Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.

HLA-BAT1 alters migration, invasion and pro-inflammatory cytokines in prostate cancer.

Prostate cancer (PCa) accounts for more than 1 in 5 diagnoses and is the second cause of cancer-related deaths in men. Although PCa may be successfully treated, patients may undergo cancer recurrence and there is a need for new biomarkers to improve the prediction of prostate cancer recurrence and improve treatment. Our laboratory demonstrated that HLA-B-associated transcript 1 (BAT1) was differentially expressed in patients with high Gleason scores when compared to low Gleason scores. BAT1 is an anti-inflammatory gene but its role in PCa has not been identified. The objective of this study is to understand the role of BAT1 in prostate cancer. In vitro studies showed that BAT1 down-regulation increased cell migration and invasion. In contrast, BAT1 overexpression decreased cell migration and invasion. RT-PCR analysis showed differential expression of pro-inflammatory cytokines (TNF-α and IL-6) and cell adhesion and migration genes (MMP10, MMP13, and TIMPs) in BAT1 overexpressed cells when compared to BAT1 siRNA cells. Our in vivo studies demonstrated up-regulation of TNF-α, IL-6, and MMP10 in tumors developed from transfected BAT1 shRNA cells when compared to tumors developed from BAT1 cDNA cells. These findings indicate that BAT1 down-regulation modulates TNF-α and IL-6 expression which may lead to the secretion of MMP-10 and inhibition of TIMP2.

Ligand-independent integrin ß1 signaling supports lung adenocarcinoma development.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin ß1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin ß1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin ß1-mediated adhesion to ECM but are dependent on integrin ß1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin ß1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

The laminin-binding integrins regulate nuclear factor κB-dependent epithelial cell polarity and inflammation.

The main laminin-binding integrins α3ß1, α6ß1 and α6ß4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

The laminin binding α3 and α6 integrins cooperate to promote epithelial cell adhesion and growth.

Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3ß1, α6ß1 and α6ß4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3ß1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.

Integrin alpha6 maintains the structural integrity of the kidney collecting system.

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.

Electrophilic adduction of ubiquitin activating enzyme E1 by N,N-diethyldithiocarbamate inhibits ubiquitin activation and is accompanied by striatal injury in the rat.

Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.

Peripheral nerve and brain differ in their capacity to resolve N,N-diethyldithiocarbamate-mediated elevations in copper and oxidative injury.

Previous studies have demonstrated that N,N-diethyldithiocarbamate (DEDC) elevates copper and promotes oxidative stress within the nervous system. However, whether these effects resolve following cessation of exposure or have the potential to persist and result in cumulative injury has not been determined. In this study, an established model for DEDC myelin injury in the rat was used to determine whether copper levels, oxidative stress, and neuromuscular deficits resolve following the cessation of DEDC exposure. Rats were exposed to DEDC for 8 weeks and then either euthanized or maintained for 2, 6 or 12 weeks after cessation of exposure. At each time point copper levels were measured by inductively coupled mass spectrometry to assess the ability of sciatic nerve, brain, spinal cord and liver to eliminate excess copper post-exposure. The protein expression levels of glutathione transferase alpha, heme oxygenase 1 and superoxide dismutase 1 in peripheral nerve and brain were also determined by western blot to assess levels of oxidative stress as a function of post-exposure duration. As an initial assessment of the bioavailability of the excess copper in brain the protein expression levels of copper chaperone for superoxide dismutase 1, and prion protein were determined by western blot as a function of exposure and post-exposure duration. Neuromuscular function in peripheral nerve was evaluated using grip strengths, nerve conduction velocities, and morphologic changes at the light microscope level. The data demonstrated that in peripheral nerve, copper levels and oxidative stress return to control levels within several weeks after cessation of exposure. Neuromuscular function also showed a trend towards pre-exposure values, although the resolution of myelin lesions was more delayed. In contrast, total copper and antioxidant enzyme levels remained significantly elevated in brain for longer post-exposure periods. The persistence of effects observed in brain suggests that the central nervous system is more susceptible to long-term cumulative adverse effects from dithiocarbamates. Additionally, significant changes in expression levels of chaperone for superoxide dismutase 1, and prion protein were observed consistent with at least a portion of the excess copper being bioactive.

