Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.     

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.     

A mass mortality of fishes caused by receding water levels in the vegetated littoral zone of the West Kleinemonde Estuary,South Africa

On 15 November 2017 the mouth of the West Kleinemonde Estuary breached following heavy catchment rains and increased river flow. The water level in the estuary following mouth opening decreased by 1.65 m within 24 h, resulting in an almost complete draining of the littoral zone where large beds of the aquatic macrophyte Ruppia cirrhosa and mats of the associated filamentous algae were present. As the water depth within the plant beds decreased, the macrophytes, together with the algal filaments, created an increasingly dense mat, trapping fish that were resident, foraging or passing through the littoral zone. By 16 November 2017 large numbers of fishes belonging to at least 20 species were trapped in pools and depressions within the littoral, as well as within the R. cirrhosa beds and filamentous algal mats in the lower reaches of this system. Other affected taxa included crustaceans, especially isopods, and large numbers of small bivalves attached to macrophyte vegetation. Beneficiaries of the fish kill, in terms of unexpected food availability, included a variety of piscivorous bird species and the Cape clawless otter Aonyx capensis. This is the first documented account of a diverse species fish kill associated with estuary mouth breaching.     

Norian-Rhaetian reefs in Argolis Peninsula,Greece

Summary Upper Triassic to Lower Jurassic shallow-water carbonate sequences of the ‘Pantokrator limestones’ are widely distributed in the Argolis Peninsula, southern Greece. Within this sequence are some reef or reefal structures. In the Mavrovouni Mountains, near Sarmeika, 6 km SE of the ancient theatre of Epidavros (Argolis Peninsula), a Norian-Rhaetian reef complex has been identified. This is the first well-documented Norian-Rhaetian reef in Greece. The main reef builders are coralline sponges (‘sphinctozoans,’ ‘inozoans’, and sclerosponges), followed by dendroid, cerioid, and solitary corals, and algae. The reef type corresponds to a ‘sponge-coral reef’.     

Temporal scaling of molecular evolution in primates and other mammals

Molecular clocks are routinely tested for linearity using a relative rate test and routinely calibrated against the geological time scale using a single or average paleontologically determined time of divergence between living taxa. The relative rate test is a test of parallel rate equality, not a test of rate constancy. Temporal scaling provides a test of rates, where scaling coefficients of 1.0 (isochrony) represent stochastic rate constancy. The fossil record of primates and other mammals is now known in sufficient detail to provide several independent divergence times for major taxonomic groups. Molecular difference should scale negatively or isochronically (scaling coefficients less than 1.0) with divergence time: where two or more divergence times are available, molecular difference appears to scale positively (scaling coefficient greater than 1.0). A minimum of four divergence times are required for adequate statistical power in testing the linear model: scaling is significantly nonlinear and positive in six of 11 published investigations meeting this criterion. All groups studied show some slowdown in rates of molecular change over Cenozoic time. The break from constant or increasing rates during the Mesozoic to decreasing rates during the Cenozoic appears to coincide with extraordinary diversification of placental mammals at the beginning of this era. High rates of selectively neutral molecular change may be concentrated in such discrete events of evolutionary diversification.      

Structure of human erythrocyte spectrin. I. Isolation of the alpha-I domain and its cyanogen bromide peptides

The alpha-I domain of human erythrocyte spectrin was produced by a mild tryptic digestion of the intact molecule and purified by a single step affinity chromatography procedure using a monoclonal antibody. A tryptic peptide representing the alpha-I domain, which migrated on polyacrylamide gels as an 80,000-dalton peptide, was subjected to automated Edman-Begg degradation. Products from automated sequencing were identified by reverse-phase high performance liquid chromatography. Two smaller proteolytic products of the alpha-I domain (T74 and T50) were also subjected to automated sequence analysis. CNBr cleavage of the alpha-I domain produced nine unique peptides which were separated by gel filtration on a high performance liquid chromatograph. Peptides were further purified by reverse-phase chromatography and characterized by amino acid analysis. Partial sequences were determined by automated NH2-terminal sequence analysis. A single aspartate-proline bond, which was partially hydrolyzed during the cyanogen bromide cleavage reaction, was also identified. These sequence data include the first 86 residues of the alpha-I domain, and the spectrin oligomer binding site has been tentatively localized within the first 39 residues. The sequence of 293 residues of a total 633 residues in the alpha-I domain is presented and represents the first structural information for this protein.     

Self-assembly of basement membrane collagen

The in vitro self-assembly of murine type IV collagen was examined by using biochemical and morphological techniques. Dimeric collagen undergoes a rapid and reversible thermal gelation at neutral pH without an appreciable lag period. The process is seen to be concentration dependent and inhibited by 2 M urea. The formed complex can be visualized by electron microscopy rotary shadowing as an irregular polygonal lattice network with extensive side by side associations within the collagenous triple-helical part of the molecules, two and three strands thick. Measurements on the matrix suggest a median stagger dimension of 170 nm, one-fifth the length of a dimer. The conversion of pepsin-generated monomers into N-terminally bound tetramers can also be demonstrated in vitro. This process is also concentration dependent and inhibited and reversed by 2 M urea but is thermally irreversible and occurs at a slow rate relative to the lateral associations. These tetramers can be seen by rotary shadowing as four-armed "spider" structures. It is proposed that lateral associations, by virtue of their faster rate of formation, precede 7S bond formation, and several models for the assembly of basement membrane collagen are discussed.     

