A megalin-like receptor is involved in protein endocytosis by Bombyx mori midgut cells in culture

Protein absorption by the insect midgut currently attracts increasing research efforts, in part fostered by the need to develop efficient strategies for oral delivery of bioinsecticides targeting haemocoelic receptors. Gut absorption of undegraded proteins has been unequivocally demonstrated in a number of insect species in vivo but only recently the mechanism involved has been clarified: we have proved that the isolated midgut of Bombyx mori larvae performs the transepithelial translocation of the model protein albumin by transcytosis. Single columnar cells in culture, isolated from the larval midgut, represent the best tool to identify the mechanism involved in protein endocytosis, the fist step of the transcytotic process. Mature columnar cells were obtained from the proliferation and differentiation of stem cells detached from B. mori midgut epithelium and maintained in culture. We investigated FITC-albumin internalisation by confocal laser scanning microscopy. The protein uptake was time-dependent and strongly reduced by low temperatures and by metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by native albumin in a concentration-dependent manner. These data prove that albumin uptake is an active process and indicate that a receptor mediates the internalisation of the protein. The internalisation takes place by clathrin-mediated endocytosis, since two specific inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. The integrity of the cytoskeletal organisation is essential for the correct functioning of the endocytic machinery because preincubations with nocodazole or cytochalasin D induced a significant reduction of FITC-albumin uptake. RT-PCR analysis and colocalisation experiments indicated that the receptor involved is a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of a variety of molecules, albumin included, in many mammalian epithelial cells. Although the specific role of the putative megalin homologue in protein absorption by the midgut of lepidopteran larvae needs to be further clarified, our study indicates that this receptor is highly conserved during evolution.