Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis.
To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca(++) and GTP-gamma-S-induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti-syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE- t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.
Martín-Martín
B
B
Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain.
Nabokina
S M
SM
Blasi
J
J
Lazo
P A
PA
Mollinedo
F
F
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D000951:Antigens, Neoplasm; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology / Q000502:physiology*; D015815:Cell Adhesion Molecules; D015550:Cell Degranulation / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000737:chemistry; D060149:Tetraspanin 30
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000951:Antigens, Neoplasm; C554586:CD63 protein, human; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D012333:RNA, Messenger; C100493:SNAP23 protein, human; D060149:Tetraspanin 30; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium