Expression of eosinophil target SNAREs as potential cognate receptors for vesicle-associated membrane protein-2 in exocytosis.
BACKGROUND
Exocytosis of eosinophil granule-derived mediators is thought to be an important effector response contributing to allergic inflammation. Secretion from many cell types has been shown to be dependent on the formation of a docking complex composed of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on the vesicle (v-SNAREs) and the target membrane (t-SNAREs). The SNARE isoforms VAMP-2, SNAP-23, and syntaxin-4 have been described in secretory processes in myeloid cells. Previously, we have demonstrated that the v-SNARE VAMP-2 is a candidate v-SNARE involved in eosinophil exocytosis and is localized to a pool of RANTES-positive vesicles that translocate to the cell periphery after IFN-gamma-induced degranulation.


OBJECTIVE
We sought to determine whether eosinophils express the t-SNARE isoforms SNAP-23 and syntaxin-4 as potential binding targets for VAMP-2 during exocytosis.


METHODS
Human peripheral blood eosinophils (>97%) from atopic subjects were subjected to RT-PCR and sequence analysis by using specific primers for SNAP-23 and syntaxin-4. Protein expression and localization was determined by means of Western blot analysis of eosinophil subcellular fractions and confirmed with confocal laser scanning microscopy.


RESULTS
Nucleotide sequences obtained from PCR products exhibited nearly identical (>95%) homology with reported sequences for human SNAP-23 and syntaxin-4. Both SNAP-23 and syntaxin-4 were present in plasma membranes, with some staining in endoplasmic reticulum and Golgi membranes. Negligible expression was detected in crystalloid and small secretory granules.


CONCLUSIONS
The plasma membrane-associated t-SNAREs SNAP-23 and syntaxin-4 are expressed in human eosinophils and are likely candidates for association with VAMP-2 during docking, which is followed by exocytosis. These findings support a role for SNARE molecules in eosinophil mediator release.
Logan
Michael R
MR
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Canada.
Lacy
Paige
P
Bablitz
Ben
B
Moqbel
Redwan
R
D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004804:Eosinophils / Q000276:immunology* / Q000378:metabolism; D005089:Exocytosis; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D013347:Subcellular Fractions
D002352:Carrier Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human