Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease / 生物工程学报

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Utilidade da concentração da elastase-1 fecal monoclonal na avaliação da função pancreática nos pacientes com fibrose cística / Use of monoclonal faecal elastase-1 concentration for pancreatic status assessment in cystic fibrosis patients

OBJETIVO: Avaliar a concentração da elastase-1 (EL-1) fecal em pacientes pediátricos com fibrose cística, portadores da mutação ∆F508. MÉTODOS: Estudo transversal com amostras colhidas consecutivamente de 51 pacientes com idade entre 4 meses e 17 anos (média 9,11±4,74), sendo 32 (62,8 por cento) pacientes do sexo masculino. Houve coleta de dados clínico-demográficos e do tipo de mutação. A insuficiência pancreática exócrina foi definida pela atividade da EL-1 fecal < 200 µg/g. A quantificação da EL-1 foi realizada pelo método ELISA monoclonal (ScheBo Biotech AG, Germany). A suplementação pancreática foi utilizada em 46 (90,2 por cento) pacientes. RESULTADOS: Quarenta e um (80,4 por cento) pacientes apresentaram insuficiência pancreática (EL-1 fecal < 100 µg/g), sendo 17 (41,5 por cento) homozigotos, 14 heterozigotos (34,1 por cento) e 10 sem ∆F508 (24,4 por cento). Ao considerar a mutação, houve associação estatisticamente significativa entre os homozigotos e a concentração da EL-1 fecal < 100 µg/g (p = 0,010). Todos os pacientes considerados insuficientes pancreáticos (n = 41) pelo teste utilizavam suplemento pancreático. Dez (19,6 por cento) apresentaram EL-1 fecal > 200 µg/g, e 5/10 (50 por cento) utilizavam enzimas. CONCLUSÕES: A atividade de EL-1 fecal < 100 µg/g, indicativa de insuficiência pancreática, apresentou-se em 17/17 (100 por cento) dos homozigotos, conforme o esperado, sendo menos frequente nos heterozigotos para ∆F508 e nos pacientes com ausência dessa mutação. Não houve relação entre a concentração da EL-1 fecal com idade e sexo dos pacientes. O teste foi padronizado, é de fácil execução e poderá ser utilizado para avaliação da função pancreática dos pacientes com fibrose cística.

OBJECTIVE: To assess the concentration of faecal elastase-1 (EL-1) in pediatric patients with cystic fibrosis with mutation ∆F508. METHODS: Cross-sectional study with samples collected consecutively from 51 patients aged 4 months to 17 years old (mean 9.11±4.74); 32 (62.8 percent) patients were male. Clinical-demographic data were collected, as well as data on the type of mutation. Exocrine pancreatic insufficiency was established by the activity of faecal EL-1 < 200 µg/g. EL-1 was quantified through the monoclonal ELISA method (ScheBo Biotech AG, Germany). Pancreatic supplements were used in 46 (90.2 percent) patients. RESULTS: Forty-one (80.4 percent) patients presented with pancreatic insufficiency (EL-1 fecal < 100 µg/g): 17 (41.5 percent) were homozygous, 14 were heterozygous (34.1 percent) and 10 were non-∆F508 (24.4 percent). Regarding the mutation, there was a statistically significant association of homozygosity with faecal EL-1 concentration < 100 µg/g (p = 0.010). All patients considered to be pancreatic insufficient (n = 41) by the test were using pancreatic supplements. Ten (19.6 percent) presented faecal EL-1 > 200 µg/g, and 5/10 (50 percent) used enzymes. CONCLUSIONS: The activity of faecal EL-1 < 100 µg/g, indicating pancreatic insufficiency, was observed in 17/17 (100 percent) of homozygous patients, as expected, and was less frequent in patients who were heterozygous for ∆F508 and in patients without the mutation. There was no association of faecal EL-1 concentration with age and sex of patients. The test was standardized, is easy to execute, and can be used to assess the pancreatic status of patients with cystic fibrosis.