Vicilin-buried peptides (VBPs) are derived from the N-terminal leader-sequence (LS) of vicilin proteins and are receiving increasing attention as novel food allergens. Characterizing the VBPs from peanuts and tree nuts may help understand comorbidity and cross-reactivity despite a distant evolutionary origin. Peptide microarrays were used to identify IgE-reactive sequences from the LS of the vicilin allergens from Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from patients diagnosed as peanut and/or pistachio/cashew allergic. The structure of four VBPs were solved using solution-NMR (cashew: AO1.1, AO1.2 and pistachio: PV3.1, PV3.2), and compared to solved VBP structures from peanut (AH1.1) and walnut (JR2.1, JR2.2, and JR2.3). Biophysical properties were assessed using in vitro proteolysis assays and circular dichroism. Hybridomas of B-cells from allergic patients were screened for cells producing IgE against VBPs. IgE binding to peptides was frequently observed in the VBP domains AH1.1, AO1.1, JR2.1, and PV3.1, and not in AO1.2, JR2.2, JR2.3, PV3.2. Comparisons of structural features suggest the VBP scaffold can support cross-reactivity despite low sequence identity. The cashew VBPs are more resistant to proteolysis than the pistachio VBPs, correlating with a slight increase in prevalence. Human IgE monoclonal antibodies were discovered with specificity for AH1.1 and JR2.1. VBPs are domains derived from vicilin allergens with variable prevalence. The conserved VBP fold could allow for cross-reactivity with peanuts and other tree-nuts including walnut. Further research is needed to correlate the VBP IgE levels to clinical diagnosis.

Background: Vicilin seed storage proteins are translated with N-terminal leader sequences (LSs) that are cleaved to yield the mature protein. These LSs were thought to be unstructured and rapidly degraded. However, Ara h 1 and Jug r 2 LS (A1LS, J2LS) have been identified in seeds, and immunodominant IgE epitopes detected. Here, common sequences containing structured CxxxC-repeat motifs were identified as potential mediators of IgE cross-reactivity despite very low (17%) sequence identity. Method: Linear IgE epitopes were identified by peptide microarrays, in which overlapping 15-mer peptides on glass slides, were incubated with sera from peanut, walnut or dual allergic individuals. Similar epitopes were computationally predicted. Peanut A1LS and walnut J2LS fragments (J2.1, J2.2, J2.3) each with a CxxxC vicilin LS motif were identified, cloned, expressed, purified and their structures solved using solution-NMR to locate and assess epitopes on the structure. Results: A1LS and J2LSs reveal similar helix-turn-helix motifs connected by disulfide bonds between adjacent CxxxC repeats forming α-hairpin structures. Peanut-allergic IgE bound more frequently to the J2LSs, regardless of walnut allergic status or A1LS binding. IgE binding pattern to peptides from both J2LS and A1LS, along with structure and computational predictions, suggest that the structure and conserved amino acid properties of peptides determine cross-reactivity. The properties of LS IgE epitopes were closely related to epitopes in 2S albumins. Conclusion: The shared α-hairpin structure is a stable scaffold that contributes to cross-reactivity despite low sequence identity. Biophysical properties are a better predictor of distant cross-reactivity than traditional measures of evolutionary conservation.

