| Part 1Expression and subcellular localization ofSNAP 29 in human mature neutrophilsBACKGROUNDS AND AIMHuman mature neutrophils contain tertiary,specific,and azurophilic granules as well as secretory vesicles.The extracellular release of granular contents from these cells plays a key role in combating infectious microorganisms during inflammatory events.The release of neutrophil granules must be tightly regulated to achieve an efficient antimicrobial response and to prevent uncontrolled release of noxious components that could lead to excessive tissue destruction.Thus,the mechanisms which underlie the release of the cytoplasmic granules come recently to be a focus.Docking and fusion of granules with plasma membrane are necessary for granule release.It is postulated that this process is mediated by a set of proteins present on granule membranes and plasma membrane. In fact,the release of neurotransmitters had been found to be mediated by a set of proteins collectively referred to as soluble N-ethylmaleimide sensitive factor attachment protein receptors(SNAREs),which are present on vesicle membranes and presynaptic membrane.Vesicle membrane-located VAMP 2,and presynaptic membrane-located syntaxin 1A and SNAP 25 bind together and form a SNARE complex, leading to docking and fusion between vesicle membranes and presynaptic membrane during neurotransmitter release.The above-mentioned findings have thrown light on the mechanism by which the exocytosis of neutrophil granules is regulated.Thus,the investigation into SNARE proteins in neutrophils is underway.So far,a number of SNARE proteins have been identified in human mature neutrophils and leukemic cell line HL60,including SNAP-23,-25, Syntaxin-1 A,-3,-4,-6,-7,-9,-11,VAMP-1,-2,-7.It was demonstrated by subcellular localization that SNAP-23,-25 and VAMP-2 were located on the membranes of tertiary and specific granules,VAMP-1 was localized on the membranes of tertiary,specific and azurophilic granules, VAMP-7 was present on the membranes of azurophilic granules,while syntaxin-4,-6 were located on plasma membrane,in resting neutriphils. Furthermore,it was found that SNARE complexes were formed to mediate docking between cytoplasmic granules and plasma membrane, leading to fusion between membranes,thus release of granules.However,the mechanisms underlying exocytosis of the cytoplasmic granules in neutrophils remain to be elucidated.Particularly,little is known about the mechanism underlying exocytosis of azurophilic granules.In this study,we aimed to investigate other SNARE proteins that are probably expressed in human mature neutrophils and their subcellular distribution.As a result,SNAP 29 was identified,for the first time,to be expressed in human neutrophils and present on the membranes of tertiary and specific granules.We also corroborated the expressions and subcellular distributions of VAMP 1,VAMP 2 and syntaxin 4 on consideration that two combinatorial SNARE complexes,SNAP 29/ VAMP 1/syntaxin 4,and SNAP 29/VAMP 2/syntaxin 4,were formed to mediate exocytosis of cytoplasmic granules in neutrophils(see part 2). It has been previously found that two isoforms for each of syntaxin 3 and SNAP 23 genes,namely,syntaxin-3A,-3B and SNAP-23A,-23B, respectively,were expressed in neutrophils.Thus,the probable isoforms for each of VAMP1,VAMP2 and syntaxin4 gene were investigated in this study by cloning and sequencing analyses using novel primers,by which a longer DNA fragments were amplified.METHODS(1) Western-blotting analysis was used to detect the expression of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) protein in human mature neutrophils.(2) Cloning and sequencing were performed to detect the expression of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) gene in human mature neutrophils.(3) Human leukemic cell line HL60,a good cell culture model for human neutrophils,was used to investigate the expressions of SNAP 29 (as well as VAMP 1,VAMP 2 and syntaxin 4) protein/gene in neutrophils.Semi-quantitative RT-PCR analysis was performed using RNA prepared from neutrophil-differentiating HL60 cells to determine the expression level of SNAP29(as well as VAMP 1,VAMP 2 and syntaxin 4) mRNA during differentiation.The above-mentioned experiments further confirmed that SNAP 29 (as well as VAMP 1,VAMP 2 and syntaxin 4) is expressed in neutrophils.