mRNAs encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells. (1/64)

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.  (+info)

Tick histamine-binding proteins: isolation, cloning, and three-dimensional structure. (2/64)

High-affinity histamine-binding proteins (HBPs) were discovered in the saliva of Rhipicephalus appendiculatus ticks. Their ability to outcompete histamine receptors indicates that they suppress inflammation during blood feeding. The crystal structure of a histamine-bound HBP, determined at 1.25 A resolution, reveals a lipocalin fold novel in containing two binding sites for the same ligand. The sites are orthogonally arranged and highly rigid and form an internal surface of unusual polar character that complements the physicochemical properties of histamine. As soluble receptors of histamine, HBPs offer a new strategy for controlling histamine-based diseases.  (+info)

Abundant secretory lipocalins displaying male and lactation-specific expression in adult hamster submandibular gland. cDNA cloning and sex hormone-regulated repression. (3/64)

We have previously identified massively expressed 24- and 20.5-kDa male-specific proteins in submandibular salivary glands (SMG) of adult hamsters. Here we report the cloning of the cDNA encoding the 24-kDa protein which we have now found to be a heterogenously N-glycosylated form of the 20.5-kDa protein. The deduced amino acid sequence indicated that the protein is a member of the lipocalin family, the two most related lipocalins being rat odorant-binding protein of nasal mucosa and aphrodisin, a pheromonal protein present in vaginal discharge and saliva of female hamsters. Northern blot analysis showed that cognate mRNA is expressed in hamster SMG and lacrimal gland (LG) displaying marked sex-hormonal repression. The sex-hormonal repression patterns showed similarities and dissimilarities between SMG and LG but they were, respectively, similar to the sex-hormonal repression pattern noted for the SMG 24/20.5-kDa male-specific proteins and for an abundant female-specific 20-kDa LG secretory protein. These SMG and LG proteins were found to be immunologically similar and secretion of the SMG proteins in saliva and their excretion in urine was detected. The male-specific and abundant expression of the SMG proteins were seen at and after sexual maturity but was not dependent on androgens. Surprisingly, a temporary male-like expression of these SMG proteins was seen in lactating females which was obliterated by oestrogen administration. Our results show that despite differences in their repression by sex hormones, the gene for SMG 24/20.5-kDa proteins is similar or identical to that of LG 20-kDa protein and their marked repression by both androgens and oestrogens might be at the transcriptional level. Moreover, they might be excellent models with which to study sex hormone repression of gene expression at the molecular level. The results of homology search and the male- and lactation-specific pressure of the SMG proteins in adult saliva and urine suggests a possibility of their involvement in olfaction-mediated chemical communication between hamsters.  (+info)

Phage display reveals a novel interaction of human tear lipocalin and thioredoxin which is relevant for ligand binding. (4/64)

Human tear lipocalin (TL) is an unusual member of the lipocalin protein family, since it is known to bind a large variety of lipophilic ligands in vivo and acts as a cysteine proteinase inhibitor in vitro. It is suggested to function as a physiological protection factor by scavenging lipophilic potentially harmful compounds. Since protein-protein interaction or macromolecular complexation is a common feature of many lipocalins, we applied phage display technology to identify TL interacting proteins. By panning of a human prostate cDNA phagemid library against purified TL we isolated a thioredoxin (Trx) encoding phage clone. Biochemical analysis revealed that TL indeed interacts with Trx and is reduced by this redox protein. Reduction of the TL-specific disulfide bond is of functional relevance, since the reduced protein shows a nine-fold increase in ligand affinity when tested with retinoic acid as ligand.  (+info)

Tear lipocalins: potential lipid scavengers for the corneal surface. (5/64)

PURPOSE: To investigate the dynamic effect of tear lipocalins (TLs), the major lipid-binding protein in tears, at aqueous-cornea and lipid-aqueous interfaces, and their potential contribution to surface tension in the tear film. METHODS: Human apo- and holo-TLs were applied to the aqueous subphase in a Langmuir trough, and changes in surface pressure were measured. Changes in the contact angle of tear components were observed on Teflon and ferric-stearate-treated surfaces. A nitroxide-labeled derivative of lauric acid and a fluorescence-labeled derivative of palmitic acid were used to monitor the dynamic interaction of lipid removed from a hydrophobic surface by the major tear components in solution. RESULTS: TLs increase the surface pressure at the aqueous-air interface by penetrating, spreading, and rearranging on the surface. Apo-TLs show a longer diffusion-dependent induction time than holo-TLs due to more extensive oligomerization of the apoprotein. Kinetic analysis of relaxation time suggests that apo-TLs have more rapid surface penetration and rearrangement than holo-TLs, indicative of a more flexible structure in apo-TLs. TLs reduce the contact angle of solutions on lipid films, a property that is greater with TLs than other tear proteins. TLs, unlike lysozyme and lactoferrin, remove labeled lipids from hydrophobic surfaces and deliver them into solution. CONCLUSIONS: TLs are potent lipid-binding proteins that increase the surface pressure of aqueous solutions while scavenging lipids from hydrophobic surfaces and delivering them to the aqueous phase of tears. These data suggest important functional roles for TLs in maintaining the integrity of the tear film.  (+info)

A novel human odorant-binding protein gene family resulting from genomic duplicons at 9q34: differential expression in the oral and genital spheres. (6/64)

Lipocalins are carrier proteins for hydrophobic molecules in many biological fluids. In the oral sphere (nasal mucus, saliva, tears), they have an environmental biosensor function and are involved in the detection of odours and pheromones. Herein, we report the first identification of human lipocalins involved in odorant binding. They correspond to a gene family located on human chromosome 9q34 produced by genomic duplications: two new odorant-binding protein genes ( hOBP (IIa) and hOBP (IIb) ), the previously described tear lipocalin LCN1 gene and two new LCN1 pseudogenes. Although 95% similar in sequence, the two hOBP (II) genes were differentially expressed in secretory structures. hOBP (IIa) was strongly expressed in the nasal structures, salivary and lachrymal glands, and lung, therefore having an oral sphere profile. hOBP (IIb) was more strongly expressed in genital sphere organs such as the prostate and mammary glands. Both were expressed in the male deferent ducts and placenta. Surprisingly, alternatively spliced mRNAs resulting in proteins with different C-termini were generated from each of the two genes. The single LCN1 gene in humans generated a putative odorant-binding protein in nasal structures. Finally, based on the proposed successive genomic duplication history, we demonstrated the recruitment of exons within intronic DNA generating diversity. This is consistent with a positive selection pressure in vertebrate evolution in the intron-late hypothesis.  (+info)

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin. (7/64)

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.  (+info)

Endonuclease activity in lipocalins. (8/64)

Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity.  (+info)