional data, they were called OBP-like. Some of them were localized in the gustatory organs and could be common carriers of odors in both olfactory and gustatory systems. Many arguments are in accordance with an active role of the OBPs in the early steps of odor discrimination. The heterogeneity of OBPs inside species and between species, the spatial segregation in their expression and the different binding affinities of PBPs towards pheromonal compounds support the hypothesis that the coding of odors is realized as soon as the level of OBPs. More, the complex OBP/odor could be the stimulus for olfactory receptors cloned in Vertebrates and still putative in Insects. This hypothesis suggests that OBPs take part as an essential element of the chemosensory transduction.     

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.     

Neutrophil Gelatinase-associated Lipocalin in Normal and Neoplastic Human Tissues. Cell Type-specific Pattern of Expression

Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions.The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.     

The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin.

Human neutrophil gelatinase-associated lipocalin (HNGAL) is a member of the lipocalin family of extracellular proteins that function as transporters of small, hydrophobic molecules. HNGAL, a component of human blood granulocytes, binds bacterially derived formyl peptides that act as chemotactic agents and induce leukocyte granule discharge. HNGAL also forms a complex with the proenzyme form of matrix metalloproteinase-9 (pro-MMP-9, or progelatinase B) via an intermolecular disulphide bridge. This association allows the subsequent formation of ternary and quaternary metalloproteinase/inhibitor complexes that vary greatly in their metalloproteinase activities. The structure and dynamics of apo-HNGAL have been determined by NMR spectroscopy. Simulated annealing calculations yielded a set of 20 convergent structures with an average backbone RMSD from mean coordinate positions of 0. 79(+/-0.13) A over secondary structure elements. The overall rotational correlation time (13.3 ns) derived from15N relaxation data is consistent with a monomeric protein of the size of HNGAL (179 residues) under the experimental conditions (1.4 mM protein, pH 6.0, 24.5 degrees C). The structure features an eight-stranded antiparallel beta-barrel, typical of the lipocalin family. One end of the barrel is open, providing access to the binding site within the barrel cavity, while the other is closed by a short 310-helix. The free cysteine residue required for association with pro-MMP-9 lies in an inter-strand loop at the closed end of the barrel. The structure provides a detailed model of the ligand-binding site and has led to the proposal of a site for pro-MMP-9 association. Dynamic data correlate well with structural features, which has allowed us to investigate a mechanism by which a cell-surface receptor might distinguish between apo and holo-HNGAL through conformational changes at the open end of the barrel.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

CD3 negative "small agranular lymphocytes" are natural killer cells

We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.     

Immunocytochemical localization of apolipoprotein D in oligodendrocyte precursor-like cells,perivascular cells,and pericytes in the human cerebral cortex

Apolipoprotein D, a lipocalin transporter of small hydrophobic molecules including sterols, steroid hormones and arachidonic acid, is a widely expressed protein in peripheral and neural tissues. It has been shown to be upregulated in the context of neural injury, and with neuronal degeneration and regeneration. Here we have used light and electron microscopic immunocytochemistry with immunogold labeling to delineate the pattern of expression of apoD in the human brain. Our results confirm previous observations that apoD is a predominantly glial protein in the nervous system. In addition we have found that apoD is present in the cytosol and outer membrane of the nuclear envelope of glial cells in the neuropil. The labeled glial cells were putatively identified as a population of oligodendrocyte precursor cells. Immunoreactivity was also associated with the cytosol of perivascular cells, and lysosomes of pericytes, in the walls of blood vessels. These observations suggest a potential role for glial cells and apoD, in the transport of sterols and small hydrophobic molecules to, or from, blood vessels in the cortex.     

Factors affecting the pheromone composition of voided boar saliva

The pheromone binding protein 'pheromaxein' which binds the pheromonal 16-androstene steroids in the saliva of the male pig (boar), was degraded and lost its binding activity in saliva incubated in air for 72 h at 21 degrees C and 37 degrees C. However, pheromaxein and its binding activity were retained in saliva incubated for 168 h at 4 degrees C. When the 3H-labelled pheromones 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol), 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 beta-ol (3 beta-androstenol) were incubated with boar saliva for 168 h at 21 degrees C, 3 alpha-androstenol was primarily converted to 5 alpha-androstenone and 5 alpha-androstenone to 3 beta-androstenol; 3 beta-androstenol was unchanged. Evidence was obtained for microorganisms being responsible for these steroid transformations.     