(4) Postnuclear protein,membrane protein and cytosol were used for western-blotting assay to determine whether SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) was membrane-bound protein.(5) Distinct subcellular fractions from resting and activated neutrophils were resolved by sucrose density gradient centrifugation,and used for western-blotting analysis to determine subcellular distribution of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) following measuring the activities of specific markers and lysozyme.(6) 15-nm gold-labeled antibodies against markers for distinct granules(The markers for tertiary,specific and azurophilic granules are gelatinase,lactoferrin and myeloperoxidase,respectively) were used to indicate different granule populations,and 10-nm gold-labeled antibody against SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) was used to indicate SNAP 29-positive(as well as VAMP 1-,VAMP 2-and syntaxin 4-positive) granules.Subcellular distribution of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) was determined by electron microscopy on basis of colocalization of SNAP 29(as well as VAMP 1, VAMP 2 and syntaxin 4) with distinct granule markers.RESULTS(1) It was demonstrated by western-blotting analysis that SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) protein was expressed in human mature neutrophils.(2) It was also shown by cloning and sequencing analysis that SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) gene was expressed in human mature neutrophils.Following gel electrophoresis using PCR products of syntaxin 4,two bands,corresponding to 1278 and 1414 bp,respectively,were shown.It was demonstrated by sequencing analysis that the nucleotide sequence of 1278-bp band was identical to that included in Genbank,while the nucleotide sequence of 1414-bp band contained an intron of 136 nucleotides.No isoforms for both VAMP 1 and VAMP 2 genes were detected.(3) The protein/gene of SNAP29(as well as VAMP 1,VAMP 2 and syntaxin 4) were expressed in HL60 cells.It was demonstrated by semi-quantitative RT-PCR assay that the mRNA level of SNAP29(as well as VAMP 1,VAMP 2 and syntaxin 4) was up-regulated during HL60 differentiation towards neutrophil,which was similar to that in mature neutrophils following DMSO treatment for 4 days,further confirming that SNAP29(as well as VAMP 1,VAMP 2 and syntaxin 4) protein/gene were expressed in human mature neutrophils,and also suggesting that SNAP29 protein is probably necessary for physiological function of mature neutrophils.(4) SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) was membrane-bound protein.(5)①Determining the activities of specific markers for distinct subcellular fractions prepared by resting neutrophils,we found that the components of distinct fractions were as follows:fraction 1,cytosol; fractions 2 and 3,plasma membrane;fractions 4 and 5,tertiary granules; fractions 5 and 6,specific granules;fractions 7 and 8,azurophilic granules.②Measuring the activities of lysozyme for distinct subcellular fractions prepared by resting neutrophils,we found that lysozyme was dominantly present in fractions 4,5,6,7 and 8,namely,tertiary,specific and azurophilic granules.Following neutrophils activation with PMA,the activities of lysozyme were significantly decreased in fractions 4,5 and 6, except for in fraction 8,which remained to be unchanged.These findings demonstrated that tertiary and specific granules,but not azurophilic granules,had translocated to plasma membrane in PMA-activated neutrophils.