Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita.

Mass spectrometric analysis revealed the presence of auxin, mainly in conjugated form, in secretions of Heterodera schachtii and Meloidogyne incognita, with or without treatment with DMT or resorcinol. M. incognita showed the highest production rates, though treatment of M. incognita with resorcinol had a negative effect on auxin production. Analysis of auxin precursor molecules in lysates of H. schachtii, M. incognita and Caenorhabditis elegans suggested that auxin is most probably a degradation product of tryptophan and that auxin may be synthesized via several intermediates, including indole-3-acetamide which is an intermediate of a pathway so far only characterized in bacteria. Furthermore, high levels of anthranilate, a degradation product of tryptophan in animals, but possibly also a precursor for auxin were detected.

Preparation and sequencing of secreted proteins from the pharyngeal glands of the plant parasitic nematode Heterodera schachtii.

summary In order to gain insight into the biology of the parasitic relationship between Heterodera schachtii and its host plant, it is important to understand the functional role of the nematode's pharyngeal secretions. These secretions presumably play a key role in establishing and maintaining a feeding site for the nematode. An optimized method was used for the in vitro production of H. schachtii pharyngeal gland secretions. These pharyngeal secretions were mainly produced in an insoluble form and could be solubilized under denaturing conditions for further analysis. The soluble fraction was concentrated with StrataClean (Stratagene, La Jolla, CA) or with a phenol/ether extraction. These methods made it possible for the first time to separate the secreted proteins on two-dimensional gels. By combining a micropreparative sample preparation with mass spectrometry, two beta-1,4-endoglucanases were identified. A third spot was identified as a novel protein by microsequencing. This is the first report on protein sequence information from pharyngeal secretions of a plant parasitic nematode.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction.

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18.

The chronic survival of many endoparasites is dependent on the ability of these organisms to escape the host immune response. Identification of the molecular mechanisms by which these organisms evade this response may yield novel approaches in the development of anti-inflammatory agents. We describe here the discovery and characterization of a novel 41-kilodalton glycoprotein from the canine hookwork (Ancylostoma caninum) that potently inhibits CD11/CD18-dependent neutrophil function in vitro. Neutrophil inhibitory factor (NIF) blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils, over a similar concentration range (IC50 10-20 nM). Studies aimed at determining the nature of the NIF binding site on neutrophils revealed selective, high affinity binding of this protein to the integrin CD11b/CD18. A cDNA encoding NIF was isolated from a canine hookworm cDNA library. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. The mature protein has 10 cysteines and has seven potential N-linked glycosylation sites. NIF has no significant sequence homologies to any previously reported protein. As such, NIF represents a prototype of a novel class of leukocyte function inhibitors.

Secondary structure of the particle associating domain of apolipoprotein B-100 in low-density lipoprotein by attenuated total reflection infrared spectroscopy.

The secondary structure of the human low-density lipoprotein (LDL) apo B-100 fragment embedded in the lipid domain of the particle has been investigated by Fourier transform attenuated total reflection infrared spectroscopy (FTIR-ATR). The solvent-exposed region of the protein was hydrolyzed by using different proteases (alpha-chymotrypsin, trypsin, proteinase K) for incubation times varying between 24 min and 48 h. Analysis of the FTIR-ATR spectra after repurification of the digested LDL particle indicates the same trend for all the hydrolysis conditions tested: the peptides remaining associated with the particle are rich in beta-sheet structure. Dichroism spectra reveal that at least part of the beta-sheets is associated with the phospholipid component of the particle.

Secondary structure and orientation of the amphipathic peptide GALA in lipid structures. An infrared-spectroscopic approach.

