Heterologous expression and characterization of a novel serine protease from Daphnia magna: A possible role in susceptibility to toxic cyanobacteria

Lange J, Demir F, Huesgen PF, Baumann U, von Elert E, Pichlo C

Aquat. Toxicol. 2018 Dec;205:140-147

PMID: 30384195

Abstract

Mass developments of toxin-producing cyanobacteria are frequently observed in freshwater ecosystems due to eutrophication and global warming. These mass developments can partly be attributed to cyanobacterial toxins, such as protease inhibitors (PIs), which inhibit digestive serine proteases of Daphnia, the major herbivore of phytoplankton and cyanobacteria. To date, mechanisms of this inhibition in the gut of the crustacean Daphnia magna are not known. Here, we characterize a single serine protease, chymotrypsin 448 (CT448), which is present in the gut of the crustacean D. magna. Sequence alignments with human serine proteases revealed that CT448 has a putative N-terminal pro-peptide which is extended compared to the mammalian homologs and within this pro-peptide two N-linked glycosylation motifs were found. CT448 was heterologously expressed in Sf21 insect cells using a baculovirus expression system for optimized protein production and secretion into the medium. The protein was purified via a one-step affinity chromatography, which resulted in a protein yield of 3.45 mg/l medium. The inactive precursor (zymogen) could be activated by tryptic digestion. This is the first example of a recombinant expression of an active crustacean serine protease, which functions in the gut of Daphnia. Proteomic identification of protease cleavage sites (PICS) and hydrolysation of various synthetic substrates showed that CT448 is a chymotrypsin-like elastase. In this study, we confirm that CT448 is a target of cyanobacterial protease inhibitors. Local evolutionary modifications of CT448 might render this proteolytic enzyme less susceptible against cyanobacterial secondary metabolites and might improve the fitness of Daphnia during cyanobacterial blooms.

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