Peanut (PN) allergy is frequently comorbid with allergy to tree-nuts (TN) such as walnuts (WN), cashews (CS) and pistachios (PS). Vicilin seed storage proteins from both PN and TN are expressed with an N-terminal leader sequence (LS) consisting of a repeated CxxxC helical-hairpin motif, the structural and immunological characterization of which may provide insights into the cross-reactivity observed between these evolutionarily distant species. The structure of the individual CxxxC motifs from PN (AH1), WN (JR21, JR22, JR23), CS (AO1, AO2), and PS (PV1, PV2) were solved using solution-NMR, while simulated gastric digestion and circular dichroism were used to assess biophysical stability. Peptide microarrays were used to identify and map IgE epitopes onto the resulting structures to identify regions responsible for both immunogenicity and cross-reactivity. PN and TN CxxxC motifs adopted a common α-helical hairpin fold with a high degree of structural similarity. Numerous PN-TN and TN-TN cross-reactive epitopes were identified within the structured regions. However, their distribution across the individual CxxxC motifs was uneven. Proteolytic resistance was similarly variable, suggesting that structural similarity and biophysical stability are both major determinants of immunogenicity and cross-reactivity within these regions. Despite the lack of sequence identity, vicilin LS may contribute to PN-TN cross reactivity. However, the immunogenicity of individual CxxxC motifs is difficult to predict using traditional bioinformatics techniques. Given the widespread prevalence of vicilin LS in other food allergens, similar interactions may contribute to cross-reactivity across a wide range of seemingly unrelated species

Recent statistical evidence suggests that allergens are more stable and more highly expressed than other proteins from an allergen source. These factors may influence human exposure and, on a smaller scale, stability may affect processing by sentinel dendritic cells skewing the immune response toward allergy. Interestingly, for cockroach allergens purified from frass, the allergen stabilities were not statistically greater than their non-allergen counterparts, likely due to the high mean stability of the latter. To investigate if this was a statistical sampling anomaly, the stability of additional recombinant cockroach allergens was studied. The stability of 6 recombinant cockroach allergens was measured as a function of guanidinium chloride concentration using HNSB and SPROX labeling. Additionally, distant relatives of Bla g 1 (MA proteins) were compared for their thermal stability using temperature dependent circular dichroism as a function of lipid ligand concentration. Bla g 1, 4, 6, and 9 showed elevated stabilities compared to the mean of non-allergen cockroach proteins. However, the differences between allergens and non-allergens was not statistically significant. Additional MA homologues of Bla g 1 demonstrated similar melting temperatures to the latter with no ligand present. However, the stability enhancement due to lipid loading was not present or not as great for other MA proteins compared to Bla g 1 loaded with lipids. Cockroach allergens are highly stable proteins compared to proteins from other allergen sources. However, compared to non-allergens from cockroaches there is not a significant difference, even with data from additional recombinant allergens.

The mechanosensitive channel of large conductance (MscL) from Escherichia coli is a prototype for the mechanosensitive class of ion channels and opens one of the largest known gated transmembrane pores. As such, MscL offers the structural framework for the development of liposomal nanovalves for biotechnological applications. Here we incorporated MscL into liposomes and investigated the effects of L-α-lysophosphatidylcholine (LPC) with varying acyl chain lengths or saturation on its pore gating. This was measured by the efflux of encapsulated 5,6-carboxyfluorescein (CF) from the MscL proteoliposomes. Efflux improved in the presence of shorter and double-bonded LPC acyl chains. It was also dependent on the detergent concentration employed during MscL purification. MscL purified in 2 mM dodecyl β-D-maltopyranoside (DDM) had a marked increase in CF efflux compared to MscL purified in 1 mM DDM when treated with LPC. The purification conditions also resulted in increased efflux from proteoliposomes containing the G22C-MscL pore mutant channel, which requires higher membrane tension for its activation compared to WT-MscL.

SIGNIFICANCE:Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis.RECENT ADVANCES:As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction.CRITICAL ISSUES:In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis.FUTURE DIRECTIONS:A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