GALA, a synthetic, amphipathic 30-amino-acid peptide, based upon a Glu-Ala-Leu-Ala motive, was designed to mimic the behavior of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH, and becomes an amphipathic alpha helix as the pH is lowered to 5, where it interacts with phospholipid bilayers. Attenuated total-reflection infrared spectroscopy, using polarized light, provides information on the structure and orientation of the peptide and the lipids, which is not subject to artifacts due to light scattering with large particles. H/2H-exchange rate of the amide N-H group and analysis of the shape of the amide I' by Fourier self-deconvolution and curve fitting indicate that the alpha-helical content increases from 19% to 69%, on lowering the pH. A further increase to 100% alpha helix is observed after interaction with palmitoyloleoylglycerophosphocholine (PamOleGroPCho) vesicles. Dichroism data obtained with oriented bilayers of the PamOleGroPCho-GALA complex demonstrate that PamOleGroPCho hydrocarbon chains and the peptide alpha helical axis are essentially perpendicular (+/- 15) to the membrane plane. At neutral pH, in the presence of dimyristoylglycerophosphocholine (Myr2GroPCho), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipoproteins AI and AII. In these discoidal complexes, the alpha-helical content was estimated to be 65%, with the rest of the structure being essentially unordered. No significant modification of the all-trans conformation of the hydrocarbon chain of Myr2GroPCho was detected upon disc formation. Dichroism measurements show that the alpha-helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is surrounded by amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoproteins AI and AII which form discoidal complexes with Myr2GroPCho, but the spectrum is quite different from that of apolipoprotein B100 in low-density lipoproteins, which does not form discoidal complexes.

Investigation of the lipid domains and apolipoprotein orientation in reconstituted high density lipoproteins by fluorescence and IR methods.

The reconstituted high density lipoproteins (rHDL) that were described in the preceding paper (Hefele Wald, J., Krul, E. S., and Jonas, A. (1990) J. Biol. Chem. 265, 20037-20043) are used in this study to analyze the organization, conformation, and dynamics of the lipid phase, as well as the relative orientation of the apolipoprotein alpha-helices and the lipid hydrocarbon chains. Two fluorescence polarization probes and a fluorescence polarity probe were used to detect the lipid phase transition behavior of the various particles, and to estimate the lipid order, mobility, and environment polarity in their gel and liquid-crystalline states. Infrared attenuated total reflection spectroscopy was used to estimate the content of secondary structure of the apolipoprotein, and the orientation of its alpha-helices with respect to the lipid hydrocarbon chains. In addition, the infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the rHDL particles. The results indicate that the overall organization and conformation of lipid molecules in a lipid bilayer is preserved in the rHDL particles, but that progressive increases in apolipoprotein content straighten the hydrocarbon chains and decrease their packing order in the gel state, and decrease their mobility in the liquid-crystalline state. The presence of apolipoprotein also affects the conformation of the lipids at the level of the ester bonds and the head group of the phospholipid. In all three particle classes the content and distribution of secondary structures of the apolipoprotein were similar, and the alpha-helical segments were parallel to the lipid hydrocarbon chains.

Mode of assembly of amphipathic helical segments in model high-density lipoproteins.

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.

Evaluation of the secondary structure of apo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy.

The secondary structure of the apo B-100 protein present in human low density lipoprotein has been investigated by transmission and attenuated total reflection infrared spectroscopy. The amount of beta-sheet (41%) is significantly higher than that determined by CD spectroscopy in the present study (12%) and elsewhere (15-16%). The high percentage of beta-sheet structure in apo B-100 supports the importance of such segments in maintaining the lipid-protein assembly in LDL. Polarized infrared spectroscopy indicates that the beta-sheet component of apo B-100 adopts a preferential orientation with respect to the phospholipid monolayer surrounding the LDL, whereas no such orientation is observed for the other secondary structure components.

Mode of organization of galactolipids: a conformational analysis.

Despite the fact that photosynthetic membranes show the conventional bilayer structure, their major lipid component monogalactosyldiacylglycerol does not form lamellar structure but takes up an hexagonal-II structure when dispersed alone in water and forms inverted lipids micelle structures when dispersed together with other lipid components of the photosynthetic membrane. We present here evidence that the mode of organization of these lipids can be predicted from a conformational approach allowing to describe the configuration of assembled amphiphilic molecules. The minimal conformational energy is calculated as the sum of the contributions resulting from the Van der Waals interactions, the torsional potentials, the electrostatic interaction and the transfer energy. Because of its calculated "cone shaped" structure monogalactosyldiacylglycerol forms inverted lipid structure with the hydrophilic groups pointing inward; for digalactosyldiacylglycerol, an other essential lipid constituent of photosynthetic membrane, its calculated cylindrical shape induces an organization in bilayer structures.