Jean-Claude Pernollet

Protein bodies of seeds: Ultrastructure, biochemistry, biosynthesis and degradation

Abstract Typical organelles for protein storage occur in seeds, protein bodies are found in haploid, diploid or triploid tissues and are single membrane bound. In some plants, they exhibit inclusions (globoid and crystalloid), but not in Gramineae endosperm or in Leguminosae cotyledons. A relationship between species and protein body ultrastructure can be put forward. The chemical composition is based mainly on storage proteins and phytic acid but, hydrolytic enzymes(protease and phytase), cations and ribonucleic acids are also present. Other minor biochemical components include oxalic acid, carbohydrates (excluding starch) and lipids. The locations of the storage proteins, enzymes and phytin are described. Protein body ontogeny during seed maturation has given rise to much controversy: are they plastidic or vacuolar? Recent studies on the location of proteosynthesis show that protein bodies are probably synthesized in endoplasmic reticulum lumen and that the Golgi apparatus plays an important role in storage protein synthesis. During germination protein bodies swell and fuse, giving rise to the cell central vacuole, while the integrity of the membrane is maintained. Protein bodies may be considered as being an example of tonoplast origin from endo-plasmic reticulum.

High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and α‐interferons

Ovine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion‐exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed‐phase column. Automated Edman degradation was then used to determine the N‐terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons α of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23–44 exhibits 82% homology. Identity between oTPB and either HuIFN‐α.9 or MuIFNα. 1 is 55%. These alignments between oTPB and IFNs occur at the N‐terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon.

Amino acid sequence of locust neuroparsins

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two‐step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N‐terminal sequence of both native neuroparsins was determined: the N‐terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N‐terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu‐C, comprises 78 residues.

Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

Structure-function relationships of α and β elicitins, signal proteins involved in the plant-Phytophthora interaction

Elicitins form a family of 10-kDa holoproteins secreted by various Phytophthora species. The large-scale purification of parasiticein, a novel elicitin secreted by P. parasitica, led to the determination of its sequence. We have compared the necrotic activities and the primary and secondary structures (determined through circular dichroism) of four elicitins. On tobacco plants, they could be classified into two classes: a, comprising capsicein and parasiticein (less necrotic), and β, comprising cryptogein and cinnamomin (very toxic with a necrosis threshold of 0.1 μg per leaf). The features of elicitin structure which might be involved in the interaction of elicitins with the leaf target cells and that could explain the different necrosis-inducing properties of the two proteins are investigated. About 75% sequence identity was observed between the four elicitins: only two short terminal regions are heterologous, while the central core is mainly conserved. The circular-dichroism spectra showed that the secondary structure of the elicitins was largely conserved. All of them consisted of approx. 50% α-helix with little or no β-structure. Comparisons of the complete sequences, amino-acid compositions, isoelectric points, hydropathy indices and the secondary-structure predictions correlated with the necrotic classification. Alpha elicitins corresponded to acidic molecules with a valine residue at position 13, while β elicitins were basic with a lysine at this position, which appeared to be a putative active site responsible for necrosis induction.

Polyglutamylation of Nucleosome Assembly Proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of ∼50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.

Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of mamestra brassicae (noctuidae)

Proteins extracted form the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88%) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria

Neurosecretory products immunologically related to either neuroparsin (NP) or ovary maturing parsin (OMP) of Locusta migratoria (Lom) were purified from the nervous corpora cardiaca of Schistocerca gregaria (Scg). The determination of both their molecular masses by mass spectrometry and their sequences by automated Edman degradation established that they are members of the NP and OMP families respectively. NP molecules of Schistocerca (Scg NPs) consisted of two major forms having about the same molecular masses as NPA and NPB of Locusta and 88% primary structure similarity. They had also the same antidiuretic activity. OMP molecules of Schistocerca (Scg OMPs) were composed in young adults of four isoforms: two long isoforms corresponding to Lom OMP, and differing by a tripeptide insertion (Pro-Ala-Ala) at position 21 and two short isoforms deprived of the 13-residue N-terminal peptide of Lom OMP and differing by the same tripeptide insertion. The PAA isoforms were observed in low amounts as compared to the other isoforms. In mature adults, only the two short isoforms were present. The complete sequence of PAA Scg OMP presents a large degree of sequence homology with Lom OMP (83%). The mixed Scg OMPs had the same biological effects as Lom OMPs. They induced precocious occurrence of both ecdysteroids and vitellogenin in the haemolymph and stimulated oöcyte growth.

Aphrodisin, an aphrodisiac lipocalin secreted in hamster vaginal secretions.

Vertebrates communicate through pheromones, which favor biological regulations within each species. Aphrodisin, a protein belonging to the lipocalin superfamily, found in hamster vaginal secretions, is detected by the male accessory olfactory system and induces or facilitates its copulatory behavior. Although much is known about aphrodisin structure, the question of whether aphrodisin bears itself the pheromonal function or is simply a carrier for hydrophobic small pheromones has not been definitely solved. Arguments based on use of recombinant aphrodisin deprived of any natural ligand and its capability to convey hamster pheromonal compounds will be discussed, together with progresses concerning putative natural ligand(s).

Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.