Inorganic polyphosphate inhibits an aspartic protease‐like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Abstract

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus.

New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining

“…14,39 Here, we measured PolyP mobilization by following DAPI-PolyP fluorescence levels in samples containing PolyP extracted from eggs of the insect R. prolixus during the early stages of development. Results showed that a strong mobilization of PolyP takes place during this period (Figure 7C) and were validated after a comparison against the well-established protocol for PolyP quantification based on the PolyP binding to silica powder in suspension followed by enzymatic PolyP hydrolysis into Pi by a recombinant yeast exopolyphosphatase, 14,26,39,53 which showed similar profiles of PolyP mobilization during the early days of development.…”

Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)

“…However, further studies will be necessary to assess a more precise function of the enzyme. In the triatomine R. prolixus, cathepsin D and acid phosphatase are involved in a programmed proteolysis of yolk protein throughout embryogenesis (Nussenzveig et al, 1992;Fialho et al, 2002Fialho et al, , 2005Gomes et al, 2010). Therefore, it is plausible that in D. maxima the same enzymatic system would be operative during atresia.…”

Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets

“…Purified recombinant R. microplus yolk cathepsin (BYC) displayed significant potential as an antitick vaccine (reviewed in [61]). In support, inorganic polyphosphate inhibits the activity of acid phosphatase(s) that activate membrane-bound APD in R. prolixus eggs [52], and was found to arrest yolk mobilization during early embryogenesis [62].…”