Raphaël Culerrier

Phylogenetic and specificity studies of two-domain GNA-related lectins: Generation of multispecificity through domain duplication and divergent evolution

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. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

A Novel Family of Lectins Evolutionarily Related to Class V Chitinases: An Example of Neofunctionalization in Legumes

A lectin has been identified in black locust (Robinia pseudoacacia) bark that shares approximately 50% sequence identity with plant class V chitinases but is essentially devoid of chitinase activity. Specificity studies indicated that the black locust chitinase-related agglutinin (RobpsCRA) preferentially binds to high-mannose N-glycans comprising the proximal pentasaccharide core structure. Closely related orthologs of RobpsCRA could be identified in the legumes Glycine max, Medicago truncatula, and Lotus japonicus but in no other plant species, suggesting that this novel lectin family most probably evolved in an ancient legume species or possibly an earlier ancestor. This identification of RobpsCRA not only illustrates neofunctionalization in plants, but also provides firm evidence that plants are capable of developing a sugar-binding domain from an existing structural scaffold with a different activity and accordingly sheds new light on the molecular evolution of plant lectins.

First case report of anaphylaxis to spirulin: identification of phycocyanin as responsible allergen.

Spirulina (Arthrospira platensis) is a cyanobacterium (formerly bluegreen algae) used as a human dietary supplement because of its high content in proteins, c-linoleic acid and vitamins (1). We present the first documented case report identifying the cyanobacterial pigment phycocyanin as a potentially allergenic protein. A 14year-old adolescent who had previously experienced urticaria, labial oedema and asthma 6 h after consumption of five spirulin tablets, exhibited positive prick test and oral challenge test to spirulin. Upon oral challenging with increasing spirulin doses corresponding to four spirulin tablets over a 3-h period, he experienced diarrhea 2 h and 5 h after the last challenge, respectively. Moreover, a diffuse erythema occurred 30 min latter the last diarrhea episode, requesting a Xyzall and Solupred medication. The offending spirulina allergen was identified on a cold acetone-precipitated protein extract prepared from spirulin tablets previously homogenized in 20 mM Tris-buffered saline (pH 7.5) and stirred for 4 h at 4 C. Upon transfer of the protein fractions separated by SDS-PAGE (2) on a nitro-cellulose membrane, a Western blotting performed in the presence of 1 : 10 diluted patient serum revealed two main IgE-reacting protein fractions (Fig. 1A,B). A digestion with trypsin of the IgE-reactive fractions in the gel, followed by mass mapping by MALDITOF analysis using Protein Prospector for identifying the peptides in the NCBI nonredundant database (3), unambiguously identified b chain of Cphycocyanin as the responsible allergen. In addition, a faint IgE-binding reaction also occurred with the protein fraction that essentially consists of a chain. C-Phycocyanin consists of a (162 amino acids) and b (172 amino acids) chains associated in a macromolecular assembly, built up from four (ab)3 trimers which aggregate face to face to form two superposed (ab)6 hexamers (Fig. 1C) (4). The IgE-reactive b chain trimers, which encircle the two central a chain trimers, occupy a nicely exposed position at the surface of C-phycocyanin.

Characterization of IgE-binding epitopes of peanut (Arachis hypogaea) PNA lectin allergen cross-reacting with other structurally related legume lectins

Sera from peanut allergic patients contain IgE that specifically interact with the peanut lectin PNA and other closely related legume lectins like LcA from lentil, PsA from pea and PHA from kidney bean. The IgE-binding activity of PNA and legume lectins was assessed by immunoblotting, surface plasmon resonance (SPR) and ELISA measurements, using sera from peanut allergic patients as a IgE source. This IgE-binding cross-reactivity most probably depends on the occurrence of structurally related epitopes that have been identified on the molecular surface of PNA and other legume lectins. These epitopes definitely differ from those responsible for the allergenicity of the major allergens Ara h 1, Ara h 2 and Ara h 3, also recognized by the IgE-containing sera of peanut allergic patients. Peanut lectin PNA and other legume lectins have been characterized as potential allergens for patients allergic to edible legume seeds. However, the clinical significance of the lectin-IgE interaction has to be addressed.

