Pierre Rougé - Academia.edu (original) (raw)

Papers by Pierre Rougé

The Plant Journal, 2003

A novel lectin has been isolated and cloned from leaves of Glechoma hederacea (ground ivy), a typ... more A novel lectin has been isolated and cloned from leaves of Glechoma hederacea (ground ivy), a typical representative of the plant family Lamiaceae. Biochemical analyses indicated that the G. hederacea agglutinin (Gleheda) is a tetrameric protein consisting of four subunits pairwise linked through an interchain disulphide bridge and exhibits a preferential speci®city towards N-acetylgalactosamine. Cloning of the corresponding gene and molecular modeling of the deduced sequence demonstrated that Gleheda shares high sequence similarity with the legume lectins and exhibits the same overall fold and three-dimensional structure as the classical legume lectins. The identi®cation of a soluble and active legume lectin ortholog in G. hederacea not only indicates that the yet unclassi®ed Lamiaceae lectins belong to the same lectin family as the legume lectins, but also sheds a new light on the speci®city, physiological role and evolution of the classical legume lectins.

European Journal of Biochemistry, 2002

Fruits of elderberry (Sambucus nigra) express small quantities of a type-2 ribosome-inactivating ... more Fruits of elderberry (Sambucus nigra) express small quantities of a type-2 ribosome-inactivating protein with an exclusive specificity towards the NeuAc(a2,6)Gal/GalNAc disaccharide and a unique molecular structure typified by the occurrence of a disulfide bridge between the B-chains of two adjacent protomers. A cDNA clone encoding this so-called Sambucus nigra fruit specific agglutinin I (SNA-If) was isolated and expressed in tobacco (Samsun NN) under the control of the 35S cauliflower mosaic virus promoter. Characterization of the purified protein indicated that the recombinant SNA-If from tobacco leaves has the same molecular structure and biological activities as native SNA-If from elderberry fruits, demonstrating that transgenic tobacco plants are fully capable of expressing and correctly processing and assembling a type-2 ribosome-inactivating protein with a complex molecular structure. None of the transformants showed a phenotypic effect, indicating that the ectopically expressed SNA-If does not affect the viability of the tobacco cells. Bioassays further demonstrated that none of the transgenic lines exhibited a decreased sensitivity to infection with tobacco mosaic virus suggesting that the elderberry type-2 RIP SNA-If does not act as an antiviral agent in planta.

Biochimie, 2003

Fruit-specific thaumatin-like proteins were isolated from cherry, apple and banana, and their enz... more Fruit-specific thaumatin-like proteins were isolated from cherry, apple and banana, and their enzymatic and antifungal activities compared. Both the apple and cherry possess a moderate endo-β1,3-glucanase activity but are devoid of antifugal activity. In contrast, the banana thaumatin-like protein inhibits the in vitro hyphal growth of Verticillium albo-atrum but is virtually devoid of endo-β1,3-glucanase activity. Both structural and molecular modeling studies showed that all three thaumatin-like proteins possess an extended electronegatively charged cleft at their surface, which is believed to be a prerequisite for endo-β1,3-glucanase activity. Docking experiments showed that the positioning of linear (1,3)-β-D-glucans in the cleft of the apple and cherry proteins allows an interaction with the glutamic acid residues that are responsible for the hydrolytic cleavage of the glucan. Due to a different positioning in the cleft of the banana thaumatin-like protein, the linear β-glucans cannot properly interact with the catalytic glutamic acid residues and as a result the protein possesses no enzymatic activity. The possible function of the fruit-specific thaumatin-like proteins is discussed in view of the observed biological activities and structural features.

Biochemical Journal, 2007

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers... more A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose.

European Journal of Biochemistry, 1997

A second NeuAc(a2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' h... more A second NeuAc(a2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sumbucus nigru) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(a2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate-binding specificity and ribosome-inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.

FEBS open bio, 2015

The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus &#39... more The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus 'Liberibacter' asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10(-3) and 0.217 × 10(-3) s(-1), respectively) as compared to JH ...

Journal of Insect Physiology, 2015

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Ana... more Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L's, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His 6 tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.

Memórias do Instituto Oswaldo Cruz, 1996

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea ... more The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Memórias do Instituto Oswaldo Cruz, 2002

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was fol... more Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.

