Knight Peter J K, Carroll Joe, Ellar David J
Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4.
Insect Biochem Mol Biol. 2004 Jan;34(1):101-12. doi: 10.1016/j.ibmb.2003.09.007.
The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to 120 kDA APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analyzed the various glycans on the 120 kDA APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of 4 possible N-glycosylation sites are occupied with fucosylated paucimannose [Man(2-3)(Fuc(1-2)GlcNAc(2)-peptide] type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal "stalk" region of the protein. It is likely that 5-6 of the 13 sites are occupied, probably with simple [GalNAc-peptide] type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc-rich, is the most likely binding site for Cry1Ac.
苏云金芽孢杆菌Cry1Ac毒素特异性结合烟草天蛾中一种120 kDa的氨肽酶N（APN）受体。这种结合相互作用由N-乙酰半乳糖胺（GalNAc）介导，推测它作为一种未明确聚糖结构的一部分共价连接到APN上。在此，我们详细介绍一种简单、快速且特异的化学去糖基化技术，该技术适用于固定在蛋白质免疫印迹上的糖蛋白。我们使用该技术直接且明确地证明，与120 kDa APN相连的碳水化合物实际上是Cry1Ac毒素的结合表位。该技术普遍适用于所有假定的Cry毒素/受体组合。我们使用碳水化合物组成分析和凝集素结合分析了120 kDa APN上的各种聚糖。数据表明，在平均APN分子中，4个可能的N-糖基化位点中有2个被岩藻糖基化的寡甘露糖[Man(2-3)(Fuc(1-2)GlcNAc(2)-肽]型N-聚糖占据。此外，我们鉴定出13个可能的O-糖基化位点，其中10个位于该蛋白质富含苏氨酸/脯氨酸的C末端“柄”区域。13个位点中可能有5 - 6个被占据，可能是简单的[GalNAc-肽]型O-聚糖。这个富含GalNAc的O-糖基化C末端柄最有可能是Cry1Ac的结合位点。