③Following western-blotting analysis using subcellular fractions prepared by resting neutrophils,we found that SNAP 29 was present in fractions 4,5 and 6,namely,on the membranes of tertiary and specific granules;VAMP 1 was localized in fractions 4,5,6,7,and 8,namely,on the membranes of tertiary,specific and azurophilic granules;VAMP 2 was located in fractions 4,5,and 6,namely,on membranes of tertiary and specific granules;while syntaxin 4 was present in fractions 2 and 3, namely,on plasma membrane.Upon neutrophils activation by PMA,we found that SNAP 29 translocated to fractions 2 and 3(plasma membrane) from fractions 4,5, and 6(membranes of TG and SG);VAMP 1 translocated to plasma membrane from fractions 4,5,and 6(membranes of TG and SG),while azurophilic-granule-located(fractions 7 and 8) VAMP1 remained unchanged;VAMP 2 also translocated to plasma membrane from fractions 4,5,and 6(membranes of TG and SG).These findings were in high correlation with the findings that tertiary and specific granules,but not azurophilic granules,translocated to plasma membrane upon neutrophils activation with PMA,further confirming the subcellular localization of SNAP 29,VAMP 1,VAMP 2 and syntaxin4.(6) Following double-labeling cytoplasmic granules with antibodies against SNARE proteins and against markers of different cytoplasmic granules,cryosections were analyzed by electron microscopy.We found that SNAP 29 was present on the membranes of tertiary and specific granules,VAMP 1 was localized on the membranes of tertiary,specific as well as azurophilic granules,VAMP 2 was located on membranes of tertiary and specific granules,while syntaxin 4 was present on plasma membrane,in resting neutrophils.CONCLUSIONS(1) The protein of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) is expressed in human mature neutrophils.(2) The gene of SNAP 29(as well as VAMP 1,VAMP 2 and syntaxin 4) is expressed in human mature neutrophils.In addition to syntaxin 4 gene of which the nucleotide sequence is identical to that included in Genbank,neutrophils express an isoform of syntaxin 4 gene,in which an intron of 136 nucleotides is inserted.No isoforms for both VAMP 1 and VAMP 2 gene were detected.(3) SNAP 29 is present on the membranes of tertiary and specific granules;VAMP 1 is located on the membranes of tertiary,specific and azurophilic granules,VAMP2 is located on the membranes of tertiary and specific granules,while syntaxin 4 is present on plasma membrane. Part 2Two Combinatorial SNARE complexes,SNAP29/VAMP1/Syntaxin4 and SNAP29/VAMP2/Syntaxin4,regulate the exocytosisof tertiary and specific granules in human neutrophilsBACKGROUNDS AND AIMHuman neutrophils contain tertiary,specific,azurophilic granules and secretory vesicles,which differ distinctly in propensity for exocytosis. Tertiary and specific granules are readily to be mobilized and exocytosed upon neutrophils activation,regulating the functions of adhesion to endothelium,diapedesis,and killing of microorganisms.Azurophilic granules,containing a large amount of lytic enzymes and proteins with bactericidal activity,are sluggishly mobilized upon neutrophils activation, and their exocytoses occur in process of phagocytosis.The fact that distinct granule populations in neutrophils differ in propensity for exocytosis indicates that secretion of neutrophil granules must be tightly regulated.The investigation into the mechanism underlying neurotransmitter release has led to SNARE(Soluble N-ethylmaleimide sensitive factor attachment protein receptor) hypothesis.According to this hypothesis, vesicle-associated membrane protein 2(VAMP 2),syntaxin 1A and 25 kDa synaptosome-associated protein(SNAP 25) share a homologous SNARE motif of～60 aa,which mediates the association of these SNARE proteins into a core complex(SNARE complex) composed of a tightly packed parallel four-helical bundle.VAMP 2 and syntaxin 1A contribute one a-helix each and SNAP 25 contributes two a-helices,leading these SNARE proteins bind tightly together and mediate docking and fusion between vesicle membrane and presynaptic membrane.All known SNARE motifs fall into two major subfamilies that contain either a conserved glutamine(Q-SNARE) or arginine(R-SNARE) in the middle of the four-helical bundle.Neuronal SNARE complex contains three Q-SNARE motifs(one contributed by syntaxin 1A and two by SNAP 25) and one R-SNARE motif(contributed by VAMP 2),namely,3 (Q-SNARE)/1(R-SNARE) type.SNARE proteins also underlie the mechanism of granular exocytosis in neutrophils,and 3(Q-SNARE)/1(R-SNARE) seems to be typical configuration for all SNARE complexes mediating docking and fusion between vesicle membrane and target membrane.In accordance with SNARE hypothesis,the SNARE proteins on granule membranes bind only to their homologous SNARE proteins on plasma membrane,forming 3(Q-SNARE)/1(R-SNARE) type,to