Denolf P, Hendrickx K, Van Damme J, Jansens S, Peferoen M, Degheele D, Van Rie J
Plant Genetic Systems, Gent, Belgium.
Eur J Biochem. 1997 Sep 15;248(3):748-61. doi: 10.1111/j.1432-1033.1997.t01-1-00748.x.
We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stringency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N (P. xylostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.
我们报道了从烟草天蛾中肠上皮细胞纯化、克隆和鉴定一种氨肽酶N的过程，该氨肽酶N能结合苏云金芽孢杆菌的一种杀虫晶体蛋白[ICP] Cry1Ab5。从这种烟草天蛾氨肽酶N获得的序列信息被用于克隆小菜蛾中肠刷状缘膜的氨肽酶N，小菜蛾的一些种群已对Cry1Ab5产生抗性。利用Cry1Ab5基质进行亲和层析，从鳞翅目烟草天蛾幼虫中肠分离出一种120 kDa的糖蛋白。在配体印迹分析中，纯化的120 kDa蛋白能区分鳞翅目特异性的Cry1Ab5和鞘翅目特异性的Cry3A δ-内毒素。120 kDa蛋白的内部氨基酸序列被用于设计简并寡核苷酸。以烟草天蛾中肠cDNA为模板进行巢式PCR，获得了一个与原核和真核氨肽酶N基因具有相似性的DNA片段。该PCR片段被用于筛选烟草天蛾和小菜蛾幼虫中肠的cDNA文库。从烟草天蛾中肠cDNA文库中克隆出一个2973 bp的核苷酸序列。该序列的开放阅读框编码一个含942个氨基酸残基的氨肽酶N（烟草天蛾Apn2），包含两个疏水区域。NH2末端疏水区域对应一个分泌信号序列，COOH末端疏水区域是糖基磷脂酰肌醇（glycosyl-PtdIns）锚定蛋白的典型特征。用小菜蛾apn2探针与小菜蛾中肠cDNA文库进行低严谨度杂交，分离出一个3118 bp的序列，其开放阅读框编码一个含946个氨基酸残基的前原蛋白。这种氨肽酶N（小菜蛾Apn1）与烟草天蛾Apn2的氨基酸同一性为61%，并含有一个用于添加糖基-PtdIns锚定的COOH末端信号肽。烟草天蛾Apn2和小菜蛾Apn1都含有四个半胱氨酸残基，在真核氨肽酶N分子中高度保守。用磷脂酰肌醇特异性磷脂酶C处理表达小菜蛾apn1基因的Sf9细胞，证明小菜蛾Apn1通过糖基-PtdIns锚定附着在昆虫细胞膜上。
