Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free ¹⁵N-labeled antigen whose ¹H-¹⁵N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.     

YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.     

A simple access to the D-mannosidase inhibitor, 1-deoxymannojirimycin

Crystalline 1,3,4,5-tetra-O-acetyl-6-bromo-6-deoxy-keto-D-fructose was prepared by reaction of 1,3,4,5-tetra-O-acetyl-D-fructopyranose with triphenylphosphane dibromide in dichloromethane. Subsequent deprotection followed by reaction of the free 6-bromodeoxyfructofuranose with sodium azide in N,N-dimethylformamide furnished the corresponding 6-azidodeoxyketose. Catalytic hydrogenation led to 1-deoxymannojirimycin in 27% overall yield from 1,3,4,5-tetra-O-acetyl-D-fructopyranose. This access is simple, inexpensive, high-yielding and clearly suitable for multigram preparations.     

Zur Physikochemie der vitalen Kern- und Plasmafluorochromierung vonAllium cepa-Epidermen

Summary Microfluorimetrically recorded fluorescence bands of vitally stained nuclei and protoplasts from the inner epidermis of yellow onion(Allium cepa) scales are compared with fluorescence bands of Tarions model solutions. The strong fluorescent staining of the nucleus and the weaker hue of the cytoplasm after application of acridine orange is mainly due to the accumulation of monomeric dye cations in polar cytoplasmic lipids. Vital fluorescent staining with neutral red shows similar effects, but in addition an accumulation of the dye base in apolar lipids can be ascertained without doubt. On the other hand, the major accumulation of the acid fluorochrome uranin (sodium fluorescein) does not take place in polar cytoplasmic lipids but, apparently, rather in the form of anions in the water phase of the ground cytoplasm and inner nuclear plasm owing to a mechanism of plasmatic ion trap.The proof that dye ions are present in vitally stained protoplasm suggests the possibility, that the familiar phenomenon of vacuole contraction may depend on a Donnan effect in the case of basic dyes and might be a consequence of the raising of osmotic values by dye anions in the case of acid dyes.Experiments with three phases (dye solution-oil-blood plasma) yield fluorescent stainings of blood plasma with acridine orange, neutral red and uranin, which may be compared to vital staining of nuclei and cytoplasm. The well-known properties of blood plasma-lipids suggest a similar function of cytoplasmic lipids, viz. as a vehicle of lipid transport in a mainly aquatic, molecular-disperse phase. A diagram (Fig. 15) illustrates the cooperation between sheetlike lipoproteid complexes (boundary layers) and lipoproteid complexes of the ground cytoplasm dispersed as particles.     

Untersuchungen an Drossel- und Teichrohrs?nger(Acrocephalus arundinaceus, A. scirpaceus): Bestandsentwicklung, Brutbiologie, ?kologie

