The gp15/400 polyprotein antigen of Brugia malayi binds fatty acids and retinoids.

Gp15/400 is a surface-proximal antigen of the filarial nematode Brugia malayi, produced as a large polyprotein precursor comprising an array of polypeptide units of approx. 14.5 kDa. Here we describe a biochemical function for gp15/400. A single 14.5-kDa unit of gp15/400 has been expressed in Escherichia coli, and found to dimerise spontaneously. This protein (designated P-RUNG) has high-affinity fatty acid and retinoid binding activity, suggesting that the parent polypeptide itself has these properties. Fluorescent fatty acid probes show significant enhancement of fluorescence intensity and shifts in emission wavelength in the presence of P-RUNG, which can be reversed by competing non-fluorescent fatty acids (oleic, palmitic, steric, arachidonic), retinoids (retinol and retinoic acid) and oleoyl Coenzyme A, but not by tryptophan, cholesterol, caproic acid, squalene, tocopherol, tocopherol acetate, succinyl CoA, 2-methylbutyric acid and 2-methylvaleric acid. Changes in intrinsic fluorescence of retinol or retinoic acid confirmed the retinoid binding function. The results of fluorescence titration experiments are consistent with stoichiometric binding to a single protein site per monomer unit with affinities (Kd) in the range 2 x 10(-6) M (for the fluorescent probe 11-((5-dansyl)amino)undecanoic acid) and 2 x 10(-7) M (for oleic acid). The extreme blue shift of the fluorescent fatty acid-protein complex suggests an unusually low polarity for the protein binding site. The intrinsic fluorescence of the single tryptophan residue of P-RUNG indicates that it also is deeply buried in a non-polar environment, but is probably not involved in ligand binding. Gp15/400, therefore, represents a new class of lipid binding protein which is possibly restricted to nematodes.

Androgen receptor in the human thymus.

An androgen binding species has been identified in partially purified cytosol from human thymic tissue, resolved from sex steroid binding globulin by gel chromatography. This putative 5 alpha-dihydrotestosterone receptor was characterised by high affinity (Kd 6.7 X 10(-10) M) and low capacity (9 fmol/mg of cytosol protein). High affinity binding was confirmed with methyltrienolone (R1881). Competition studies were carried out with a number of androgenic compounds and 5 alpha-19 nordihydrotestosterone was shown to possess the highest affinity for the androgen receptor.

Rheumatoid factor induction in the mouse: sex differences and the effect of the sex steroids.

Female CBA mice produced a significantly higher plasma rheumatoid factor (RF) response to Salmonella typhosa lipopolysaccharide than did male mice. The peak level in females was observed on day 5-6 after injection and in males on day 7-8. Elevated RF levels continued to be detected more than 30 days later. A second injection of LPS, 38 days after the first, to assess the secondary response, had no more than an additive effect on plasma RF concentration, although the day of peak response was earlier by two days in both sexes. Administration of oestradiol-17 beta by Silastic implant brought forward the day of peak response by two days in both sexes although it reduced its amplitude considerably. Testosterone had little effect on the peak concentrations achieved in both sexes, but did produce a slower decay in plasma RF level. This investigation indicates that the sex hormones can influence the response to LPS, a polyclonal B cell activator. This may have implications for the sex differences seen in autoimmune diseases.

Androgen binding cytosol receptors in the rat thymus: physicochemical properties, specificity and localisation.

Normal rat-thymus cytosol was shown to contain a 5 alpha-dihydrotestosterone-binding species characterised by low capacity and high affinity (Kd-6 X 10(-10) M). The principal binding moiety had a sedimentation coefficient of 8 S at low ionic strength. Competitive binding studies with 61 compounds showed that the specificity was highly androgenic while comparison with similar data from the prostate indicated that the thymic androgen receptor had a different specificity pattern. The thymus was not found to be capable of 5 alpha-reduction; unlike the prostate, it rapidly metabolised testosterone to androstenedione. Androgen receptor levels were significantly higher in intact females than males and the binding species was positively identified only in medullary tissue--thymocytes were devoid of the receptor. This data supports the idea that the thymus is an androgen-responsive tissue and that at least part of the immunoregulatory effects of androgens are mediated through the thymus.

Oestrogen receptors in macrophages.

The presence of receptors for oestradiol-17 beta and 5 alpha-dihydrotestosterone (5 alpha-DHT) in the human monocytic leukaemia cell line J111 and rat peritoneal macrophages was investigated using whole-cell assays. For both cell types, high-affinity binding species for oestrogen were detected, whereas no indication of specific binding was observed for 5 alpha-DHT. Analysis of the data according to Scatchard showed curved lines, indicating the presence of two different oestrogen-binding species. The dissociation constant (Kd) values for the receptors of the rat peritoneal macrophages were calculated to be 1.4 x 10(-10) M and 3.3 x 10(-9) M, while for the J111 cells, the Kd values were 8.7 x 10(-11) M and 2.5 x 10(-9) M. Sucrose-gradient ultracentrifugation identified one oestrogen-binding species of 7.1S. The receptors had a relatively high affinity for diethylstilboestrol (DES) but did not bind to a monoclonal antibody specific for the classical oestrogen receptor, suggesting that oestrogen receptors in macrophages could be of a different type.

Hormonal manipulation of the immune response in systemic lupus erythematosus: a drug trial of an anabolic steroid, 19-nortestosterone.

Ten patients (8 female, 2 male) with systemic lupus erythematosus (SLE) were entered into an open trial with the anabolic steroid 19-nortestosterone (19-nor). Their clinical condition did not improve, nor were significant changes observed in the majority of laboratory data. However, overall the platelet count rose, and in patients with abnormal levels of T lymphocytes bearing receptors for IgG Fc treatment returned the values to normal. Despite this latter result, suppressor cell activity remained slightly below the normal range throughout the study period.

The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterization.

We report here on the structure and function of the ABA-1 allergen protein of the parasitic nematode Ascaris, the first nematode allergen to be characterized in detail. Using the fluorescent fatty acid analog 11-(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino)undecanoic acid (DAUDA), it was demonstrated that ABA-1 is a fatty acid binding protein (FABP) with a high affinity for the fluorescent analog (8.8 x 10(-8) M) and for oleic acid in competition experiments (1.3 x 10(-8) M), with a single binding site for ligand per monomer unit. Blue-shifting of fluorescence emission of DAUDA upon binding was unprecedented in degree among FABPs, being equivalent to that occurring in cyclohexane. A similarly blue-shifted spectrum was obtained with a probe in which the fluorophore was bound to the alpha carbon of a fatty acid, indicating that the carboxylate group of bound fatty acids is probably not exposed to solvent. In competition experiments and by observation of changes in their intrinsic fluorescence, retinol and retinoic acid were also found to bind in the fatty acid binding site. Circular dichroism (CD) of the ABA-1 protein revealed a high alpha-helix content (59%) which was consistent with the four-helix structure for the protein predicted from sequence algorithms. Fluorescence measurements showed that the single, highly conserved tryptophan residue is deeply buried in an unusually apolar environment and that this was unaffected by ligand binding. DSC studies of thermal stability indicate that unfolding of the ABA-1 dimer is cooperative and biphasic (Tm approximately 71 and 89 degrees C), suggesting a two-domain thermal unfolding process, again consistent with the predicted structure. Only the folding of the high-temperature domain is reversible on cooling. DSC confirmed the gel filtration analysis, which indicated that ABA-1 forms a dimer. Aside from being the first nematode allergen for which structure or function has been elucidated, ABA-1 provides a highly manipulable model for investigation of the interaction between hydrophobic ligands and alpha-helical proteins.