SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils.
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.
Allergy and Lung Research Laboratory, University of Aarhus, Aarhus C, DK-8000, Denmark.
H J
HJ
Hoffmann
T
T
Bjerke
M
M
Karawajczyk
R
R
Dahl
M A
MA
Knepper
S
S
Nielsen
D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism*; D018076:DNA, Complementary; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D050683:R-SNARE Proteins; D051381:Rats; D017208:Rats, Wistar; D012260:Ribonucleases
D001798:Blood Proteins; D018076:DNA, Complementary; D047091:Eosinophil Granule Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D012260:Ribonucleases