Journal of Insect Physiology, 2014

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti ... more We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzymatic activity and the Michaelis Menten kinetic parameters K m , V max , k cat (turn over number) and k cat /K m (catalytic efficiency) using JHA and its analogues as substrates. AeaJHAMT methylates JHA III 5-fold faster than farnesoic acid (FA). Significant differences in lower methyl transferase (MT) activities towards the cis/trans/trans, cis/trans/cis and the trans/cis/cis isomers of JHA I (1.32, 4.71 and 156-fold, respectively) indicate that substrate chirality is important for proper alignment at the catalytic cavity and for efficient methyl transfer by S-adenosyl methionine (SAM). Our 3D model shows a potential binding site below the main catalytic cavity for JHA analogues causing conformational change and steric hindrance in the transfer of the methyl group to JHA III. These, in silico, observations were corroborated by, in vitro, studies showing that several JHA analogues are potent inhibitors of AeaJHAMT. In vitro, and in vivo studies using [ 3 H-methyl]SAM show that the enzyme is present and active throughout the adult life stage of A. aegypti. Tissue specific expressions of the JHAMT gene of A. aegypti (jmtA) transcript during the life cycle of A. aegypti show that AeaJHAMT is a constitutive enzyme and jmtA transcript is expressed in the corpora allata (CA), and the ovary before and after the blood meal. These results indicate that JH III can be synthesized from JHA III by the mosquito ovary, suggesting that ovarian JH III may play an important physiological role in ovarian development and reproduction. Incubating AeaJHAMT with highly pure synthetic substrates indicates that JHA III is the enzyme's preferred substrate, suggesting that AeaJHAMT is the ultimate enzyme in the biosynthetic pathway of JH III.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2002

Among the very homologous family of K-and L-thionins, known for their antimicrobial activity, the... more Among the very homologous family of K-and L-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles. ß

Acta Crystallographica Section D Biological Crystallography, 1999

The -amylase from Tenebrio molitor larvae (TMA) has been crystallized in complex with the -amylas... more The -amylase from Tenebrio molitor larvae (TMA) has been crystallized in complex with the -amylase inhibitor (-AI) from the bean Phaseolus vulgaris. A molecular-replacement solution of the structure was obtained using the re®ned pig pancreatic -amylase (PPA) and -AI atomic coordinates as starting models. The structural analysis showed that although TMA has the typical structure common to -amylases, large deviations from the mammalianamylase models occur in the loops. Despite these differences in the interacting loops, the bean inhibitor is still able to inhibit both the insect and mammalian -amylase.

Planta, Jan 10, 2000

The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Char... more The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-beta-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed.

Eur J Biochem, 1997

The Journal of Biological Chemistry, 1990

Advances in Experimental Medicine and Biology, 2011

O-glycosylation is a widely distributed posttranslational modification initiated by the addition ... more O-glycosylation is a widely distributed posttranslational modification initiated by the addition of GalNAc to serine or threonine residues of polypeptide chains. The GalNAc may be further substituted to form linear/branched sugar chains. Some proteins, like the mucins of vertebrates, are extensively O-glycosylated and consist predominantly of carbohydrate. T antigens, which were originally designated as Thomsen–Friedenreich or TF antigens, are of

European Journal of Biochemistry, 2000

Abbreviations: a-AI1, isoform 1 of Phaseolus vulgaris a-amylase inhibitor; CVA, Crocus vernus agg... more Abbreviations: a-AI1, isoform 1 of Phaseolus vulgaris a-amylase inhibitor; CVA, Crocus vernus agglutinin; GNA, Galanthus nivalis agglutinin; HCA, hydrophobic cluster analysis; LECCVA, cDNA encoding the lectin from Crocus vernus; PHA-E, erythroagglutinating lectin of Phaseolus vulgaris; PHA-L: leucoagglutinating lectin of Phaseolus vulgaris; PLA, Phaseolus limensis agglutinin; SPR, surface plasmon resonance; RU, resonance units; ASA, Allium sativum agglutinin; ASRA, Allium sativum lectin-related protein; PMLRP, Polygonatum multiflorum lectin-related protein; di1,2Man, a-1,2-mannobiose; di1,3Man, a-1,3-mannobiose; di1,4Man, a-1,4-mannobiose; di1,6Man, and a-1,6-mannobiose; triMan, tri-(a-1,3,a-1,6-mannotriose); pentaMan, pentamannosides (a-1,3,a-1,6-mannopentaose).

European Journal of Biochemistry, 1996

The molecular structure of the Sambucus nigra agglutinin V (SNAV), which has been described previ... more The molecular structure of the Sambucus nigra agglutinin V (SNAV), which has been described previously as a type-2 ribosome-inactivating protein called nigrin b, has been studied in detail by analysis of the purified protein combined with cDNA cloning and molecular modelling. Native SNAV is a dimer of two [A-s-s-B] pairs. Hapten inhibition assays indicated that GalNAc is a 20-fold more potent inhibitor of SNAV than Gal. A cDNA clone encoding SNAV was isolated from a cDNA library constructed with mRNA from the bark. Sequence analysis of this cDNA revealed a striking similarity to the recently cloned NeuAc a-2,6-gal/GalNAc-specific S. nigra bark agglutinin I (SNAI) and to the previously sequenced type-2 ribosome-inactivating proteins from Ricinus communis and Abrus precatorius. In addition, molecular modelling of SNAV further suggested that its structure closely resembles that of ricin. The Nterminal sequence of the B chain of SNAV also shows a marked similarity with the polypeptide of the previously described GalNAc-specific S. nigra bark agglutinin I1 (SNAII), which unlike SNAV and SNAI has no ribosome-inactivating activity. It appears, therefore, that elderbeny bark contains at least two different type-2 ribosome-inativating proteins and a lectin built up of subunits which are closely related to the B chain of SNAV.