Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces alpha-helical structures above the cmc. Both types of aggregates contain significant amounts of beta-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce alpha-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form alpha-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles.     

Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3β, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3β loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3β in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3β dependent functional hierarchy of human iNKT cells.     

A comparative ecological investigation of two related mayfly nymphs

Zusammenfassung Heptagenia sulphurea wird auf Steinen, H. fuscogrisea in der Vegetation von Fließgewässern gefunden (Fig. 2).In Experimenten wählte H. sulphurea Steine und groben Kies. H. fuscogrisea wählte Batrachium als Substrat, nur ein geringer Anteil der Larven fand sich auf Callitriche (Fig. 3).Die Zeit, die zwischen Loslassen und erneutem Anhaften auf dem Substrat im strömenden Milieu verstreicht, war unterschiedlich bei den zwei Spezies. Die auf Steinen lebende Art reagierte schneller als die aus der Vegetation.H. sulphurea verhielt sich, wenn sie von der Strömung ergriffen worden war, ziemlich passiv, doch trug die turbulente Strömung das Tier dem Substrat zu, so hatte es gewöhnlich sicheren Halt.Im Strömungsaquarium bevorzugt H. sulphurea die strömungsexponierten Bereiche, aber viele waren auch im ruhigen Wasser (Fig. 5).Wurde H. fuscogrisea von der Strömung ergriffen, so führte sie Schwimmbewegungen aus. Erreicht sie eine Haltmöglichkeit an strömungsexponiertem Platz, so wird sie oft weitergetragen. Die Tiere lassen sie meist in ruhigem Wasser nieder.Im Strömungsaquarium bevorzugt H. fuscogrisea überwiegend die Regionen ruhigen Wassers (Fig. 5).Die Zeit zwischen Loslassen und Festsetzen war deutlich durch die Temperatur beeinflußt (Fig. 6).Der dichte Haarbesatz an der hinteren Kante der Femur von H. sulphurea wird als Anpassung an das Leben in der Strömung angesehen, weil er die Wirbelbildung herabsetzen kann. Der Haarbesatz entlang der Tibia bei H. fuscogrisea wird als Anpassung zum Schwimmen angesehen, zu dem these Spezies befähigt ist, wenn sie von der Strömung ergriffen wird (Fig. 8).Die Spezies von Steinen ist etwas mehr abgeflacht als die aus der Vegetation (Fig. 7).     

Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano,Hellenic Volcanic Arc

Over the last decades, there has been growing interest about the ecological role of hydrothermal sulfide chimneys, their microbial diversity and associated biotechnological potential. Here, we performed dual-index Illumina sequencing of bacterial and archaeal communities on active and inactive sulfide chimneys collected from the Kolumbo hydrothermal field, situated on a geodynamic convergent setting. A total of 15,701 OTUs (operational taxonomic units) were assigned to 56 bacterial and 3 archaeal phyla, 133 bacterial and 16 archaeal classes. Active chimney communities were dominated by OTUs related to thermophilic members of Epsilonproteobacteria, Aquificae and Deltaproteobacteria. Inactive chimney communities were dominated by an OTU closely related to the archaeon Nitrosopumilus sp., and by members of Gammaproteobacteria, Deltaproteobacteria, Planctomycetes and Bacteroidetes. These lineages are closely related to phylotypes typically involved in iron, sulfur, nitrogen, hydrogen and methane cycling. Overall, the inactive sulfide chimneys presented highly diverse and uniform microbial communities, in contrast to the active chimney communities, which were dominated by chemolithoautotrophic and thermophilic lineages. This study represents one of the most comprehensive investigations of microbial diversity in submarine chimneys and elucidates how the dissipation of hydrothermal activity affects the structure of microbial consortia in these extreme ecological niches.