mediate docking and fusion between granule membranes and plasma membrane.Difference in varieties and density of SNARE proteins on granule membrane could account for their respective propensities for exocytosis in neutrophils.SNAP 29 has been found to be expressed in human neutrophils,and is located on the membranes of tertiary and specific granules in the latest study(see part 1).A large number of other SNARE proteins,such as VAMP 1(located on the membranes of tertiary,specific and azurophilic granules),VAMP 2(present on the membranes of tertiary and specific granules),and syntaxin 4(localized on plasma membrane),have also been found to be expressed in neutrophils.This study aimed to further investigate whether SNAP29 is involved in exocytosis of cytoplasmic granules in neutrophils,as well as to find its homologous SNARE proteins with which the SNARE complexes are formed to mediate exocytosis of cytoplasmic granules.METHODS(1) Electropermeabilized neutrophils were prepared,and incubated with antibody against SNAP 29.Subsequently,the electropermeabilized neutrophils were activated with Ca2++ GTP-γ-S.Following fixation with paraformaldehyde,the activated electro-permeabilized neutrophils were incubated with antibody against either CD66b(marker for tertiary and specific granules) or CD63(marker for azurophilic granules),then incubated with FITC-conjugated anti-mouse immunoglobulin.Inhibitory effect of anti-SNAP 29 Ab on activation-evoked cell surface expression of either CD66b or CD63 was measured by flow cytometry to investigate whether SNAP 29 was involved in exocytosis of tertiary,specific and azurophilic granules.Likewise,the antibodies against VAMP 1,VAMP 2,and syntaxin 4 were also used to investigate whether VAMP 1,VAMP 2,and syntaxin 4 were involved in exocytosis of tertiary,specific and azurophilic granules, respectively.(2) The antibody against SNAP 29 was also used in combination with other antibodies(such as anti-VAMP 1,-VAMP 2,or-syntaxin 4) to incubate electropermeabilized neutrophils,and synergistically inhibitory effect on activation-evoked expression of either CD66b or CD63 on cell surface was measured to investigate the probable SNARE complexes with SNAP 29 during exocytosis of cytoplasmic granules.(3) Coimmunoprecipitation assay was also used to investigate the probable SNARE complexes involved in exocytosis.Homogenate was prepared from PMA-or fMLP-activated neutrophils,from which the SNARE complexes with SNAP 29 were immunoprecipitated by antibody against SNAP 29 precoupled to protein A-Sepharose.The immunoprecipitates were boiled at 95℃and subjected to polyacrylamide gel electrophoresis,followed by western-blotting analysis using antibodies,such as anti-VAMP 1,-VAMP 2,and-syntaxin 4,to detect the homologous SNARE proteins of SNAP 29.Homogenate prepared from resting neutrophils was used as control.Likewise,the homologous SNARE proteins for each of VAMP 1, VAMP 2,and syntaxin 4 were also investigated.RESULTS(1) Both antibodies,anti-SNAP 29 and-VAMP 2,inhibited activation-evoked cell surface expression of CD66b in dose-dependent manner,but had no effect on the expression of CD63.These findings indicated that both SNAP29 and VAMP2 mediated exocytosis of tertiary and specific granules.In contrast,neither SNAP 29 nor VAMP 2 mediated exocytosis of azurophilic granules.Both antibodies,anti-VAMP1 and-syntaxin 4,inhibited activation- evoked cell surface expression of both CD66b and CD63 in dose-dependent manner,indicating that both VAMP 1 and syntaxin 4 mediated exocytosis of tertiary,specific and azurophilic granules.(2) In combination with anti-SNAP 29 and anti-VAMP 1 Abs, synergistically inhibitory effect on activation-evoked cell surface expression of CD66b was shown.In contrast,their inhibitory effect on the expression of CD63 was equivalent to that of anti-VAMP 1 Ab used alone.These findings suggested that a SNARE complex could be formed by SNAP 29 and VAMP 1 to mediate the exocytosis of tertiary and specific granules.Additionally,VAMP 1,but not SNAP 29 regulated exocytosis of azurophilic granules.In combination with anti-SNAP 29 and anti-VAMP 2 Abs, synergistically inhibitory effect on activation-evoked cell surface expression of CD66b was demonstrated.In