Zusammenfassung Von 1973–1978 wurden systematische Beringungen und regelmäßige Nestkontrollen einer Drs-Population im Fränkischen Weihergebiet (Nordbayern) durchgeführt (Auswertung von 487 Nestkarten). Die vorhandene Trs-Population wurde nicht systematisch erfaßt (645 Nestkarten).Der Bestand der einzelnen Teilpopulationen des Drs schwankte im Untersuchungszeitraum; die gesamte Population blieb annähernd konstant.Die Nestabstände benachbarter Drs-Bruten innerhalb eines günstigen Schilfstreifens lagen zwischen 7 m und ca. 300 m. Das kolonieartige Brüten der Trs wird mit Beispielen belegt.Medianer Legebeginn des Drs war der 29. Mai, der des Trs der 13. Juni. Der nach Erreichen des Maximums im Legemuster folgende Abfall war beim Drs deutlich steiler als beim Trs. Die mittlere Gelegegröße des Drs betrug 4,73, die des Trs 3,85 Eier. Bei beiden Arten fand eine Gelegegrößenreduktion mit fortschreitender Brutzeit statt.Das Schlüpfen der Jungen erfolgte beim Drs überwiegend am 12. bis 14. Tag nach Ablage des letzten Eies, beim Trs am 11. bis 13. Die Brutdauer betrug meist 14 (Drs) bzw. 13 (Trs) Tage.Beim Drs waren 59,7 % der Nester erfolgreich. Der Ausfliegeerfolg, bezogen auf erfolgreiche Nester, betrug 73,2 %, der Gesamtbruterfolg demnach 43,7 %. Beim Trs ergaben sich entsprechend die Werte 66,6 %, 82,9 % und 55,2 %. Auch die durchschnittliche Anzahl flügger Jungvögel pro Brutnest lag beim Drs mit 2,00 etwas niedriger als beim Trs mit 2,15. Als Reproduktions-rate des Drs wurde ein Wert von 2,24 flüggen Jungen pro errechnet. Für den Trs wird die Reproduktionsrate wesentlich höher geschätzt.Die Verluste wurden nach Ursachen aufgeschlüsselt, wobei besonders verglichen mit gleichaltrigen Trs die vielfach größere Empfindlichkeit nestjunger Drs gegen Regen und Kälte auffiel.In zwei Fällen konnten für Drs- Zweitbruten mittels Beringung nachgewiesen werden. Beobachtungen an Trs gaben zu Vermutungen von Zweitbruten bei dieser Art Anlaß.Bigamie wurde beim Drs mehrfach mittels Farbberingung nachgewiesen. Aus den Nestabständen konnte im Untersuchungsgebiet nicht auf monogames oder polygames Verhalten der Drs geschlossen werden. Es werden Angaben über Alter und Verhalten polygamer in verschiedenen Jahren gemacht. Paarzusammenhalt am Vorjahresbrutplatz wurde beim Drs mittels Beringung einmal nachgewiesen.Die meisten überlebenden der als Brutvögel beringten Drs kehrten ins Untersuchungsgebiet zurück, von den überlebenden nestjung beringten dagegen etwa ein Drittel. 3 nestjung beringte Trs wurden nach 1 bzw. 2 Jahren im Untersuchungsgebiet zur Brutzeit kontrolliert.Ein Drittel der Drs-Population stammte aus dem Untersuchungsgebiet. 2 nestjung beringte aus 78 km und 500 km Entfernung wurden als Brutvögel kontrolliert. Alter der Brutvögel 1–9 Jahre.An Beispielen wird die räumliche und zeitliche Einnischung beider Arten beschrieben.

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Studies onAcrocephalus arundinaceus andscirpaceus: Population trends, breeding biology, and ecology

Summary Results of 6 years (1973–1978) of systematic ringing and regular nest controls of a great reed warbler population in Northern Bavaria are given (data of 487 nests) as well as results on a population of the reed warbler (data of 645 nests).Numbers of great reed warblers fluctuated in different parts of the study area. The whole population, however, remained fairly constant.Data on the arrival of males in the breeding area are given.Distances between neighbouring nests of great reed warblers varied from 7 to about 300 m. Differences in nest densities in the reed warbler could be found.The mean first egg laying dates in the great reed warbler and the reed warbler were May 29 and June 13 respectively. The great reed warbler showed a distinct steeper decrease in its egg laying pattern than the smaller species. Average clutch size in the great reed warble was 4.73 eggs and 3.85 eggs in the reed warbler. In both species clutch size decreased during the season.Great reed warbler nestlings hatched on the 12th to 14th day after the last egg had been laid, reed warbler nestlings on the 11th to 13th day. The incubation period was mainly 14 days in the great reed warbler and 13 days in the reed warbler.In the great reed warbler 59.7 % of the nests were successful. The fledging success of successful nests was 73.2 %, accordingly the total nest success was 43.7 %. The corresponding data in reed warbler were 66.6 %, 82.9 % and 55.2 %. On average great reed warblers produced 2.00 fledglings per clutch, reed warblers 2.15. In the great reed warbler a reproduction rate of 2.24 fledglings per female was calculated. The reproduction rate in reed warbler was estimated substantially higher.Great reed warbler nestlings were much more sensitive to rain and cold weather than reed warbler nestlings of the same age.Two great reed warbler males were proved to make a second brood. The same is supposed for the other species but could not be proved so far.Polygyny was proved several times in the great reed warbler. Age and behaviour of polygynous males in different years are reported. Two great reed warblers were found breeding with their former mates at last year's breeding place.Most of the great reed warblers ringed as breeding birds returned into the study area. Nearly one third of the great reed warbler nestlings returned for breeding. Three reed warblers ringed as nestlings could be controlled in the study area during breeding period after 1 year and 2 years respectively.Distances of returned great reed warblers in relation to their birth places and their former breeding places are specified.One third of the great reed warbler population originates from the study area. Two females breeding in the study area were ringed as nestlings 78 km and 500 km apart.One-year-old to nine-year-old great reed warblers were found breeding. Data on the age composition of breeding birds are given.Plant species supporting the nests of great reed warbler and reed warbler were investigated. Differences in the spatial and temporal habitat selection of both species are described.

     

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.     

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.     

Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.