Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Molecular cloning and primary structure of cDNA fragment for alpha-latrocrustatoxin from black widow spider venom

A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.     

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein

Venom from Pimpla hypochondriaca, an endoparasitoid of pupae, was size-fractionated using gel filtration chromatography and analysed by SDS-PAGE in the presence and absence of reducing agent. A complex mixture of more than 20 venom constituents was identified which ranged in M(r) between approximately 5 and 100 kDa. Venom from a wide range of size fractions inhibited the motility of larval haemocytes and prevented the formation of cell aggregates when analysed in vitro, indicating that anti-haemocytic activity is mediated by multiple venom components. Sephadex A25 beads injected into the haemocoel of pupae were encapsulated within 24h. This reaction was abolished when the pupae were injected with 30 microg of venom protein, equivalent to one-fifth of a venom sac, 1h prior to implantation of the beads, confirming that venom suppresses encapsulation in pupae. Using random 5' end sequencing of a P. hypochondriaca venom gland cDNA library, we have isolated a cDNA encoding a 25.3 kDa protein containing a signal peptide and having sequence similarity to serine proteases. The N-terminal sequence of six residues from two venom proteins of 28 and 30 kDa was the same and identical to amino acids encoded by the cDNA, confirming that two mass forms of the protein are secreted into the venom sac. The N-terminal sequence of both venom proteins began nine residues towards the C terminus following the predicted signal sequence cleavage site, suggesting that the proteins are proteolytically processed before or during storage in the venom sac. The general applicability of using random 5' sequencing to identify cDNAs encoding secretory products is discussed.     

Nematicidal and Molecular Docking Investigation of Essential Oils from Pogostemon cablin Ecotypes against Meloidogyne incognita

Root-knot nematode, Meloidogyne incognita is one of the most destructive nematodes worldwide. Essential oils (EOs) are being extensively utilized as eco-benign bionematicides, although the precise mechanism of action remains unclear. Pogostemon cablin Benth. is well-known as “Patchouli”. It is native to South East Asia and known for ethno-pharmacological properties. In this study, chemical composition and potential nematicidal effect of EOs hydrodistilled from the leaves of P. cablin grown at three different locations in India were comprehensively investigated to correlate their mechanism of action for target specific binding affinities toward nematode proteins. Aromatic volatile Pogostemon essential oils (PEO) from Northern India (PEO-NI), Southern India (PEO-SI) and North Eastern India (PEO-NEI) were analyzed by Gas Chromatography-Mass Spectrometry (GC/MS) to characterize forty volatile compounds. Maximum thirty-three components were identified in PEO-NEI. Sesquiterpenes were predominant with higher content of α-guaiene (2.3–24.4 %), patchoulol (6.1–32.7 %) and α-bulnesene (5.9–27.1 %). Patchoulol was the major component in PEO-SI (32.7±1.2 %) and PEO-NEI (29.2±1.1 %), while α-guaiene in PEO-NI (24.4±1.2 %). In vitro nematicidal assay revealed significant nematicidal action (LC₅₀ 44.6–87.0 μg mL⁻¹) against juveniles of M. incognita within 24 h exposure. Mortality increases with increasing time to 48 h (LC₅₀ 33.6–71.6 μg mL⁻¹) and 72 h (LC₅₀ 27.7–61.2 μg mL⁻¹). Molecular modelling and in silico studies revealed multi-modal inhibitive action of α-bulnesene (−22 to −13 kJ mol⁻¹) and α-guaiene (−22 to −12 kJ mol⁻¹) against three target proteins namely, acetyl cholinesterase (AChE), odorant response gene-1 (ODR1), odorant response gene-3 (ODR3). Most preferable binding mechanism was observed against AChE due to pi-alkyl, pi-sigma, and hydrophobic interactions. Structure nematicidal activity relationship suggested the presence of hydroxy group for nematicidal activity is nonessential, rather highly depends on synergistic composition of sesquiterpene hydrocarbons.     

Molecular cloning and characterisation of a Rab-binding GDP-dissociation inhibitor from Medicago truncatula.

We have isolated and sequenced the full-length cDNA of a GDP-dissociation inhibitor (GDI) from the model legume Medicago truncatula L. The cDNA (MtGDI) contains an open reading frame of 1335 bp, coding for a protein of 444 amino acids with a calculated molecular mass of 49,785 kDa. The deduced amino acid sequence shows significant homology to other plant GDIs, the highest homology being found to GDI from the legume Cicer arietinum (96% identity). The MtGDI was expressed as a N-terminal FLAG-fusion protein in Escherichia coli BL21 (DE3). Its direct interaction with a small G protein of Rab type from Medicago sativa, MsRab11f, was demonstrated in vitro by co-immunoprecipitation using a peptide-specific antibody raised against MtGDI. The dissociation constant of the MtGDI-MsRab11f complex (4 muM) was determined by a surface plasmon resonance (SPR) assay. Real-time RT-PCR and Western blot analyses suggested that MtGDI is ubiquitously expressed in M. truncatula. High levels of MtGDI mRNA were detected in uninfected roots, leaves and root nodules. In etiolated seedlings and cell cultures, the amount of MtGDI mRNA was much lower. In all tissues tested, the peptide-specific anti-MtGDI antibody detected the expected 50 kDa protein in the total protein extracts. MtGDI was found in the cytosol; however, a significant fraction was associated with the intracellular membranes in seedlings and roots indicating a membrane localisation of the protein. A second immunoreactive band was detected in leaves suggesting that more than one GDI isoform exist in M. truncatula.     

Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.     

Expressed sequence tag analysis generated from a normalized full-length cDNA library of the root-knot nematode (Meloidogyne incognita)

Root-knot nematodes are obligatory sedentary endoparasites that require a plant host to complete their life cycle. To understand the functions of Meloidogyne incognita nematode genes transcribed from eggs and second-stage juveniles (J2), we have constructed a normalized full-length M. incognita cDNA library and analyzed the ESTs using Pendant-Pro Sequence Analysis Suite. The 5,832 M. incognita ESTs formed 3,263 clusters and 2,241 singletons. The sequences ranged from 51 to 1,740 base pairs, and their average size was 699 base pairs. The protein length of M. incognita ESTs ranged from 150 to 299 amino acids. Forty contigs of predicted proteins that were grouped by BLASTP identity values had significant homology to the genes expressed in their organelle structures (cuticle, epidermis, extracellular matrix and muscle). Using the gomerger method of contigs, we could functionally assign GO terms to 2,147 (53.4%) of 4,024 contigs. Following the E.C. numbers method using UniProt database hits, we could functionally classify E.C. numbers to 288 (7.2%) of 4024 contigs. Also, the taxonomy was classified to 2,329 (57.9%) of 4,024 contigs. We could predict transmembrane regions of 4,024 clusters using the TMpred algorithm. Of the 4,024 contigs with transmembrane regions, 1,457 (36.2%) were assigned more than one domain, and 2,567 (63.8%) could not be assigned a transmembrane domain. The M. incognita ESTs will provide a foundation for developing novel target genes for parasite control and contribute to accelerating the research of biologically-related species.     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.