Pollen Proteases Play Multiple Roles in Allergic Disorders.

Allergic diseases are a major health concern worldwide. Pollens are important triggers for allergic rhinitis, conjunctivitis and asthma. Proteases released upon pollen grain hydration appear to play a major role in the typical immunological and inflammatory responses that occur in patients with allergic disorders. In this study, we aimed to identify specific proteolytic activity in a set of pollens with diverse allergenic potential. Diffusates from Chenopodium album, Plantago lanceolata and Eucalyptus globulus were added to a confluent monolayer of Calu-3 cells grown in an air-liquid interface system. We identified serine proteases and metalloproteinases in all pollen diffusates investigated. Proteases found in these pollen diffusates were shown to compromise the integrity of the lung epithelial barrier by disrupting transmembrane adhesion proteins E-cadherin, claudin-1 and Occludin, as well as, the cytosolic complex zonula occludens-1 (ZO-1) resulting in a time-dependent increase in transepithelial permeability. Tight junction disruption and increased transepithelial permeability facilitates allergen exposure to epithelial sub-layers contributing to the sensitization to a wide range of allergens. These pollen extracts also induced an increase in the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) cytokines measured by flow cytometry possibly as a result of the activation of protease-activated receptors 2 (PAR-2).

Cardoon as a Sustainable Crop for Biomass and Bioactive Compounds Production.

Cardoon is a multi-purpose and versatile Mediterranean crop, adapted to climate change, with a wide spectrum of potential applications due its added value as a rich source of fibers, oils and bioactive compounds. The Cynara species are a component of the Mediterranean diet and have been used as food and medicine since ancient times. The important role of cardoon in human nutrition, as a functional food, is due to its high content of nutraceutical and bioactive compounds such as oligofructose inulin, caffeoylquinic acids, flavonoids, anthocyanins, sesquiterpenes lactones, triterpenes, fatty acids and aspartic proteases. The present review highlights the characteristics and functions of cardoon biomass which permits the development of innovative products in food and nutrition, pharmaceutics and cosmetics, plant protection and biocides, oils and energy, lignocellulose materials, and healthcare industries following the actual trends of a circular economy.

Selected Cardoon (Cynara cardunculus L.) Genotypes Suitable for PDO Cheeses in Mediterranean Regions.

Cardoon flower extract is a traditional and exclusive rennet used for some PDO cheeses in several Mediterranean regions, due to its extremely high concentration in cardosins. In this preliminary study, six individual cardoon genotypes (1M - 6M) were selected because they revealed a wide and consistent diversity of total and specific cardosin concentrations in flowers. During three growing seasons, the stability of 12 biochemical characteristics of flower extracts and 26 plant morphological descriptors was confirmed. Surprisingly, the cardosin profiles of each genotype, based on four main groups A0, A1, A and B, were stable during the annual flower harvesting period and over all three years using ion-exchange chromatography and native-PAGE electrophoresis. This knowledge will allow an improvement in the quality and standardization of cardosin profiles from cardoon flowers used for cheese production and other innovative applications. The results obtained are promising for the development of a plant breeding program based on biochemical and morphological characteristics in order to obtain the most adapted plant architecture for combined purposes related to specific cardosins composition, flower and plant biomass production, and ease of harvesting.

Functional and conformational changes in the aspartic protease cardosin A induced by TFE.

Conformational and functional changes of cardosin A, an aspartic protease of vegetal origin, in the presence of 2,2,2-trifluoroethanol (TFE), were assessed. TFE induced alterations of cardosin activity and conformation that differed with the solvent concentration. MD simulations showed that there are significant local alterations in protein flexibility and TFE molecules were found to replace several hydration molecules in the active site of the enzyme. This may explain some of the activity loss observed in the presence of TFE, especially at low TFE concentrations, as well as the recovery of enzyme activity upon aqueous dilution, indicating the release of the TFE molecules from the active site.

Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10(4) s(-1) m(-1) against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study.

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine-glycine-aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.

Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L.

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.

Purification of a novel aminopeptidase from the pollen of Parietaria judaica that alters epithelial integrity and degrades neuropeptides.

BACKGROUND: Parietaria judaica pollen is a common cause of pollinosis in the Mediterranean area. OBJECTIVE: This study sought to purify and characterize the peptidase responsible for the majority of proteolytic activity present in the pollen extract of P judaica, and to investigate its contribution to the allergic response. METHODS: A serial of chromatographic steps was applied to isolate the peptidase from P judaica's pollen, and its biochemical properties were determined. Bioactive peptides present in the airways were incubated with the peptidase, and their degradation was visualized by direct protein sequencing. In addition, we measured the cellular detachment, by methylene blue binding assay, of an airway-derived epithelial cell line (A549) in the presence of the peptidase, and visualized, by Western blot, the degradation of proteins from intercellular junctions. RESULTS: We purified a 98-kDa peptidase from the pollen of P judaica that was classified as an aminopeptidase on the basis of its biochemical properties and internal amino acid sequence. The aminopeptidase was able to degrade bioactive peptides. Moreover, the aminopeptidase caused cellular detachment of A549 cell line and degradation of occludin and E-cadherin. CONCLUSION: Our results suggest that the P judaica aminopeptidase can alter the integrity of the epithelium barrier by degrading occludin as well as E-cadherin. In addition, P judaica aminopeptidase can degrade bioactive peptides, which can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs. CLINICAL IMPLICATIONS: The novel aminopeptidase described here could constitute a relevant therapeutic target in the treatment of allergic disorders induced by the pollen of P judaica.

Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains.

Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains.

Molecular cloning of a potato leaf cDNA encoding an aspartic protease (StAsp) and its expression after P. infestans infection.

Aspartic proteinases (EC 3.4.23) are widely distributed in the plant kingdom, and a number of cDNAs have been isolated from different plants. Here we report the isolation an expression analysis of a cDNA from Solanum tuberosum L. (cv. Pampeana) named StAsp. The StAsp cDNA clone was obtained using a reverse transcriptase-polymerase chain reaction (RT-PCR) and degenerated primers encoding to plant aspartic proteinases conserved domains. The coding region of the gene is 1494 bp long encoding 497 amino acids of a predicted 54 kDa molecular mass and with a pI of 5.5. The gene shares a high homology with an aspartic proteinase cDNA of tomato, 97% and 94% homology on the level of DNA and protein, respectively. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert. Northern blot analysis indicated that StAps transcripts are differentially accumulated in potato leaves after Phytophthora infestans infection in two potato cultivars with different degree of field resistance to this pathogen. In the resistant cultivar (Pampeana), induction was higher and more durable than in the susceptible cultivar (Bintje), suggesting that the StAsp level expression are associated with the resistance degree of potato cultivars to P. infestans. Results obtained previously about the induction of StAP proteins in stress conditions and these results suggest that potato aspartic proteinases are components of the plant defense response.

Activation, proteolytic processing, and peptide specificity of recombinant cardosin A.

Cardosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Here we describe the activation and proteolytic processing of recombinant procardosin A. The cleavage sites involved in this multi-step autocatalytic process were determined, some of them using a novel method for C-terminal sequence analysis. Even though the two-chain recombinant enzyme displayed similar properties as natural cardosin A, a single-chain mutant form was engineered based on the processing results and produced in Escherichia coli. Determination of its primary specificity using two combinatorial peptide libraries revealed that this mutant form behaved like the natural enzyme. The primary specificity of the enzyme closely resembles those of cathepsin D and plasmepsins, suggesting that cardosin A shares the same peptide scissile bond preferences of its vacuolar/lysosomal mammalian and protozoan homologues.

The characterisation of the collagenolytic activity of cardosin a demonstrates its potential application for extracellular matrix degradative processes.

