SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils

The SNARE hypothesis, describing a protein assembly-disassembly pathway, wa s recently proposed for the sequential steps of synaptic vesicle docking, a ctivation, and fusion. To determine if SNARE proteins are involved in regul ated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fracti onation, and immunocytochemistry documented that vesicle-associated membran e protein-a (VAMP-S), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR prod ucts amplified from a domain conserved among VAMP isoforms revealed identit y only to VAMP-S but not to VAMP-I or cellubrevin. Functional experiments r evealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophil s, significantly inhibited both IgE receptor- and phorbol ester-mediated ex ocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabil ized eosinophils. Thus, these results strongly suggest a critical role of S NAREs in regulated exocytosis in eosinophils. (C) 2001 Academic Press.