关键词: 褐飞虱； 氨肽酶N, 中肠； S2细胞； 酶活性； 膜结合蛋白

Abstract: 【Aim】 Aminopeptidases N (APNs) are a class of important proteases in the digestive system in insects. This study aims to verify the expression of two apn genes (nlapn1 and nlapn4) with high transcription level in the midgut epithelium of the brown planthopper, Nilaparvata lugens, and to identify and analyze the characteristics of their proteins. 【Methods】 Phylogenetic analysis of both NLAPN1 and NLAPN4 of N. lugens was conducted by maximum likelihood method. Western blotting and LC-ESI-MS/MS were respectively conducted to localize and identify NLAPN1 and NLAPN4 in the midgut brush border membrane vesicles (BBMVs) of N. lugens. NLAPN1 and NLAPN4 were respectively expressed in Drosophila S2 cells. Lacolization of both NLAPN1 and NLAPN4 in S2 cells was analyzed by Western blot and immunofluorescence. The enzymatic activities of NLAPN1 and NLAPN4 were determined through enzyme assays using leucine-p-NA, Ala-p-NA, Met-p-NA and Lys-p-NA as the substrate, respectively. 【Results】 Phylogenetic tree analysis showed that both NLAPN1 and NLAPN4 of N. lugens were clustered together with the APN proteins highly expressed in the midgut of other hemipteran insects. NLAPN1 and NLAPN4 with the molecular weight of ~160 kD were identified in the midgut BBMV of N. lugens by Western blot and LC-ESI-MS/MS. Western blot and immunofluorescence analysis showed that NLAPN1 and NLAPN4 were expressed on the cytomembrane of transfected S2 cells, while that of NLAPN4lackG without glycosylphosphatidylinositol (GPI) anchor site at the C-terminal end was distributed in the cytoplasm. Enzyme assay results revealed that both NLAPN1 and NLAPN4 showed certain enzymatic activity using Ala-p-NA and Lys-p-NA as the substrate, while using leucine-p-NA as the substrate, NLAPN1 showed extremely high enzymatic activity (＞60 U/mg). 【Conclusion】 NLAPN1 and NLAPN4 are both highly expressed GPI-anchored membrane-bound aminopeptidases N located on the epithelial membrane of the midgut of N. lugens. Both NLAPN1 and NLAPN4 show similar structure and enzymatic characteristics to the previous identified membrane-bound APN proteins in lepidotperans, coleopterans and dipterans. Physiological and biochemical functions of membrane-bound APNs in the midgut of N. lugens and their interaction with exogenous pathogenic microorganisms need to be further studied.

Key words: Nilaparvata lugens, aminopeptidase N, midgut, S2 cell, enzymatic activity, membrane-bound protein