Receptor mediated endocytosis of vicilin in Callosobruchus maculatus (Coleoptera: Chrysomelidae) larval midgut epithelial cells

Abstract

The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin.

Insight into Cellular Uptake and Transcytosis of Peptide Nanoparticles in Spodoptera frugiperda Cells and Isolated Midgut

“…Review of literature suggests that one cell type can endocytose the same nanoparticle using multiple pathways, as nanoparticle formulations are often made up of a group of heterogeneous particles with different sizes, which makes the uptake process more diverse . Clathrin-mediated endocytosis, macropinocytosis, and caveolin-mediated endocytosis have been documented before in insect cells, including Sf9 cells for the uptake of dsRNA, viruses, proteins and lipoproteins. − Nonetheless, this is the first study that demonstrates the implication of these pathways in the internalization of dsRNA and dsRNA associated with peptide nanoparticles.…”

Vicilin from Anadenanthera colubrina Seeds: An alternative tool to combat Callosobruchus maculatus

“…This toxic action of vicilins may explain the prolongation of its life cycle and/or the low rate of emergence of adults after contact with AcV. The cytotoxic effect of C. maculatus vicilins depends on the glycoprotein binding in the microvilli on the surface of the intestinal epithelium, mediated by caveolin, which takes the fragments of vicilin to the hemolymph and mainly deposits them in the adipose body ( Alexandre et al, 2011 , Kunz et al, 2017 ). Miranda et al (2020) , showed a strong link in C. maculatus between the vicilin of V. unguiculata IT81D 1045 (cultivar) and acetylated chitin and that chemical modifications in the amino acid residues tryptophan, lysine and tyrosine decreased this affinity to chitin binding, consequently attenuating the toxicity of vicilin in relation to the insect.…”

Prospects, challenges and current status of RNAi through insect feeding

“…Our review of the current literature suggests the uptake process of dsRNA–nanoparticle complexes has not yet been elucidated in insect cells. The cellular internalization process has been studied only with naked dsRNA (not associated with nanoparticles) . From research done in mammalian cells, we can infer that most likely these complexes are being taken up by cells through endocytosis, although some polymers can be internalized by non‐endocytic pathways through direct translocation of the cell membrane, with its subsequently diffusion into the cell (Fig.…”