DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes. (February 2018)

• Record Type: Journal Article
• Authors: Leyria, Jimena; Fruttero, Leonardo L.; Ligabue-Braun, Rodrigo; Defferrari, Marina S.; Arrese, Estela L.; Soulages, José L.; Settembrini, Beatriz P.; Carlini, Celia R.; Canavoso, Lilián E.
• Is Part Of: Journal of insect physiology. Volume 105(2018:Oct.)
• Page Start: 28
• Page End: 39
• Publication Date: 2018-02
• Subjects: β-ATPase β-chain of ATP synthase -- OCT Optimal Cutting Temperature -- BSA Bovine serum albumin -- DMP dimethylpimelimidate -- FBS fetal bovine serum -- PMSF phenylmethyl-sulfonyl fluoride -- TLCK N-α-p-tosyl-L-lysine chloromethyl ketone -- PBS phosphate buffered saline -- NaPB sodium phosphate buffer -- SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis -- MS/MS tandem mass spectrometry -- RT-PCR Reverse Transcription – Polymerase chain reaction -- TBS Tris buffered saline -- anti-Lp anti-lipophorin antibody -- anti-Vt anti-vitellin antibody -- FITC fluorescein isothiocyanate -- LpR lipophorin receptor -- LDLR low-density lipoprotein receptor -- M6Pr mannose-6-phosphate receptor -- LRP1 LDL receptor-related protein-1 -- Co-IP co-immunoprecipitation -- Lp lipophorin -- Vg vitellogenin; Triatominae -- Cathepsin D -- Lipophorin -- Vitellogenesis -- Oocyte; Insects -- Physiology -- Periodicals; Insectes -- Physiologie -- Périodiques; Insects -- Physiology; Periodicals; 571.157
• DOI: 10.1016/j.jinsphys.2018.01.002
• Languages: English
• ISSNs: 0022-1910

Abstract

Graphical abstract: Highlights: DmCatD was purified from eggs and its identity was confirmed by mass spectrometry. DmCatD has 86% identity with cathepsin D from the triatomine Triatoma infestans. DmCatD has features that suggest distancing from "classical" cathepsin D enzymes. Different approaches support the interaction between DmCatD and lipophorin. Lipophorin carries DmCatD, facilitating its internalization in the oocytes. Abstract: DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from "classical" cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.