Pig meat quality from entire males

This paper constitutes an updated review of the production and meat quality aspects of rearing entire male pigs. Since a major obstacle in rearing entire males is the incidence of boar taint, possible methods for detection are also summarised. Safe and fast methods for detection of boar taint would be valuable in avoiding complaints from consumers. Pig meat quality is determined by many aspects, among which odour and taste are the most important attributes. Odour may be negatively affected by the presence of a pheromonal steroid, androstenone, and a fermentation product of l-tryptophan, skatole. Male pigs are surgically castrated in many countries to minimise the risk of accumulation of high levels of androstenone and skatole. Raising entire male pigs is more profitable because they have superior production characteristics and improved meat quality due to leaner carcasses and higher protein content, as compared to castrated pigs. Furthermore, surgical castration is negative from an animal welfare point of view. In most studies, no differences in sensory quality have been found between lean meat from entire male pigs with low levels of androstenone and skatole and pork from castrates and females. The question that remains is: which substances are responsible for boar taint besides androstenone and skatole and whether they need to be considered? The threshold values used for androstenone and skatole might also be too high for highly sensitive persons. Recent research shows that a human odorant receptor, ORD7D4, is involved in sensitivity to androstenone. If the ORD7D4 genotypes of consumer and expert panels are known, this might facilitate consumer studies in the future. There is still a great need for rapid on/at-line detection methods in abattoirs for identifying carcasses with unacceptable levels of boar taint compounds. Several emerging rapid technologies with a potential for boar taint detection have been investigated. They represent various measurement principles such as chemical sensor arrays (electronic noses), mass-spectrometry fingerprinting, ultra-fast gas chromatography, gas-phase spectrometry and biosensors. An industrial detection method should allow 100% correct classification of both acceptable and not-acceptable samples with regard to boar taint sorting criteria. There are, however, still too high a percentage of false negatives ranging from 5% to 20%. In addition, these methods do not yet seem to fulfil the industrial specifications with regard to cost efficiency, simplicity and analysis time. There is still no dedicated measurement technology available for on/at-line detection of boar-tainted carcasses that measures both androstenone and skatole.     

Natural ligands of hamster aphrodisin

The chemical nature of vertebrate pheromones remains largely to be deciphered. Hamster aphrodisin is a rare instance of mammal proteinaceous sexual pheromone. This protein, found in vaginal secretions, facilitates the mounting behaviour of males via activation of a specialized sensory structure named the vomeronasal organ, which activates the accessory olfactory bulb. Since it might carry small pheromonal ligands due to its lipocalin structure, we analysed organic extracts from natural aphrodisin. We identified five predominant compounds specifically bound onto natural aphrodisin as 1-hexadecanol (44.7%), 1-octadecanol (19.5%), Z-9-octadecen-1-ol (18.2%), E-9-octadecen-1-ol (15.4%) and hexadecanoic acid (2.2%). Interestingly, these compounds are also described as part of insect pheromone blends, disclosing the continuing story of amazing coincidences of chemical communication shared by mammals and insects.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

The tryptophan phosphorescence of porcine and mutant bovine odorant-binding proteins: a probe for the local protein structure and dynamics

Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs.     

Biochemical, nutritional and genetic effects on boar taint in entire male pigs

Pork odour is to a great extent affected by the presence of malodorous compounds, mainly androstenone and skatole. The present review outlines the current state of knowledge about factors involved in the regulation of androstenone and skatole in entire male pigs. Androstenone is a pheromonal steroid synthesised in the testes and metabolised in the liver. Part of androstenone accumulates in adipose tissue causing a urine-like odour. Skatole is produced in the large intestine by bacterial degradation of tryptophan and metabolised by hepatic cytochrome P450 enzymes and sulphotransferase. The un-metabolised part accumulates in adipose tissue, causing faecal-like odour. Androstenone levels are mostly determined by genetic factors and stage of puberty, whereas skatole levels in addition to genetic background and hormonal status of the pigs are also controlled by nutritional and environmental factors. To reduce the risk of tainted carcasses entering the market, male pigs are surgically castrated in many countries. However, entire males compared to castrates have superior production characteristics: higher growth rate, better feed efficiency and leaner carcasses. Additionally, animal welfare aspects are currently of particular importance in light of increasing consumers' concerns. Nutrition, hormonal status, genetic influence on boar taint compounds and the methods to develop genetic markers are discussed. Boar taint due to high levels of skatole and androstenone is moderately heritable and not all market weight entire males have boar taint; it should thus be possible to select for pigs that do not have boar taint. In these studies, it is critical to assess the steroidogenic potential of the pigs in order to separate late-maturing pigs from those with a low genetic potential for boar taint. A number of candidate genes for boar taint have been identified and work is continuing to develop genetic markers for low boar taint. More research is needed to clarify the factors involved in the development of boar taint and to develop additional methods to prevent the accumulation of high concentrations of skatole and androstenone in fat. This review proposes those areas requiring further research.