Mark C. Prescott

Odorranalectin Is a Small Peptide Lectin with Potential for Drug Delivery and Targeting

Background Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein–sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectins interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting. Principal Findings Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. 125I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a β-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form. Conclusion These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection.

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone.

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.

Isolation and characterisation of intact steryl ferulates from seeds

Abstract Approaches to the isolation and characterisation of intact steryl ferulates were investigated. Alkaline washing of the total lipid extract obtained from seeds allowed preliminary separation of a phenolic ester containing fraction from the abundant neutral lipids (comprising largely triacylglycerides, sterols and steryl fatty acyl esters). Subsequent thin-layer chromatography (silica gel) afforded purified steryl ferulates. Separation of individual molecular species was achieved by reversed-phase (ODS) high-performace liquid chromatography (HPLC) using the acetate derivatives of the steryl ferulates, or by high-temperature gas chromatography (GC) of the underivatised steryl ferulates or their acetates or trimethylsilyl ethers. Characterisation of intact individual molecular species was based on the electron impact mass spectra obtained by GC—mass spectrometry (MS) of the underivatised ferulates or their trimethylsilyl ethers, or by direct insertion probe MS of the acetates of the steryl ferulates obtained by preparative HPLC. The feasibility of the above approach was established using the well characterised steryl ferulate mixture γ-oryzanol obtained from rice bran oil. The methods were subsequently applied to the analysis of the steryl ferulates present in seeds of maize ( Zea mays ).

Differential Proteomic Analysis of Arabidopsis thaliana Genotypes Exhibiting Resistance or Susceptibility to the Insect Herbivore, Plutella xylostella

A proteomic study was conducted to investigate physiological factors affecting feeding behaviour by larvae of the insect, Plutella xylostella, on herbivore-susceptible and herbivore-resistant Arabidopsis thaliana. The leaves of 162 recombinant inbred lines (Rils) were screened to detect genotypes upon which Plutella larvae fed least (P. xylostella-resistant) or most (P. xylostella-susceptible). 2D-PAGE revealed significant differences in the proteomes between the identified resistant and susceptible Rils. The proteomic results, together with detection of increased production of hydrogen peroxide in resistant Rils, suggest a correlation between P. xylostella resistance and the production of increased levels of reactive oxygen species (ROS), in particular H2O2, and that this was expressed prior to herbivory. Many of the proteins that were more abundant in the Plutella-resistant Rils are known in other biological systems to be involved in limiting ROS damage. Such proteins included carbonic anhydrases, malate dehydrogenases, glutathione S-transferases, isocitrate dehydrogenase-like protein (R1), and lipoamide dehydrogenase. In addition, patterns of germin-like protein 3 isoforms could also be indicative of higher levels of reactive oxygen species in the resistant Rils. Consistent with the occurrence of greater oxidative stress in the resistant Rils is the observation of greater abundance in susceptible Rils of polypeptides of the photosynthetic oxygen-evolving complex, which are known to be damaged under oxidative stress. The combined results suggest that enhanced production of ROS may be a major pre-existing mechanism of Plutella resistance in Arabidopsis, but definitive corroboration of this requires much further work.

Negative ion ammonia chemical ionization and electron impact ionization mass spectrometric analysis of steryl fatty acyl esters

Synthesis of steryl palmitates, varied in the nature of the steryl moiety, provided model compounds for investigation of the mass spectrometric behavior of steryl long-chain fatty acyl esters. The structure of the steryl moiety was varied according to: (i) position and degree of unsaturation in the steroid nucleus and C-17 side-chain, (ii) position and degree of methylation, (iii) presence or absence of a 9 beta, 19-cyclopropane ring. Compounds were chosen so as to be representative of biochemically important steryl esters. Electron impact (EI) behavior of steryl palmitate esters closely resembles that of their short-chain (e.g. acetate) counterparts. M+.ions were generally weak or absent and the major high mass ions arose from characteristic fragmentations of the steroid nucleus following loss of the acyl moiety ([M-RCO2H]+.). Fragment ions characteristic of the acyl moiety were lacking. Negative ion chemical ionization (NICI) using ammonia as reagent gas, on the other hand, afforded spectra containing characteristic fragment ions [RCO2]-, [RCO2-18]-, and [RCO2-19]- from which the nature of the fatty acyl moiety can be readily deduced. Hence, NICI and EI provide complementary means of ionization for the mass spectrometric determination of structures of steryl esters.

The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica

Background Liver fluke infection of livestock causes economic losses of over US

Mass Spectrometric Determination of Methyl Farnesoate Profiles and Correlation with Ovarian Development in the Edible Crab, Cancer pagurus

3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. Methodology/Principal Findings In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. Conclusions/Significance We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity.

Steryl esters in a cell suspension culture of celery (Apium graveolens)

Evidence indicates that methyl farnesoate (MF), produced by the mandibular organs (MOs), is the crustacean equivalent of the juvenile hormones of insects. Gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) was used to measure the titers of MF in the MOs of the crab, Cancer pagurus. Comparison of the amounts of MF detected in groups of left and right MOs from crabs at different stages of ovarian development showed no statistically significant variation. The amounts of MF detected in MOs were in the range of 230 ng/MO in crabs with immature ovaries to 400 ng/MO in crabs with fully developed ovaries. Application of GC-MS-SIM to the measurement of MF in hemolymph was unsuccessful, leading to the development of a high-performance liquid chromatography-MS-SIM (HPLC-MS-SIM) method for analysis of such titers. MF concentrations in hemolymph varied throughout ovarian development, exhibiting a peak at the beginning of secondary vitellogenesis (140 ng/ml), falling to basal levels thereafter.

Proteomic analysis of embryonic Fasciola hepatica: Characterization and antigenic potential of a developmentally regulated heat shock protein

Arange of analytical techniques was used to investigate the composition of the steryl fatty acyl esters in a cell suspension culture of celery (Apium graveolens). Gas chromatography (GC) and GC-mass spectrometry (GC-MS), using electron ionization (EI) and negative ion chemical ionization (NICI), were employed to characterize the intact steryl esters. Assignments were supported by analysis of the sterol and fatty acid moieties released from the intact molecular species by alkaline hydrolysis. A selectivity for sterol esterification was noted, with the major free sterol, stigmasterol, occurring only in a very small amount in the esterified form. Instead, the precursors to Δ5-phytosterols, particularly cycloartenol, predominated in the ester fraction. The pentacyclic triterpene, β-amyrin, was also found as the palmitate and linoleate esters. Changes in composition and abundance of the steryl esters during the different growth phases of a celery cell suspension culture were investigated. The total amount of esterified sterols exceeded that of free sterols throughout the growth cycle. The changes observed during growth highlighted differences between the esters of precursor sterols and those of the 4-desmethyl-sterols, and it is postulated that the various steryl esters perform different functions in cell metabolism.

Characterization of carotenoid acyl este rs produced in drought-stressed barley seedlings

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.