Hoffmann H J, Bjerke T, Karawajczyk M, Dahl R, Knepper M A, Nielsen S
Allergy and Lung Research Laboratory, University of Aarhus, Aarhus C, DK-8000, Denmark.
Biochem Biophys Res Commun. 2001 Mar 23;282(1):194-9. doi: 10.1006/bbrc.2001.4499.
The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation, and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.
最近提出的SNARE假说描述了一种蛋白质组装-拆卸途径，用于解释突触小泡对接、激活和融合的连续步骤。为了确定SNARE蛋白是否参与嗜酸性粒细胞的调节性胞吐作用，研究人员检测了人血嗜酸性粒细胞中SNARE蛋白的存在及其功能作用。免疫印迹、亚细胞分级分离和免疫细胞化学结果表明，囊泡相关膜蛋白2（VAMP-2），一种囊泡-SNARE，在人嗜酸性粒细胞中表达。同时也检测到了Syntaxin 4和SNAP-25。对从VAMP异构体中保守结构域扩增得到的克隆RT-PCR产物进行测序，结果显示仅与VAMP-2相同，而与VAMP-1或细胞ubrevin不同。功能实验表明，破伤风毒素预处理可切割嗜酸性粒细胞中的VAMP-2，显著抑制来自链球菌溶血素-O通透化嗜酸性粒细胞的IgE受体和佛波酯介导的嗜酸性粒细胞阳离子蛋白（ECP）胞吐作用。因此，这些结果强烈表明SNARE蛋白在嗜酸性粒细胞的调节性胞吐作用中起关键作用。