Type I collagen is the major fibrous protein of mammals being needed to strengthen and organise the extracellular matrix (ECM). Connective tissue components are modulated by matrix metalloproteinases, which are critical for disintegration and remodelling of ECM under physiological and pathological conditions. Cardosin A is an abundant aspartic proteinase (AP) from Cynara cardunculus L. that has been shown to be able to hydrolyse fibrillar collagen within the alpha-chains. The aim of this work is the characterisation of collagen degradation by cardosin A, since in the native state fibrillar collagen is resistant to most proteolytic enzymes. The pattern of type I collagen hydrolysis by cardosin A is defined and maintained for at least 24 hours of digestion, suggesting that cardosin A can hydrolyse collagen at a small number of specific peptide bonds. N-terminal sequencing of hydrolysis products identified one cleavage site as being Phe464-Gln465 in the alpha2 chain of collagen I. Two peptides were synthesised correspondent to collagen I specific sequences, in order to produce two polyclonal antibodies, that allowed the identification of three collagen fragments following cardosin A cleavage. Defining the mechanism of collagen cleavage by collagenases and other enzymes, like cardosin A, is important for the comprehension of physiological and pathological processes affecting the ECM. To our knowledge, this is the first study of in vitro collagenolytic activity of a plant AP. Therefore, in view of the cardosin A restricted specificity towards collagen this enzyme may be proposed for an eventual medical or technical procedures assisting ECM remodelling.

The Helix aspersa (brown garden snail) allergen repertoire.

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.

Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression.

Glutamate receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. It has been reported that protein kinase A (PKA) can phosphorylate the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR4 on Ser842, both in vitro and in vivo. Here, we studied the regulation of GluR4 phosphorylation and intracellular trafficking by PKA and by metabotropic receptors coupled to adenylyl cyclase (AC), in cultured chick retinal amacrine-like neurones, which are enriched in GluR4. The regulation of AMPA receptor activity by PKA and by metabotropic AC-coupled receptors was also investigated by measuring the [Ca2+]i response to kainate in Na(+)-free medium. Stimulation of AC with forskolin (FSK), or using the selective agonist of dopamine D1 receptors (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF38393), increased the [Ca2+]i response to kainate, GluR4 phosphorylation at Ser842 and GluR4 surface expression. Pre-incubation of the cells with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist of group II metabotropic glutamate receptors (mGluR), which are coupled to inhibition of AC, inhibited the effect of FSK and of SKF38393 on AMPA receptor activity, GluR4 phosphorylation and expression at the plasma membrane. These results indicate that there is a functional cross-talk between dopamine D1 receptors and group II mGluR in the regulation of GluR4 phosphorylation and AMPA receptor activity. Our data show that GluR4 phosphorylation at Ser842 by PKA, and its recruitment to the plasma membrane upon phosphorylation, is regulated by metabotropic receptors.

Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation.

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.

Phosphorylation of GluR4 AMPA-type glutamate receptor subunit by protein kinase C in cultured retina amacrine neurons.

We have previously reported that the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is potentiated by protein kinase C (PKC) in cultured chick retina amacrine neurons, and that constitutive PKC activity is necessary for basal AMPA receptor activity (Carvalho et al., 1998). In this study, we evaluated the phosphorylation of the GluR4 subunit, which is very abundant in cultured amacrine neurons, to correlate it with the effects of PKC on AMPA receptor activity in these cells. 32P-labelling of GluR4 increased upon AMPA receptor stimulation or cell treatment with phorbol 12-myristate 13-acetate (PMA) before stimulating with kainate. By contrast, phosphorylation of GluR4 was not changed when PKC was inhibited by treating the cells with the selective PKC inhibitor GF 109203X before stimulation with kainate. We conclude that GluR4 is phosphorylated upon PKC activation and/or stimulation of AMPA receptors in cultured amacrine cells. Additionally, AMPA receptor activation with kainate in cultured chick amacrine cells leads to translocation of conventional and novel PKC isoforms to the cell membrane, suggesting that PKC could be activated upon AMPA receptor stimulation in these cells.