Morniga G: a plant lectin as an endocytic ligand for photosensitizer molecule targeting toward tumor-associated T/Tn antigens.

Porphyrins are used as photosensitizer (PS) in photodynamic therapy in cancer treatment. Nevertheless, the development of photochemotherapy in oncology remains limited, because of the low selectivity of PSs. In order to allow PS targeting toward tumor‐associated antigens, for the first time a white‐light activatable porphyrin, [5‐(4‐(5‐carboxy‐1‐butoxy)‐phenyl)‐10,15,20‐tris(4‐N‐methyl)‐pyridiniumyl)‐porphyrin] (TrMPyP) was covalently linked to Morniga G (MorG), a galactose‐specific binding plant lectin, known to recognize with high‐affinity tumor‐associated T/Tn antigen in cell‐free systems. Firstly, using fluorescein‐labeled MorG, the sugar‐dependent binding and uptake of lectin by Tn‐positive (Jurkat lymphoid leukemia) cells was demonstrated. Secondly, the TrMPyP–MorG conjugate was molecularly characterized. Cytometric and confocal microscopic analysis demonstrated that PS covalent linking to MorG preserved sugar‐dependent specific binding and uptake of lectin by Jurkat leukemia lymphocytes. Thirdly, the conjugate (with a 1:1 PS:lectin ratio) that was bound and quickly (5 min) taken‐up, induced greater than 90% cytotoxicity upon irradiation at 10 nm concentration, whereas the free PS was absolutely nontoxic. On the contrary, normal lymphocytes strongly resisted to the conjugate‐mediated phototoxicity. Thus, owing to their binding and endocytosis capacities, plant lectins represent promising molecules for targeting of tumor glycan alteration and to enhance the efficiency of specific delivery of PSs to tumor cells.

A case of severe anaphylaxis to kidney bean: phaseolin (vicilin) and PHA (lectin) identified as putative allergens

isoforms showed high identities among beta-1 (96%) and beta-2 homologs (97%). IgE immunoblot analysis with purified native parvalbumins, recombinant parvalbumins (salmon, trout) and food-grade fish gelatin (Rousselot, Courbevoie, France) was performed using patient serum. Salmon and trout parvalbumins were strongly positive, but cod and carp parvalbumins were negative (Fig. 1A). Furthermore, IgE did not recognize mackerel, redfish and herring parvalbumin or fish gelatin. IgE binding to salmon parvalbumin was abolished by preincubation of the serum with trout parvalbumin, but not with cod or carp parvalbumin (data not shown). Specific IgE binding was quantified by enzyme-linked immunosorbent assay (ELISA) using purified parvalbumins in the presence of 0.5 mM calcium chloride. IgE binding was similar for salmon parvalbumin and trout homologs (Fig. 1B). IgE titers decreased to <0.1 kU/l upon calcium depletion. No IgE reactivity was found for other parvalbumins or fish gelatin. In conclusion, we could show that the IgE antibodies of a patient clinically mono-allergic to salmon and trout bound to parvalbumins of these two salmonid fishes, but not to parvalbumin of cod, carp, mackerel, redfish and herring. Comparison of salmon parvalbumin to the trout homolog showed that the beta-1 isoforms differed by four and the beta-2 isoforms by three amino acids only. When aligning the salmonid beta-1 parvalbumins to those of carp, cod and mackerel, the C-terminal regions at positions 3–10 and 27–38 were identical for salmonid parvalbumins, but different compared to the proteins of the other fishes. These two regions could possibly be part of the B-cell epitopes recognized specifically by the patient’s IgE antibodies on salmon and trout parvalbumin. This report illustrates also the importance of using a panel of parvalbumins from different fish species to perform component-resolved diagnostic work-up of fish allergy. In clinical practice, this will help advising fish-allergic patients which fish species to consume, preferably after confirmation by in-vivo provocation tests. *Laboratory of Immunogenetics and Allergology CRP-Santé 84, Val Fleuri L-1526 Luxembourg Tel.: +352 26 97 03 35 Fax: +352 26 97 03 90 E-mail: annette.kuehn@crp-sante.lu

Two structurally identical mannose‐specific jacalin‐related lectins display different effects on human T lymphocyte activation and cell death

