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Midgut aminopeptidase N isoforms from Ostrinia nubilalis: Activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis |
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| Authors: | Cristina M Crava Yolanda Bel Agata K Jakubowska Juan Ferré Baltasar Escriche |
| Institution: | 1. College of Life Sciences, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing 210095, China;2. Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China;1. Department of Entomology, Louisiana State University Agricultural Center, 404 Life Sciences Building, Baton Rouge, LA 70803, USA;2. Department of Entomology and Nematology, University of Florida, IFAS SWFREC, Immokalee, FL 34142, USA;1. Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, P.O. Box ‘1177’, 3018 Sfax, Tunisia;2. Department of Biological & Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box ‘2713’, Doha, Qatar;1. Department of Physical Chemistry, Faculty of Pharmacy, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), University of Granada, C. U. Cartuja, 18071, Granada, Spain;2. Department of Organic Chemistry, Faculty of Sciences, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), University of Granada, C. U. Fuentenueva s/n, 18071, Granada, Spain;3. Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), University of Granada, Cartuja Campus, 18071, Granada, Spain;1. Department of Entomology and Acarology, “Luiz de Queiroz” College of Agriculture (ESALQ), University of São Paulo (USP), Av. Pádua dias 11, Piracicaba, São Paulo 13418-900, Brazil;2. Department of Entomology, University of Minnesota, 219 Hodson Hall, 1980 Folwell Ave., 55108 Saint Paul, MN, USA;3. Dow AgroSciences LLC, 9330 Zionsville Rd, 46268 Indianapolis, IN, USA |
| Abstract: | Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis. |
| Keywords: | European corn borer Cry receptor Sf21 cells Immunocytochemistry Lepidoptera ALP"} {"#name":"keyword" "$":{"id":"kwrd0050"} "$$":[{"#name":"text" "_":"alkaline phosphatase APN"} {"#name":"keyword" "$":{"id":"kwrd0060"} "$$":[{"#name":"text" "_":"aminopeptidase N BSA"} {"#name":"keyword" "$":{"id":"kwrd0070"} "$$":[{"#name":"text" "_":"bovine serum albumin BBMV"} {"#name":"keyword" "$":{"id":"kwrd0080"} "$$":[{"#name":"text" "_":"brush border membrane vesicles GFP"} {"#name":"keyword" "$":{"id":"kwrd0090"} "$$":[{"#name":"text" "_":"green fluorescent protein GPI"} {"#name":"keyword" "$":{"id":"kwrd0100"} "$$":[{"#name":"text" "_":"glycosylphosphatidylinositol hpi"} {"#name":"keyword" "$":{"id":"kwrd0110"} "$$":[{"#name":"text" "_":"hours post infection O/N"} {"#name":"keyword" "$":{"id":"kwrd0120"} "$$":[{"#name":"text" "_":"overnight LC"} {"#name":"keyword" "$":{"id":"kwrd0130"} "$$":[{"#name":"text" "_":"liquid chromatography MOI"} {"#name":"keyword" "$":{"id":"kwrd0160"} "$$":[{"#name":"text" "_":"multiplicity of infection MS"} {"#name":"keyword" "$":{"id":"kwrd0170"} "$$":[{"#name":"text" "_":"mass spectrometry RT"} {"#name":"keyword" "$":{"id":"kwrd0180"} "$$":[{"#name":"text" "_":"room temperature |
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