Midgut aminopeptidase N isoforms from Ostrinia nubilalis: activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacillus thuringiensis.
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.
Department of Genetics, University of Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain. Electronic address: m.cristina.crava@uv.es.
Cristina M
CM
Crava
Yolanda
Y
Bel
Agata K
AK
Jakubowska
Juan
J
Ferré
Baltasar
B
Escriche
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity; D018826:CD13 Antigens / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism* / Q000633:toxicity; D041981:Gastrointestinal Tract / Q000201:enzymology; D006460:Hemolysin Proteins / Q000378:metabolism* / Q000633:toxicity; D007527:Isoenzymes / Q000378:metabolism; D009036:Moths / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding; D061987:Sf9 Cells; D013379:Substrate Specificity; D018675:Toxicity Tests
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007527:Isoenzymes; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens
ALP; APN; Ala-p-NA; BBMV; BSA; Cry receptor; European corn borer; GFP; GPI; Immunocytochemistry; LC; Lepidoptera; MOI; MS; O/N; RT; Sf21 cells; alanine-p-nitroanilide; alkaline phosphatase; aminopeptidase N; bovine serum albumin; brush border membrane vesicles; glycosylphosphatidylinositol; green fluorescent protein; hours post infection; hpi; leu-p-NA; leucine-p-nitroanilide; liquid chromatography; lys-p-NA; lysine-p-nitroanilide; mass spectrometry; multiplicity of infection; overnight; room temperature