title: Biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode Steinernema carpocapsae.
abstract: A trypsin-like serine protease was purified by gel filtration and anion-exchange chromatography from the excretory-secretory products of parasitic phase Steinernema carpocapsae. The purified protease exhibited a molecular mass of about 29 kDa by SDS-PAGE and displayed a pI of 6.3. This protease exhibited high activity with trypsin-specific substrate N-Ben-Phe-Val-Arg-p-nitroanilide and was highly sensitive to aprotinin and benzamidine. The purified trypsin protease digested the chromogenic substrate N-Ben-Phe-Val-Arg-p-nitroanilide with K(m), V(max) and k(cat) values of 594.2 mum, 0.496 mum/min and 22.8/s, respectively. The optimal pH and temperature for protease activity were 9 and 30 degrees C, respectively. Internal amino acid sequencing yielded 150 amino acids and these were homologous to other trypsin sequences. In vitro investigation was carried out to monitor prophenoloxidase suppression in Galleria mellonella by the purified protease; about 38.9-52.6% suppression of prophenoloxidase was observed. The purified protease affected insect haemocyte spreading, causing cells to become spherical or round. Protease-treated actin filaments were highly disorganized in haemocytes. In vitro, G. mellonella haemocytes recognized infective juveniles of Heterorhabditis bacteriophora; however, S. carpocapsae and Steinernema glaseri were not recognized. We provide experimental evidence that the purified trypsin has the potential to alter host haemocytes, actin filaments and to inhibit host haemolymph melanization.
authors.affiliation: CIRN and Department of Biology, University of Azores, Ponta Delgada, Portugal. natesanbala@uac.pt
authors.forename: N
authors.initials: N
authors.lastname: Balasubramanian
authors.forename: D
authors.initials: D
authors.lastname: Toubarro
authors.forename: N
authors.initials: N
authors.lastname: Simões
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001550:Benzamidines / Q000494:pharmacology; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors; D048430:Cell Shape / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors; D015801:Helminth Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D006434:Hemocytes / Q000187:drug effects / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007108:Immune Tolerance; D007526:Isoelectric Point; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D017168:Rhabditida / Q000201:enzymology*; D017172:Rhabditoidea / Q000276:immunology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D057057:Serine Proteases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D001550:Benzamidines; D004792:Enzyme Precursors; D015801:Helminth Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; C006164:S 2160; D007611:Aprotinin; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases; C032157:benzamidine