N,N-diethyldithiocarbamate promotes oxidative stress prior to myelin structural changes and increases myelin copper content.

Dithiocarbamates are a commercially important class of compounds that can produce peripheral neuropathy in humans and experimental animals. Previous studies have supported a requirement for copper accumulation and enhanced lipid peroxidation in dithiocarbamate-mediated myelinopathy. The study presented here extends previous investigations in two areas. Firstly, although total copper levels have been shown to increase within the nerve it has not been determined whether copper is increased within the myelin compartment, the primary site of lesion development. Therefore, the distribution of copper in sciatic nerve was characterized using synchrotron X-ray fluorescence microscopy to determine whether the neurotoxic dithiocarbamate, N,N-diethyldithiocarbamate, increases copper levels in myelin. Secondly, because lipid peroxidation is an ongoing process in normal nerve and the levels of lipid peroxidation products produced by dithiocarbamate exposure demonstrated an unusual cumulative dose response in previous studies the biological impact of dithiocarbamate-mediated lipid peroxidation was evaluated. Experiments were performed to determine whether dithiocarbamate-mediated lipid peroxidation products elicit an antioxidant response through measuring the protein expression levels of three enzymes, superoxide dismutase 1, heme oxygenase 1, and glutathione transferase alpha, that are linked to the antioxidant response element promoter. To establish the potential of oxidative injury to contribute to myelin injury the temporal relationship of the antioxidant response to myelin injury was determined. Myelin structure in peripheral nerve was assessed using multi-exponential transverse relaxation measurements (MET(2)) as a function of exposure duration, and the temporal relationship of protein expression changes relative to the onset of changes in myelin integrity were determined. Initial assessments were also performed to explore the potential contribution of dithiocarbamate-mediated inhibition of proteasome function and inhibition of cuproenzyme activity to neurotoxicity, and also to assess the potential of dithiocarbamates to promote oxidative stress and injury within the central nervous system. These evaluations were performed using an established model for dithiocarbamate-mediated demyelination in the rat utilizing sciatic nerve, spinal cord and brain samples obtained from rats exposed to N,N-diethyldithiocarbamate (DEDC) by intra-abdominal pumps for periods of 2, 4, and 8 weeks and from non exposed controls. The data supported the ability of DEDC to increase copper within myelin and to enhance oxidative stress prior to structural changes detectable by MET(2). Evidence was also obtained that the excess copper produced by DEDC in the central nervous system is redox active and promotes oxidative injury.

Nitrogen substituent polarity influences dithiocarbamate-mediated lipid oxidation, nerve copper accumulation, and myelin injury.

Dithiocarbamates have a wide spectrum of applications in industry, agriculture, and medicine, with new applications being investigated. Past studies have suggested that the neurotoxicity of some dithiocarbamates may result from copper accumulation, protein oxidative damage, and lipid oxidation. The polarity of a dithiocarbamate's nitrogen substituents influences the lipophilicity of the copper complexes that it generates and thus potentially determines its ability to promote copper accumulation within nerve and induce myelin injury. In the current study, a series of dithiocarbamate-copper complexes differing in their lipophilicity were evaluated for their relative abilities to promote lipid peroxidation determined by malondialdehyde levels generated in an ethyl arachidonate oil-in-water emulsion. In a second component of this study, rats were exposed to either N,N-diethyldithiocarbamate or sarcosine dithiocarbamate; both generated dithiocarbamate-copper complexes that were lipid- and water-soluble, respectively. Following the exposures, brain, tibial nerve, spinal cord, and liver tissue copper levels were measured by inductively coupled mass spectroscopy to assess the relative abilities of these two dithiocarbamates to promote copper accumulation. Peripheral nerve injury was evaluated using grip strengths, nerve conduction velocities, and morphologic changes at the light microscope level. Additionally, the protein expression levels of glutathione transferase alpha and heme-oxygenase-1 in nerve were determined, and the quantity of protein carbonyls was measured to assess levels of oxidative stress and injury. The data provided evidence that dithiocarbamate-copper complexes are redox active and that the ability of dithiocarbamate complexes to promote lipid peroxidation is correlated to the lipophilicity of the complex. Consistent with neurotoxicity requiring the formation of a lipid-soluble copper complex, significant increases in copper accumulation, oxidative stress, and myelin injury were produced by N,N-diethyldithiocarbamate but not by sarcosine dithiocarbamate.

