Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants.

Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15-30% residue identity) short-chain dehydrogenase/reductase family (SDR). It is the only reported member that oxidizes mainly ethanol and 2-propanol among other alcohols. To confirm the role of Ser139 we constructed two site-directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity. Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156. Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase.

Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gln, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156 are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.

Immune complexes in retinitis pigmentosa.

In a group of 46 patients with retinitis pigmentosa (RP) we studied the presence of circulating immune complexes (CIC) and the alterations in the complement system. Our results showed the presence of CIC in 43.5% of the patients studied, reduced levels of the complement components C3 and C4 (p less than 0.001), and of the haemolytic activity CH50 (p less than 0.001) when compared with a control group consisting of a 100 healthy subjects. We found a statistically significant correlation between the values of C3 and CIC (p less than 0.01), C4 and CIC (p less than 0.01), and between CH50 and CIC (p less than 0.001). These findings indicate that the CIC may play a role in the pathogenesis of primary retinitis pigmentosa.

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain.

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

Secretion of mouse-metallothionein by engineered E. coli cells in metal-enriched culture media.

Heterologous Escherichia coli expression systems were designed and assayed for the synthesis of functional mouse metallothionein (MT) as a secreted fusion protein. MT secretion was compared among different systems, and the optimum vector/host/medium combination was tested for metal removal. In this case, the Cu content of the medium decreased by up to 34% after growth of recombinant bacteria. The potential use of these genetically-engineered bacteria for water bioremediation is discussed as an alternative to cytoplasmic MT or membrane-bound MT heterologous expression systems.

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains.

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.

T cell subsets in allergic respiratory disease using monoclonal antibodies.

Fifty patients with perennial allergic rhinitis and extrinsic allergic asthma were studied to determine the relative percentages of OKT3+, OKT4+ and OKT8+ cells as well as the OKT4+/OKT8+ ratios. The percentages of the OKT3+ cells (66 +/- 7) were reduced when compared with those of 50 healthy controls (72 +/- 6), (p less than 0.001). The mean values and deviations of the OKT4 marker produced no statistically significant differences between the patient group (44 +/- 6) and the control group (44 +/- 6). A reduction in the OKT8+ marker in the patient group was registered (20 +/- 6), (p less than 0.001). The OKT4+/OKT8+ ratio was, as a result, raised in the patient group (2.3 +/- 0.6) in relation to the control group (1.6 +/- 0.3). The reduction in the OKT3+ cells together with the imbalance in the subset inducer-helper/cytotoxic-suppressor in allergic respiratory disease could lead to non-regulation of B cells and to hyperproduction of IgE.

Immune complexes in retinitis pigmentosa.

In a group of 46 patients with retinitis pigmentosa (RP), we studied the levels of IgM, the presence of a rheumatoid factor (RF), the circulating immune complexes (CIC) and the alterations in the complement system. Our results showed increased IgM levels in two patients (226 and 260 mg/dl), the presence of RF in three patients (205, 187 and 80 IU/ml), the existence of CIC in 43.5% of the subjects, reduced levels of the complement components C3 and C4 (p less than 0.001) and of the haemolytic activity CH50 (p less than 0.001) when compared with a control group consisting of 100 healthy subjects. We found a statistically significant correlation between the values of C3 and CIC (p less than 0.01), C4 and CIC (p less than 0.01) and between CH50 and CIC (p less than 0.001).

Recombinant synthesis of mouse Zn3-beta and Zn4-alpha metallothionein 1 domains and characterization of their cadmium(II) binding capacity.

Genetic engineering, coupled with spectroscopic analyses, has enabled the metal binding properties of the alpha and beta subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn4-alpha MT and Zn3-beta MT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to alpha fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the native protein. Titration of Zn3-beta MT with CdCl2 results in the formation of Cd3-beta MT. The addition of excess Cd(II) leads to Cd4-beta MT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-beta MT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd3-beta MT species is stable in the presence of this strong metal-chelating agent.

In vivo copper- and cadmium-binding ability of mammalian metallothionein beta domain.

The beta domain of mouse metallothionein 1 (betaMT) was synthesized in Escherichia coli cells grown in the presence of copper or cadmium. Homogenous preparations of Cu-betaMT and Cd-betaMT were used to characterize the corresponding in vivo-conformed metal-clusters, and to compare them with the species obtained in vitro by metal replacement to a canonical Zn3-betaMT structure. The copper-containing betaMT clusters formed inside the cells were very stable. In contrast, the nascent beta peptide, although it showed cadmium binding ability, produced a highly unstable species, whose stoichiometry depended upon culture conditions. The absence of betaMT protein in E. coli protease-proficient hosts grown in cadmium-supplemented medium pointed to drastic proteolysis of a poorly folded beta peptide, somehow enhanced by the presence of cadmium. Possible functional and evolutionary implications of the bioactivity of mammalian betaMT in the presence of monovalent and divalent metal ions are discussed.

Replacement of terminal cysteine with histidine in the metallothionein alpha and beta domains maintains its binding capacity.

To generate novel forms of metal-binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli. The spectroscopic and analytical results showed that the alphaMT (C33H, C36H, C41H, C57H) and betaMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected. In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd-mutated MT forms with 4 and 3 Cd(II), respectively. However, although Cys-to-His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild-type proteins. While C13H, C36H and C57H seem to contain Zn(II)-aggregates that are closely related to those of the wild-type proteins, only C41H and C57H gave rise to Cd(II)-aggregates similar to those of Cd4-alphaMT, where the His residue plays the role of the substituted Cys. Despite the structural implications of the Cys-to-His replacement, the dissociation constants showed no major decrease in the Cd-binding affinity in any of the mutants assayed compared with the wild-type.

Identification of foetal brain proteins by two-dimensional gel electrophoresis and mass spectrometry comparison of samples from individuals with or without chromosome 21 trisomy.

Protein expression in foetal brain with or without chromosome 21 trisomy (Down's syndrome) was analyzed by two-dimensional gel electrophoresis and mass spectrometry. Data generated by in-gel digestion and matrix-assisted laser desorption/ionization mass spectrometry allowed identification of 40 proteins. Most of these are common to syndrome and healthy subjects and represent different types of protein. However, a few proteins, identified as truncated structural proteins (tubulin, actin), were present in part of the trisomy samples but absent from the controls. This is interpreted to indicate increased proteolysis in the syndrome samples but could also reflect some altered expression or processing. Independent of the apparently increased proteolysis in the syndrome samples, and in spite of the use of total brain tissues, the results show that two-dimensional protein separation patterns are largely similar between the syndrome and control samples upon silver-staining, but that differences associated with structural components can be detected and identified.