Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study

Arylsulfatase A (ASA) hydrolyzes sulfate esters with a pH optimum of 5. Interactions between p-nitrocatechol sulfate (NCS, artificial substrate) and active site residues of ASA are revealed from their co-crystal structure. Since equivalent ASA interactions with its natural substrates, sulfogalactosylceramide (SGC) and sulfogalactosylglycerolipid (SGG), are not known, we computationally docked SGC/SGG to the ASA crystal structure. Our dockings suggested that Cys69 was the active site residue, and Lys302 & Lys123 as residues anchoring the sulfate group of SGC/SGG to the active site, as observed for NCS. We further confirmed these results using 2 recombinant ASA mutants: C69A and CKK (Cys69, Lys302 and Lys123-all mutated to Ala). Both ASA mutants failed to desulfate SGC/SGG, and CKK showed minimal binding to [¹⁴C]SGC, although C69A still had affinity for this sulfoglycolipid. However, our dockings suggested additional intermolecular hydrogen bonding and hydrophobic interactions between ASA and SGC/SGG, thus contributing to the specificity of SGC/SGG as natural substrates.     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.     

Membrane bilayer properties of sphingomyelins with amide-linked 2- or 3-hydroxylated fatty acids

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(D)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5-10 °C) gel-liquid transition temperatures (T(m)) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(D)-palmitoyl SM, however, had lower T(m) (5 °C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner.     

Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes

The light organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis, but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like haemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host's environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri . Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri , increased binding by haemocytes from uncured animals to the level observed for haemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting that they produce a factor that complements the mutant's defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting indicated that once binding to haemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of haemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host haemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association.     

Draft genome of Phaeobacter gallaeciensis ANG1, a dominant member of the accessory nidamental gland of Euprymna scolopes

Phaeobacter gallaeciensis strain ANG1 represents the dominant member of the bacterial consortium within the reproductive accessory nidamental gland (ANG) of the squid Euprymna scolopes. We present a 4.59-Mb assembly of its genome, which may provide clues as to how it benefits its host.     

Cephalopod genomics: A plan of strategies and organization

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, “Paths to Cephalopod Genomics- Strategies, Choices, Organization,” held in Durham, North Carolina, USA on May 24-27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper.     

Small changes in timing of breeding among subarctic passerines over a 32‐year period

Many bird populations in temperate regions have advanced their timing of breeding in response to a warming climate in recent decades. However, long‐term trends in temperature differ geographically and between seasons, and so do responses of local breeding populations. Data on breeding bird phenology from subarctic and arctic passerine populations are scarce, and relatively little data has been recorded in open‐nesting species. We investigated the timing of breeding and its relationship to spring temperature of 14 mainly open‐nesting passerine species in subarctic Swedish Lapland over a period of 32 years (1984–2015). We estimated timing of breeding from the progress of post‐juvenile moult in mist‐netted birds, a new method exploring the fact that the progress of post‐juvenile moult correlates with age. Although there was a numerical tendency for earlier breeding in most species (on average ?0.09 days/year), changes were statistically significant in only three species (by ?0.16 to ?0.23 days/year). These figures are relatively low compared with what has been found in other long‐term studies but are similar to a few other studies in subarctic areas. Generally, annual hatching dates were negatively correlated with mean temperature in May. This correlation was stronger in long‐distance than in short‐distance migrants. Although annual temperatures at high northern latitudes have increased over recent decades, there was no long‐term increase in mean temperature in May over the study period at this subarctic site. This is probably the main reason why there were only small long‐term changes in hatching dates.     

Conformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2: molecular mechanics calculations show a compact helical structure exposing an epitope which potentially mimics galabiose

Conformational analyses of the branched repeating unit of the O-antigenic polysaccharide of Shigella dysenteriae type 2 have been performed with molecular mechanics MM3. A filtered systematic search on the trisaccharide alpha-D-GalNAc-(1-->3)-[alpha-D-GlcNAc-(1-->4)]-alpha-D-GalNAc forming the branch, shows essentially a single favored conformation. Also, the downstream alpha-D-GalNAc-(1-->4)-alpha-D-Glc linkage is sterically constrained. The alpha-D-Glc-(1-->4)-beta-D-Gal moiety, however, forms a more flexible link region between the branch points, and shows a 90 degrees bend similar to what is known for the galabiose moiety occurring in globo-glycolipids. The calculations indicate that consecutive repeating units in their minimum energy conformation arrange in a helical structure with three repeating units per turn. This helix is very compact and appears to be stabilized by hydrophobic interactions involving the N-acetyl groups at the branch points. Random conformational search suggests the existence of another helical structure with four repeating units per turn. It appears possible that the alpha-D-Glc-(1-->4)-beta-D-Gal moiety, which is exposed on the surface of the helical structures, can evade recognition by the immune system of the host by the mimicry of globo structures.     

The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.