contrast,no effect on activation-evoked cell surface expression of CD63 was shown.These findings suggested that one SNARE complex could be formed between SNAP 29 and VAMP 2 to mediate the exocytosis of tertiary and specific granules.Neither SNAP 29 nor VAMP 2 regulated exocytosis of azurophilic granules.In combination with anti-SNAP 29 and anti-syntaxin 4 Abs, synergistically inhibitory effect on activation-evoked cell surface expression of CD66b was shown,while their inhibitory effect on expression of CD63 was equal to that of anti-syntaxin 4 Ab used alone. The above-mentioned findings suggested that a SNARE complex could be formed by SNAP 29 and syntaxin 4 to mediate the exocytosis of tertiary and specific granules.Moreover,syntaxin 4 regulated exocytosis of azurophilic granules.In combination with anti-syntaxin 4 and anti-VAMP 1 Abs, synergistically inhibitory effect on activation-evoked cell surface expression of both CD66b and CD63 was demonstrated,suggesting that a SNARE complex could be formed by syntaxin 4 and VAMP 1 to mediate the exocytosis of tertiary,specific,and azurophilic granules.In combination with anti-syntaxin 4 and anti-VAMP 2 Abs, synergistically inhibitory effect on activation-evoked cell surface expression of CD66b was shown.In contast,their inhibitory effect on expression of CD63 was equivalent to that of anti-syntaxin 4 Ab used alone.These findings suggested that a SNARE complex could be formed by VAMP 2 and syntaxin 4 to mediate the exocytosis of tertiary and specific granules.In addition,syntaxin 4 regulated exocytosis of azurophilic granules.Together,two SNARE complexes,SNAP 29/VAMP 1/syntaxin 4 and SNAP 29/VAMP 2/syntaxin4,could be formed to mediate exocytosis of tertiary and azurophilic granules,and another SNARE complex could be also formed by VAMP 1 and syntaxin 4 to modulate the release of azurophilic granules.(3) It was demonstrated by coimmunoprecipitation assay that the antibody against SNAP 29 immunoprecipitated each of VAMP 1,VAMP 2 and syntaxin 4 in homogenate prepared by PMA-activated neutrophils, suggesting that SNARE complexes could be formed by SNAP 29 and each of VAMP 1,VAMP 2 and syntaxin 4 to mediate the exocytosis of tertiary and specific granules.The antibody against VAMP 2 immunoprecipitated either SNAP 29 or syntaxin 4,but not VAMP 1,in homogenate prepared by PMA-activated neutrophils,indicating that SNARE complexes could be formed by VAMP 2 and either SNAP 29 or syntaxin 4 to mediate the exocytosis of tertiary and specific granules.Notably,VAMP 2 didn't bind to VAMP 1.The antibody against syntaxin 4 immunoprecipitated each of SNAP 29,VAMP 1,and VAMP 2 in homogenate prepared by PMA-activated neutrophils,demonstrating that SNARE complexes could be formed by syntaxin 4 and each of SNAP 29,VAMP 1,and VAMP 2 to mediate exocytosis of tertiary and specific granules.The findings that anti-syntaxin 4 immunoprecipitated much more VAMP 1 from the homogenate prepared by fMLP-activated neutrophils than that from the homogenate prepared by PMA-activated neutrophils indicated that syntaxin 4 bound to tertiary-granule-/specific-granule-located VAMP 1,mediating exocytosis of these granules,as well as to azurophilic-granules-located VAMP 1,mediating exocytosis of azurophilic granules.Together,two combinatorial SNARE complexes,SNAP 29/VAMP 1/syntaxin 4 and SNAP 29/VAMP 2/syntaxin 4,could be formed to mediate exocytosis of tertiary and specific granules,and another SNARE complex,VAMP 1/syntaxin 4 could be also formed to mediate exocytosis of azurophilic.VAMP 1 and VAMP 2 didn't coexist in one SNARE complex.CONCLUSIONS(1) Each of SNAP 29,VAMP 1,VAMP 2 and syntaxin 4 mediates exocytosis of cytoplasmic granules.(2) SNAP 29 as well as VAMP 2 regulates exocytosis of tertiary and specific granules;Both VAMP 1 and syntaxin 4 regulate exocytosis of tertiary,specific and azurophilic granules.(3) Two combinatorial SNARE complexes,SNAP 29/VAMP 1/ syntaxin 4 and SNAP 29/VAMP 2/syntaxin 4,are formed to mediate exocytosis of tertiary and specific granules,and another SNARE complex, VAMP 1/syntaxin 4 is also formed to mediate exocytosis of azurophilic. VAMP 1 and VAMP 2 don't coexist in one SNARE complex. |
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