Copper accumulation and lipid oxidation precede inflammation and myelin lesions in N,N-diethyldithiocarbamate peripheral myelinopathy.

Dithiocarbamates have a wide spectrum of applications in industry, agriculture and medicine with new applications being actively investigated. One adverse effect of dithiocarbamates is the neurotoxicity observed in humans and experimental animals. Results from previous studies have suggested that dithiocarbamates elevate copper and promote lipid oxidation within myelin membranes. In the current study, copper levels, lipid oxidation, protein oxidative damage and markers of inflammation were monitored as a function of N,N-diethyldithiocarbamate (DEDC) exposure duration in an established model for DEDC-mediated myelinopathy in the rat. Intra-abdominal administration of DEDC was performed using osmotic pumps for periods of 2, 4, and 8 weeks. Metals in brain, liver and tibial nerve were measured using ICP-MS and lipid oxidation assessed through HPLC measurement of malondialdehyde in tibial nerve, and GC/MS measurement of F(2) isoprostanes in sciatic nerve. Protein oxidative injury of sciatic nerve proteins was evaluated through quantification of 4-hydroxynonenal protein adducts using immunoassay, and inflammation monitored by quantifying levels of IgGs and activated macrophages using immunoassay and immunohistochemistry methods, respectively. Changes in these parameters were then correlated to the onset of structural lesions, determined by light and electron microscopy, to delineate the temporal relationship of copper accumulation and oxidative stress in peripheral nerve to the onset of myelin lesions. The data provide evidence that DEDC mediates lipid oxidation and elevation of total copper in peripheral nerve well before myelin lesions or activated macrophages are evident. This relationship is consistent with copper-mediated oxidative stress contributing to the myelinopathy.

Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity.

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant (P < 0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T(0) plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.

Peripheral nerve protein expression and carbonyl content in N,N-diethlydithiocarbamate myelinopathy.

Human exposure to dithiocarbamates results from their uses as pesticides, in manufacturing, and as pharmaceutical agents. Neurotoxicity is an established hazard of dithiocarbamate exposure and has been observed in both humans and experimental animals. Previous studies have shown that the neurotoxicity of certain dithiocarbamates, including N,N-diethyldithiocarbamate (DEDC), disulfiram, and pyrrolidine dithiocarbamate, can manifest as a primary myelinopathy of peripheral nerves. Because increased levels of copper in peripheral nerves and elevated levels of lipid peroxidation products accompany DEDC-induced lesions, it has been suggested that the disruption of copper homeostasis and increased oxidative stress may contribute to myelin injury. To further assess the biological impact of DEDC-mediated lipid peroxidation in nerves, the changes in protein expression levels resulting from DEDC exposure were determined. In addition, protein carbonyl content in peripheral nerves was also determined as an initial assessment of protein oxidative damage in DEDC neuropathy. Rats were exposed to DEDC by intra-abdominal osmotic pumps for eight weeks and proteins extracted from the sciatic nerves of DEDC-exposed animals and from non-exposed controls. The comparison of protein expression levels using two-dimensional difference gel electrophoresis demonstrated significant changes in 56 spots of which 46 were identified by MALDI-TOF/MS. Among the proteins showing increased expression were three isoforms of glutathione transferase, important for the detoxification of reactive alpha,beta-unsaturated aldehydes generated from lipid peroxidation. The increased expression of one isoform, glutathione transferase pi, was localized to the cytoplasm of Schwann cells using immunohistochemistry. An immunoassay for nerve protein carbonyls demonstrated a significant increase of approximately 2-fold for the proteins isolated from DEDC-exposed rats. These data support the ability of DEDC to promote protein oxidative damage in peripheral nerves and to produce sufficient lipid peroxidation in either myelin or another component of the Schwann cell to elicit a protective cellular response to oxidative stress.

Screening of a peanut (Arachis hypogaea L.) cDNA library to isolate a Bowman-Birk trypsin inhibitor clone.