MscL acts as an osmotically-activated nanovalve, allowing bacteria to respond to hypo-osmotic stress by opening nanometer-size channel pores. The underlying mechanism of channel activation by membrane tension has been obtained for MscL channels reconstituted into artificial liposomes using patch clamp, EPR and FRET spectroscopy in combination with computational modeling of channel dynamics during channel opening. Given the large size of the MscL pore (>25 Å), we have investigated its suitability for use as a nanovalve enabling controlled release of liposome-encapsulated compounds. Liposomes present one of the major forms of particulate drug carriers and provide an excellent method of encapsulation of highly toxic drugs, for example. In this study we describe methods for generating small liposomes of uniform size based on a combination of liposome extrusion techniques and continuous sucrose gradient centrifugation. We also demonstrate that MscL reconstituted into these liposomes may be used as a nanovalve for controlled release of small molecules including the self-quenching fluorescent dye 5,6-carboxyfluorescein (CF). CF release is regulated by the MscL-activating amphipath L-α-Lysophosphatidylcholine and exhibits a dependence on liposome size, amphipath concentration and protein-to-lipid ratio (see Figure). Supported by the NHMRC project grant 635513.

The bacterial mechanosensitive ion channel of large conductance (MscL) acts as an emergency release valve to protect against osmotic stress1,2; its pore diameter is estimated to increase by >25Å on opening3,4. To investigate mechanisms for controlling the gating of this channel for use in targeted drug delivery systems, we have encapsulated the fluorescent dye 5,6-carboxyfluorescein (CF) into liposomes (diam. 100 nm) via sonication and extrusion using the LiposoFast system, followed by incorporation of MscL protein. Functional assays of MscL by patch-clamping confirmed that the dye did not affect the protein incorporation, and unencapsulated dye was removed by column purification. Liposomes were incubated with different concentrations of the amphipath L-α-Lysophosphatidylcholine (LPC) dissolved in ∼3% MeOH for 30 min to induce stress on liposomal membranes5. Dye release from the liposomes via MscL was monitored as increased fluorescence in the external medium. The percentage fluorescence change quantified using a BMG Omega Polarstar Reader (excitation at 485nm and emission at 535nm). Based on these results, we demonstrate cargo release by MscL by means of manipulating the curvature of liposome membranes4. 1. Martinac, B., J. Cell Sci., 117, 2449-2460 (2004). 2. Perozo, E., Nat. Rev. Mol. Biol. , 7, 109-119 (2006). 3. Cruickshank, C.C., Minchin, R., Le Dain, A., and Martinac, B. Biophys J 73, 1925-1931 (1997) 4. Perozo, E., Cortes, D.M., Sompornpisut, P. Kloda, A. and Martinac, B. Nature 418: 942-948 (2002) 5 Perozo, E., Kloda, A., Cortes, D.M., Martinac, B., Nat. Str. Biol. 9, 696-703(2002) Supported by grants from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC). A.Foo is a UQ Scholarship recipient.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

The main function of the photosynthetic process is to capture solar energy and to store it in the form of chemical 'fuels'. Increasingly, the photosynthetic machinery is being used for the production of biofuels such as bio-ethanol, biodiesel and bio-H2. Fuel production efficiency is directly dependent on the solar photon capture and conversion efficiency of the system. Green algae (e.g. Chlamydomonas reinhardtii) have evolved genetic strategies to assemble large light-harvesting antenna complexes (LHC) to maximize light capture under low-light conditions, with the downside that under high solar irradiance, most of the absorbed photons are wasted as fluorescence and heat to protect against photodamage. This limits the production process efficiency of mass culture. We applied RNAi technology to down-regulate the entire LHC gene family simultaneously to reduce energy losses by fluorescence and heat. The mutant Stm3LR3 had significantly reduced levels of LHCI and LHCII mRNAs and proteins while chlorophyll and pigment synthesis was functional. The grana were markedly less tightly stacked, consistent with the role of LHCII. Stm3LR3 also exhibited reduced levels of fluorescence, a higher photosynthetic quantum yield and a reduced sensitivity to photoinhibition, resulting in an increased efficiency of cell cultivation under elevated light conditions. Collectively, these properties offer three advantages in terms of algal bioreactor efficiency under natural high-light levels: (i) reduced fluorescence and LHC-dependent heat losses and thus increased photosynthetic efficiencies under high-light conditions; (ii) improved light penetration properties; and (iii) potentially reduced risk of oxidative photodamage of PSII.

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007