RESUMO
Cysteine proteinases are required for a wide range of physiological processes in all living organisms. In parasitic nematodes, they are particularly crucial for the digestion of host tissues and evasion of host immune responses. Therefore, in general, these are identified as primary targets for the control of parasitic nematodes. Herein, cathepsin S-like cysteine proteinase of Heterodera avenae (Hacp-s) has been cloned and analysed for the first time. The predicted protein is 298 amino acids long and showed significant similarity with cathepsin S of Heterodera glycines (Hgcp-s). The sequence of cathepsin S contains a signal peptide of 30 amino acids which suggests its role in extracellular functions. Multiple sequence alignment revealed the presence of ERFNIN motif and conserved catalytic residues. Three dimensional structure (3D) of Hgcp-s was modelled using homology modelling. In order to illustrate the plausible mode of interaction of cathepsin S (Hgcp-s), docking analysis was performed with E-64 cysteine proteinase inhibitor. Docking studies revealed the hydrogen bonding of E-64 with Gln153, His299 and Gly203 as well as close interaction with catalytic residues Cys159 and Asn320. Expression analysis of Hacp-s using qRT-PCR showed high expression of cathepsin S in pre parasitic J2s and female stages suggesting its significant role in both pre-parasitic and parasitic stages of the nematode life cycle.
Assuntos
Sequência de Aminoácidos/genética , Animais , Catepsinas/química , Catepsinas/genética , Catepsinas/metabolismo , Grão Comestível/parasitologia , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Simulação de Acoplamento Molecular , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Alinhamento de Sequência , Tylenchoidea/genética , Tylenchoidea/metabolismo , Tylenchoidea/patogenicidadeRESUMO
Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 ug and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93 percent of the alleles found on MSRR03 were detected, and 87-88 percent of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population.
Assuntos
Animais , Feminino , Repetições de Microssatélites , Técnicas de Amplificação de Ácido Nucleico , Técnicas de Genotipagem/métodos , Tylenchoidea/genética , DNA de Helmintos/genética , Variação Genética , Nematoides/genética , OviparidadeRESUMO
Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.