Peanut crop losses due to insect and pest infestation cost peanut farmers nearly 20% of their annual yields. The conventional use of chemicals to combat this problem is costly and toxic to humans and livestock and leads to the development of resistance by target insects. Transgenic plants expressing a trypsin inhibitor gene in tobacco and cowpea have proven to be efficient for resistance against insects. Therefore, a transgenic peanut overexpressing a trypsin inhibitor gene could be an alternative solution to the use of toxic chemicals. Five Bowman-Birk trypsin inhibitor (BBTI) proteins were previously isolated from peanut. However, to date, neither cDNA nor genomic DNA sequences are available. The objective of this research was to screen a peanut cDNA library to isolate and sequence at least one full-length peanut BBTI cDNA clone. Two heterologous oligonucleotides were constructed on the basis of a garden pea (Pisum sativa) trypsin inhibitor nucleotide sequence and used as probes to screen a peanut lambda gt-11 cDNA library. Two positive and identical cDNA clones were isolated, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length BBTI cDNA of about 243 bp, with a start codon ATG at position +1 and a stop codon TGA at position +243. In the 3' end, two poly adenylation signals (AATAAA) were identified at positions +261 and +269. The isolated cDNA clone encodes a protein of 80 amino acid residues including a leader sequence of 11 amino acids. The deduced amino acid sequence is 100% identical to published sequences of peanut BBTI AI, AII, BI, and BIII and 81% identical to BII.

Genomic organization of peanut allergen gene, Ara h 3.

Type 1 hypersensitivity to peanut proteins is a well-recognized health problem. Several peanut seed storage proteins have been identified as allergens. Ara h 3, a glycinin protein, is one of the important peanut allergens. Although amino acid and cDNA sequences are available for Ara h 3, there is not information at the genomic level. The objectives of this study were to isolate, sequence, and characterize the genomic clone of peanut allergen, Ara h 3. A peanut genomic library was screened, using two [32P] end-labeled oligonucleotide probes designed based on cDNA sequences of Ara h 3 and Ara h 4. Four positives lambda FIX II clones were obtained after four rounds of screenings. Digestion with Sac I resulted in two fragments of 1.5 and 10 kb hybridizing to the probes. Both fragments were subcloned into p-Bluescript vector and sequenced. The Ara h 3 gene spans 3.5 kb and consists of four exons, three introns, 5' and 3' flanking regions. The open reading frame is 2008 bp long and can encode a polypeptide of 538 amino acids residues. Sequences analogous to a TATA-box (TATAAAT), CAAT-box (AGGA), G-box (TCCTACGTGTCC) and several cis-elements were found in the promoter region. In the 3' downstream region, three polyadenylation signals (AATAAA) were identified.

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4.

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.

Structure and organization of the genomic clone of a major peanut allergen gene, Ara h 1.

Peanut is one of the most allergenic foods. It contains multiple seed storage proteins identified as allergens, which are responsible for triggering IgE-mediated allergic reactions. Ara h 1 is a major peanut allergen recognized by over 90% of peanut sensitive population. The objectives of this study were to isolate, sequence, and determine the structure and organization of at least one genomic clone encoding Ara h 1. Two 100 bp oligonucleotides were synthesized and used as probes to screen a peanut genomic library constructed in a Lambda FIX II vector. After three rounds of screening, four putative positive clones were selected and their DNA digested with SacI. A unique 12-13 kb insert fragment was released, confirmed positive by Southern hybridization, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length Ara h 1 gene of 4447 bp with four exons of 721, 176, 81 and 903 bp and three introns of 71, 249 and 74 bp. The deduced amino acid encodes a protein of 626 residues that is identical to the Ara h 1 cDNA clone P41b. Several well characterized elements for promoter strength were found in the promoter region of Ara h 1 and include two TATA-boxes (TATATAAATA and TTATATATAT) at positions -89 and -348, respectively; a CAAT-box (CAAT) at position -133, a GC-box (CGGGACCGGGCCGG GCCTTCGGGCCGGGCCGGGT) at position -475, two G-boxes (TAACACGTACAC and ATGGACGTGAAA) at positions -264 and -1808, respectively; two RY elements (CATGCAC and CATGCAT) at positions -235 and -278, respectively; and other cis-element sequences. In the 3' UTR, a poly-A signal (AATAAA) was found at +2350, two additional stop codons (TAA) at +2303 and +2306, and TTTG/CTA/G motifs. Three introns and a potentially strong promoter could explain the high expression of the Ara h 1 gene. Amino acid sequence comparisons revealed high sequence similarity with other plant vicilins, member of the cupin superfamily.