Kunz Daniele, Oliveira Gabriel B, Uchôa Adriana F, Samuels Richard I, Macedo Maria Lígia R, Silva Carlos P
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, C.P. 476, 88040-900 Florianópolis, SC, Brazil.
Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN, Brazil.
Comp Biochem Physiol B Biochem Mol Biol. 2017 Aug;210:39-47. doi: 10.1016/j.cbpb.2017.06.003. Epub 2017 Jun 17.
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.
蛋白质穿过昆虫肠道上皮的运输过程仍未得到充分理解。有证据表明，豇豆种子（Vigna unguiculata）的主要储存蛋白豆球蛋白在豆象Callosobruchus maculatus的幼虫体内被内化。据报道，这种豆球蛋白与幼虫消化道柱状细胞微绒毛膜中存在的蛋白质相互作用。在本研究中，我们通过体外实验研究了豆象幼虫中豆球蛋白内吞作用所涉及的细胞途径。在体外实验方法中，我们将异硫氰酸荧光素标记的豆球蛋白与分离的中肠整装片在有无内吞作用抑制剂的情况下进行孵育。通过共聚焦显微镜和荧光测量监测标记或未标记球蛋白的命运。我们的结果表明，豆球蛋白的内化是由于受体介导的内吞作用。在此，我们报告了一种微绒毛豆球蛋白结合蛋白的身份，该蛋白是通过在豆球蛋白琼脂糖柱上进行亲和层析纯化得到的。推测的豆球蛋白受体与具有CRAL-TRIO结构域的蛋白质高度同源，特别是Sec14超家族成员α-生育酚转移蛋白。通过使用内吞途径的特异性抑制剂确定了豆球蛋白内化所涉及的精确机制。抑制剂制霉菌素III和制霉菌素显著抑制了豆球蛋白的内吞作用，而氯丙嗪和氧化苯胂对内吞作用的影响要小得多，这表明内吞途径主要由小窝蛋白介导。