Plant lectins displaying similar single sugar‐binding specificity and identical molecular structure might present various biological effects. To explore this possibility, the effects on human lymphocytes of two mannose‐specific and structurally closely related lectins, Morniga M from Morus nigra and artocarpin from Artocarpus integrifolia were investigated. In silico analysis revealed that Morniga M presents a more largely open carbohydrate‐binding cavity than artocarpin, probably allowing interactions with a broader spectrum of carbohydrate moieties. In vitro, Morniga M interacted strongly with the lymphocyte surface and was uptaken quickly by cells. Morniga M and artocarpin triggered the proliferation and activation of human T and NK lymphocytes. A minority of B lymphocytes was activated in artocarpin‐treated culture, whereas Morniga M favored the emergence of CD4+ CD8+ T lymphocytes. Moreover, cell death occurred in activated PBMC, activated T lymphocytes, and Jurkat T leukemia cells incubated with Morniga M only. The biological effects of both lectins were dependent on carbohydrate recognition. The Morniga M‐induced cell death resulted, at least in part, from caspase‐dependent apoptosis and FADD‐dependent receptor‐mediated cell death. Finally, Morniga M, but not artocarpin, triggered AICD of T lymphocytes. In conclusion, both lectins trigger lymphocyte activation, but only Morniga M induces cell death. In spite of similar in vitro mannose‐binding specificities and virtually identical structure, only Morniga M probably interacts with carbohydrate moieties bound to molecules able to induce cell death. The present data suggest that subtle alterations in N‐glycans can distinguish activation and cell death molecules at the lymphocyte surface.

IgE-binding epitopic peptide mapping on a three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum)

The three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) consists of three protomers exhibiting the cupin motif, arranged in a homotrimer around a three-fold symmetry axis. Using the SPOT technique, 11 continuous IgE-binding epitopic peptides were characterized on the molecular surface of the 13S globulin allergen of buckwheat. Except for one of them, they all correspond to well exposed regions containing electropositiveley and/or electronegatively charged residues, which cover up to 40% of the molecular surface of the allergen. Some of these epitopes come in close contact to probably create more extended discontinuous epitopes, especially those located on the edge of the 13S globulin homotrimer. Half of the identified epitope peptides remain unaltered in a core structure protected against hydrolysis by digestive proteases and are thus assumed to promote the allergenicity of the 13S globulin. In addition, a few of these epitopes coincide with sequential IgE-binding epitopes previously characterized in soybean 11S globulins, that could account for the IgE-binding cross-reactions observed between soybean and buckwheat in Western blot experiments.

Targeting of T/Tn Antigens with a Plant Lectin to Kill Human Leukemia Cells by Photochemotherapy

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy.

Les allergies alimentaires aux fruits

Resume Les fruits comestibles renferment de nombreux allergenes qui correspondent essentiellement a des proteines de defense ou proteines PR (pour Pathogenesis Related). Ces proteines, tres repandues chez les vegetaux, sont des panallergenes. Les principaux allergenes appartiennent aux groupes des PR-2 (1,3β-glucanases), des PR-3 (chitinases de classe I), des PR-4 (transporteurs d’auxine ou germines), des PR-5 (proteines thaumatine-like ou TLP), des PR-10 (proteines Bet v 1-like) et des PR-14 (proteines de transfert des lipides ou LTP). S’y ajoutent des profilines et des pectine esterases. En raison de la presence de ces allergenes dans les pollens, les aeroallergenes sont le plus souvent responsables de la sensibilisation primaire. Ainsi, la plupart des allergies a la pomme dependent d’une sensibilisation prealable au pollen de bouleau, l’allergene Mal d 1 croisant avec Bet v 1. L’allergie au kiwi ou a la banane sont souvent associees aux allergenes croisants (chitinases, 1,3β-glucanases) du syndrome latex-fruits. La sensibilisation directe par contact ou ingestion (LTP de peche) est possible et peut donner lieu a des reactions anaphylactiques generalisees, notamment dans les pays du Sud de l’Europe (Espagne). Le diagnostic biologique des allergies aux fruits est souvent delicat en raison du petit nombre d’allergenes recombinants disponibles (rPru p 1, rPru p 3 et rPru p 4 de peche) et de l’instabilite des extraits de fruits.