Characterisation of the Genes Encoding Mannuronan-C5-Epimerase in the Brown Alga Lessonia Variegata

<p>Alginate is known to be a commercially valuable polysaccharide, of great importance in industries such as food, cosmetics, medicine and pharmaceuticals. It is obtained commercially by harvesting brown algae. The final step in the alginate biochemical pathway involves the epimerization of D-mannuronic residues into L-guluronic residues, catalyzed by the enzyme mannuronan-C5-epimerase. This final step has been found to be responsible for controlling the physicochemical properties of the produced alginate. This study is the first to characterize the genes encoding for the enzyme mannuronan-C5- epimerase in the Northern, Southern and Wellington lineages of the brown alga Lessonia variegata (Phaeophyceae). The gene of interest was amplified by standard PCR and cloning. Cloning PCR results revealed the presence of two distinct copies of the gene in Lessonia variegata. The coding region of the copies was found to be very conserved with very little sequence variation. The Lessonia variegata sequences were compared with those of Laminaria digitata and Saccharina japonica, which indicated that at least one gene duplication event has occurred in Lessonia variegata, leading to the formation of two gene duplicates. The possible mechanisms by which the gene paralogs may control the structure and function of the produced alginate have been discussed.</p>

Characterisation of the Genes Encoding Mannuronan-C5-Epimerase in the Brown Alga Lessonia Variegata

<p>Alginate is known to be a commercially valuable polysaccharide, of great importance in industries such as food, cosmetics, medicine and pharmaceuticals. It is obtained commercially by harvesting brown algae. The final step in the alginate biochemical pathway involves the epimerization of D-mannuronic residues into L-guluronic residues, catalyzed by the enzyme mannuronan-C5-epimerase. This final step has been found to be responsible for controlling the physicochemical properties of the produced alginate. This study is the first to characterize the genes encoding for the enzyme mannuronan-C5- epimerase in the Northern, Southern and Wellington lineages of the brown alga Lessonia variegata (Phaeophyceae). The gene of interest was amplified by standard PCR and cloning. Cloning PCR results revealed the presence of two distinct copies of the gene in Lessonia variegata. The coding region of the copies was found to be very conserved with very little sequence variation. The Lessonia variegata sequences were compared with those of Laminaria digitata and Saccharina japonica, which indicated that at least one gene duplication event has occurred in Lessonia variegata, leading to the formation of two gene duplicates. The possible mechanisms by which the gene paralogs may control the structure and function of the produced alginate have been discussed.</p>

Mutually Exclusive Expression of Closely Related Odorant-Binding Proteins 9A and 9B in the Antenna of the Red Flour Beetle Tribolium castaneum

Olfaction is crucial for insects to find food sources, mates, and oviposition sites. One of the initial steps in olfaction is facilitated by odorant-binding proteins (OBPs) that translocate hydrophobic odorants through the aqueous olfactory sensilla lymph to the odorant receptor complexes embedded in the dendritic membrane of olfactory sensory neurons. The Tribolium castaneum (Coleoptera, Tenebrionidae) OBPs encoded by the gene pair TcasOBP9A and TcasOBP9B represent the closest homologs to the well-studied Drosophila melanogaster OBP Lush (DmelOBP76a), which mediates pheromone reception. By an electroantennographic analysis, we can show that these two OBPs are not pheromone-specific but rather enhance the detection of a broad spectrum of organic volatiles. Both OBPs are expressed in the antenna but in a mutually exclusive pattern, despite their homology and gene pair character by chromosomal location. A phylogenetic analysis indicates that this gene pair arose at the base of the Cucujiformia, which dates the gene duplication event to about 200 Mio years ago. Therefore, this gene pair is not the result of a recent gene duplication event and the high sequence conservation in spite of their expression in different sensilla is potentially the result of a common function as co-OBPs.

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

Conservation and divergence of ancestral AGAMOUS/SEEDSTICK subfamily genes from the basal angiosperm Magnolia wufengensis

AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon–intron structures, expression patterns, protein–protein interaction patterns and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis (Magnoliaceae). MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of the STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of the SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves differ from those found in monocots and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon–intron structural changes of the AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon–intron structures, expression and interaction patterns, and functions, and provide strong evidence for the significance of gene duplication in the expansion and evolution of the AG/STK subfamily.

Early Archean origin of heterodimeric Photosystem I

When and how oxygenic photosynthesis originated remains controversial. Wide uncertainties exist for the earliest detection of biogenic oxygen in the geochemical record or the origin of water oxidation in ancestral lineages of the phylum Cyanobacteria. A unique trait of oxygenic photosynthesis is that the process uses a Type I reaction centre with a heterodimeric core, also known as Photosystem I, made of two distinct but homologous subunits, PsaA and PsaB. In contrast, all other known Type I reaction centres in anoxygenic phototrophs have a homodimeric core. A compelling hypothesis for the evolution of a heterodimeric Type I reaction centre is that the gene duplication that allowed the divergence of PsaA and PsaB was an adaptation to incorporate photoprotective mechanisms against the formation of reactive oxygen species, therefore occurring after the origin of water oxidation to oxygen. Here I show, using sequence comparisons and Bayesian relaxed molecular clocks that this gene duplication event may have occurred in the early Archean more than 3.4 billion years ago, long before the most recent common ancestor of crown group Cyanobacteria and the Great Oxidation Event. If the origin of water oxidation predated this gene duplication event, then that would place primordial forms of oxygenic photosynthesis at a very early stage in the evolutionary history of life.

Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms

Alx1 is a pivotal transcription factor in a gene regulatory network that controls skeletogenesis throughout the echinoderm phylum. We performed a structure-function analysis of sea urchin Alx1 using a rescue assay and identified a novel, conserved motif (Domain 2) essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of Domain 2 conferred robust skeletogenic function on Alx4. We used cross-species expression experiments to show that Alx1 proteins from distantly related echinoderms are not interchangeable, although the sequence and function of Domain 2 are highly conserved. We also found that Domain 2 is subject to alternative splicing and provide evidence that this domain was originally gained through exonization. Our findings show that a gene duplication event permitted the functional specialization of a transcription factor through changes in exon-intron organization and thereby supported the evolution of a major morphological novelty.

The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.