Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.     

Synthesis,Characterization and Applications of a Perdeuterated Amphipol

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization–ion mobility spectrometry–mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120 % D₂O. In ¹H-¹H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6 % of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.     

Isolation and characterization of the first microsatellite markers for the endangered swan mussel <Emphasis Type="Italic">Anodonta cygnea</Emphasis> L. (Bivalvia: Unionoidea)

Freshwater molluscs are globally critically threatened, yet little is known about their genetic diversity. We describe the isolation and characterization of the first microsatellite markers for the European swan mussel, Anodonta cygnea. Nine loci were polymorphic in a panel of 110 individuals from seven German and Estonian populations. Number of alleles ranged from 2 to 14 and heterozygosity levels ranged from 0.021 to 0.498 for H _o and from 0.069 to 0.634 for H _e. Deficiency of heterozygous genotypes was observed in six loci. The length of the microsatellite repeat motives was positively correlated with the allele numbers in the respective loci.     

The three desulfoglucosinolate sulfotransferase proteins in Arabidopsis have different substrate specificities and are differentially expressed

Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3'-phosphoadenosine 5'-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 microM) and low (50 microM) sulfate concentrations in the medium did not influence the levels of expression.     

Cathepsin D deficiency is associated with a human neurodegenerative disorder

Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.     

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.     

X-linked adrenoleukodystrophy: clinical, biochemical and pathogenetic aspects

X-linked adrenoleukodystrophy (X-ALD) is a clinically heterogeneous disorder ranging from the severe childhood cerebral form to asymptomatic persons. The overall incidence is 1:16,800 including hemizygotes as well as heterozygotes. The principal molecular defect is due to inborn mutations in the ABCD1 gene encoding the adrenoleukodystrophy protein (ALDP), a transporter in the peroxisome membrane. ALDP is involved in the transport of substrates from the cytoplasm into the peroxisomal lumen. ALDP defects lead to characteristic accumulation of saturated very long-chain fatty acids, the diagnostic disease marker. The pathogenesis is unclear. Different molecular mechanisms seem to induce inflammatory demyelination, neurodegeneration and adrenocortical insufficiency involving the primary ABCD1 defect, environmental factors and modifier genes. Important information has been derived from the X-ALD mouse models; species differences however complicate the interpretation of results. So far, bone marrow transplantation is the only effective long-term treatment for childhood cerebral X-ALD, however, only when performed at an early-stage of disease. Urgently needed novel therapeutic strategies are under consideration ranging from dietary approaches to gene therapy.