Rac1 Is Essential for Basement Membrane-Dependent Epiblast Survival

During murine peri-implantation development, the egg cylinder forms from a solid cell mass by the apoptotic removal of inner cells that do not contact the basement membrane (BM) and the selective survival of the epiblast epithelium, which does. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Rac1 ablation in embryonic stem cell-derived embryoid bodies (EBs) leads to massive apoptosis of epiblast cells in contact with the BM. Expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 additionally blocks the apoptosis of inner cells and cavitation, indicating that the spatially regulated activation of Rac1 is required for epithelial cyst formation. We further show that Rac1 is activated through integrin-mediated recruitment of the Crk-DOCK180 complex and mediates BM-dependent epiblast survival through activating the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Our results reveal a signaling cascade triggered by cell-BM interactions essential for epithelial morphogenesis.All epithelial sheets and tubes rest upon a basement membrane (BM), a thin mat of specialized extracellular matrix (ECM) consisting of laminins, type IV collagens, perlecan, and nidogens. The BM provides essential survival signals to protect epithelial cells from apoptosis, in addition to its role in cell adhesion, migration, proliferation, and polarity orientation. In the developing chick retina, removal of the retinal BM by collagenase digestion resulted in severe apoptosis of retinal neuroepithelial cells (17). In mice, targeted deletion of the genes for the BM component laminins or perlecan caused BM defects and various degrees of apoptosis of cells that attach to the BM (34, 41, 42). Also, mammary epithelial cells can survive for a long period of time when grown on a reconstituted basement membrane derived from Engelbreth-Holmof Swarm (EHS) tumor (Matrigel), but they die by apoptosis when grown on plastic, fibronectin, or type I collagen despite their firm attachment on these substrates (2, 11, 36). A similar response of keratinocytes to BM type IV collagen versus non-BM matrix proteins was observed in bioengineered human skin equivalents (40). These results suggest that the BM provides a unique microenvironment for the survival of associated epithelial cells.Embryoid body (EB) differentiation has been used to study epithelial morphogenesis and early embryogenesis. When cultured in suspension as small aggregates, mouse embryonic stem (ES) cells adhere strongly together and form spherical EBs. The outer cells of the EB differentiate to become endoderm cells, which secrete laminins, type IV collagen, perlecan, and other BM components that assemble into an underlying BM equivalent to the embryonic BM separating extraembryonic endoderm from the epiblast. Integrin α6β1 in the epiblast cells and integrin α5β1 in the endoderm cells redistribute from a pericellular location to a predominantly sub-basement membrane location (28). Following BM formation, the epiblast cells adjacent to the BM polarize to become a pseudostratified columnar epithelium (the epiblast epithelium), whereas the inner cells not in contact with the BM undergo apoptosis and are selectively removed by phagocytosis/autophagy, creating a proamniotic-like cavity. That the BM is essential for these sequential processes is evidenced by the observation that targeted deletion of the laminin γ1 gene in EBs blocks BM assembly, subsequent epiblast epithelialization, and then apoptosis-dependent cavitation (32, 42). These differentiation processes recapitulate peri-implantation development and provide a tractable in vitro model for the study of apoptosis and BM-dependent cell survival during epithelial morphogenesis.While BM-dependent cell survival is often coupled with apoptotic removal of centrally located cells not in contact with the BM during morphogenesis of epithelial cysts such as mammary glandular acini and embryonic mouse egg cylinders (7, 29), the molecular mechanisms underlying this fundamental process are poorly understood. Elegant studies on teratocarcinoma cell-derived EBs have suggested that formation of an epithelial cyst as they develop is the result of the interplay of two signals (7). One is a death signal from the endoderm that induces apoptosis of the centrally located cells to create a cavity; the other is a rescue signal mediated by contact with the BM and is required for the survival of the newly formed epiblast epithelium. Subsequent studies have revealed that bone morphogenetic protein 2 (BMP-2) is highly expressed in the endoderm and that expression of a dominant-negative (DN) BMP receptor in EBs blocked cavitation, suggesting BMP-2 to be a death factor (6). The survival signals from the interaction of the epiblast cells with the BM were studied by treating the EBs with polyclonal antiserum against membrane glycoproteins consisting of ECM adhesion receptors. The antiserum treatment induced programmed cell death in the BM-contacting epiblast layer. However, the identities of the receptors and the downstream signaling molecules involved have not been explored.In this study, we utilized EBs differentiated from genetically modified ES cells to investigate the mechanisms of BM-dependent cell survival. We show that targeted deletion of the Rac1 gene in EBs leads to massive apoptosis of epiblast cells in contact with the BM. Rac1 is activated in a BM- and integrin-dependent fashion. Stable expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 also blocks the apoptosis of inner cells and cavitation. These results suggest that the spatial activation of Rac1 is essential not only for BM-dependent epiblast survival but also for apoptosis-mediated cavitation. We further show that Crk mediates Rac1 activation by recruiting the Rac1-specific activator DOCK180 to the cell-BM adhesions and that the phosphatidylinositol 3-kinase (PI3K)-Akt pathway acts downstream of Rac1 to promote BM-dependent survival. Collectively, our results have established a key role for Rac1 in embryonic epithelial morphogenesis and have uncovered a signaling pathway that mediates BM-dependent epithelial survival.