Single chain human chorionic gonadotropin,hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.     

Polymorphisms in the PMP-22 gene region (17p11.2–12) are crucial for simplified diagnosis of duplications/deletions

DNA duplications and deletions of a 1.5-Mb region in chromosome 17p11.2–12 comprising the gene encoding peripheral myelin protein 22 (PMP-22) are the common mutations in Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP). A 1.7-kb recombination hotspot region has been identified within misaligned flanking repeats (CMT1-REP elements) by detection of CMT- and HNPP-specific junction fragments in Southern blot analyses. In order to simplify routine diagnosis we introduce a polymerase chain reaction-based method to identify directly specific REP junction fragments. Using this test, specific fragments were detected in ∼ 67% of both CMT duplication and HNPP deletion cases. Polymorphism within a specific restriction enzyme recognition site is crucial for both Southern blot and PCR analyses of junction fragments. Received: 25 October 1996 / Revised: 16 December 1996     

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i+ ,K i+ -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na⁺,K⁺-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na⁺,K⁺-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of ⁸⁶Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na⁺,K⁺-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na⁺]_i/[K⁺]_i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na⁺,K⁺-ATPase-mediated Na⁺ and K⁺ fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

Non-covalent associations of cyclomaltooligosaccharides (cyclodextrins) with trans-β-carotene in water: evidence for the formation of large aggregates by light scattering and NMR spectroscopy

Light scattering and NMR experiments provide evidence for the formation of large aggregates, like micelles, from β-carotene complexes with β- and γ-cyclodextrin in water. High-resolution NMR spectra of the system γ-cyclodextrin/β-carotene in D₂O point out guest-induced chemical shift variation of the sugar protons, thus suggesting host–guest interaction in solution.     

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.     

Method for the complete separation of radioactively labeled human γ-globin chains from pre-βA chains on CM-cellulose chromatography: Use of cold carrier globin chains

Carboxymethyl (CM)-cellulose chromatography of globins is the technique used most frequently in analysis of hemoglobin synthesis. However, if this method is to be reliable in cases where only small amounts of fetal hemoglobin (α₂γ₂) compared to adult hemoglobin (α₂β₂^A) are synthesized, it is important to obtain a clean separation of γ chains from pre-β^A chains. In the past, it has been found that small amounts of pre-β^A chains tend to elute with the γ chains. Radioactively labeled γ chains can be completely and reproducibly separated from small amounts of labeled pre-β^A chains by the addition of unlabeled β^J chains (Hb J Baltimore = β16 Gly → Asp) which elute at the same position as the pre β^A chains, thus increasing the quantity of this peak and allowing a clean separation from the γ chains.     

Use of [Cit312,314] filaggrin (306–324) analogue for the diagnosis of rheumatoid arthritis. Conformational study by circular dichroism and fourier transformed infrared spectroscopy

Antifilaggrin autoantibodies (AFA) have described to be the most specific markers of rheumatoid arthritis (RA) and epitopes containing citrulline within the sequence of filaggrin identified as major antigenic sites recognised by AFA. In this paper we confirm that citrulline is an essential constituent of filaggrin related antigenic determinants recognised by RA specific autoantibodies, thus having an important role for the development of filaggrin peptides based serological tests. Moreover, we describe our findings on the comparative conformational analysis of filaggrin (306–324) peptide sequence and an analogue in which two arginines were simultaneously substituted by citrulline carried out by Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR). Results might contribute to the understanding of the structural features that may be important to explain the enhanced binding characteristics of citrullinated peptides to the autoantibodies in rheumatoid arthritis.