IDENTIFICATION AND LOCALISATION OF COMPONENTS OF THE SNARE COMPLEX IN GRANULOCYTES

4.1 Introduction

It is clear that all vesicular transport steps require close apposition between

membranes before fusion can occur. The proteins o f the SNARE complex partially fulfil

this function (Sollner et al, 1993a; Sudhof, 1995). The SNARE complex is composed o f a vesicular v-SNARE protein VAMP and two t-SNAREs localised to the target organelle,

namely SNAP25 and syntaxin (Sollner et a l, 1993a). These proteins form a ternary complex through their coiled-coil domains and the formation o f this complex with the

hydrolysis o f ATP by the cytosolic NSF protein is thought to provide the energy that

drives the fusion process (Sollner et a l, 1993b). The cytosolic components o f the complex, NSF and a-SN A P also have the ability to dissociate the SNARE complex,

allowing their recycling after fusion (Sollner et a l, 1993b). Throughout the secretory pathway, almost every vesicular transport step is carried out by a distinct SNARE complex and the SNAREs that mediate a given transport step are conserved from yeast to

mammals (Ferro-Novick and Jahn, 1994). Among the t-SNARES o f the SNAP25/23/29

family, SNAP23 is ubiquitously expressed and is involved in the exocytic pathway o f

non-neuronal cells (Sadoul et a l, 1997; Low et a l, 1998). W ithin the syntaxin family, only syntaxins 1,2,3 and 4 have been detected at the plasma membrane and are reported

to regulate exocytosis (Bennet et a l, 1993; Volchuk et a l, 1996; Tellam et a l, 1997). The v-SNARE homologues VA M Pl and VAMP2 function in regulated exocytosis

(Chilcote et a l, 1995) and cellubrevin (VAMP3), VAMP7 and VAMPS have been suggested to play a role in the endocytic pathway (Galli et a l, 1994; Advani et a l, 1999; Wong et al, 1998).

Biochemical approaches to the identification o f haematopoietic cell secretory

granule fusion complex components have until recently, with the possible exception o f

identification o f Rab3 isoforms in this study proved unsuccessful (Lacy et a l, 1995). The classical approach for testing for the involvement o f SNARE proteins in neuronal

systems, that is toxin treatment, has not proved fruitful in studying degranulation in cells

o f the haematopoietic lineage (Arora et a l, 1994; P. Lacy, personal communication). However, evidence from studies using human neutrophils has demonstrated the presence

o f components o f the fusion complex (Brumell et a l, 1995). It was notable that those components identified, syntaxin 4, VAMP2 and a 39kda isofoim o f the secretory carrier

membrane protein (SCAMP), were found predominantly on secondary and tertiary

granules and secretory vesicles, but were absent from primary (lysosomal) granules. This

evidence and the inability to identify fusion complex components in the eosinophil which

lacks secondary and tertiary granules, raises the possibility that the secretory lysosome

utilizes different components to regulate its fusion with the plasma membrane. The

involvement o f SNAP23 in the regulation o f compound exocytosis, linking granule

plasma membrane and granule-granule fusion began to shed light on the involvement o f

this SNARE family in haematopoietic cell degranulation (Guo et a l, 1998). More recently, syntaxin3 and VAMP7 have been shown to be expressed in RBL-2H3 cells by

PGR (Hibi et a l, 2000). The authors suggest that both VAMP7 and syntaxin3 are localised to secretory granules, moving to the plasma membrane upon degranulation.

This was presented as evidence for the involvement o f these SNARES in the degranulation o f RBL-2H3 cells, although colocalisation and functional data was not

presented. VAMP7 has also been shown to mediate vesicular transport from endosomes

to lysosomes (Advani et a l, 1999), and is required for homotypic lysosome fusion in alveolar macrophages (Ward et a l, 2000). Given the lysosomal nature o f the secretory granule o f haematopoietic cells this could also be a possible explanation for the

observations o f Hibi et al, and not a role in degranulation, highlighting the need for functional data. Recently, VAMPS has been localised to RBL-2H3 cell granules and

overexpression o f syntaxin 4 demonstrated to inhibit degranulation (Paumet et a l, 2000). Because o f the problems associated with protein expression levels and antibody

specificity, a molecular biological approach was taken to try to identify components o f

the vesicle docking and fusion machinery in eosinophils and RBL-2H3 cells, the chosen

model system. It was hoped that this approach, probing for components o f the

docking/fusion complex either by PGR, or library screening at low stringency could also

4.2 Identification o f components of the vesicle docking and fusion machinery by PCR and cDNA library screening

4.2.1 Introduction

In an effort to identify components involved in the regulated release o f

haematopoietic cell granule contents, it was hoped that a molecular biological approach

could prove successful. This approach made use o f oligonucleotides designed to regions

o f homology o f known components o f the SNARE complex and the use o f SNARE PCR

products to screen RBL-2H3 and eosinophil cDNA libraries at low stringency. This

approach has been used with some success previously, for example to identify

homologues o f Rab3 (Oberhauser et a l, 1994) and RabS (Armstrong et al, 1996). Since the initiation o f this study, numerous SNARE homologues have been identified

predominantly through database searching (Wong et a l, 1998; Zeng et a l, 1998). However, for the purpose o f this study screening was limited to the major neuronal v-

SNARE transmembrane components o f the complex identified when this study began, namely, V A M Pl, VAMP2 and cellubrevin.

4.2.1 Results

In order to investigate which members o f the SNARE complex were expressed in

granulocytes, cDNA libraries were constructed from human eosinophils and the RBL-

2H3 cell line (a gift from J. Armstrong). Libraries mass excised from the lambdaZap II

phagemid vector were amplified with pairs o f degenerate primers (table 4.1) designed to

correspond to highly conserved regions o f members o f the v-SNARE family (Fig. 4.1).

The resultant DNA fragments generated by PCR (Fig. 4.2, lanes 3,4) were compared to

the PCR products obtained from a human brain cDNA template (Fig. 4.2, lanes 4,5).

Eosinophil PCR products corresponding in size to those obtained using human brain

cDNA were purified, subcloned into the TA cloning vector & sequenced. All clones

generated from the eosinophil cDNA library were found to be VAMP2. The resultant DNA fragment obtained using the oligonucleotide primer pair 2899/2897 (Fig. 4.2, lane

3) was then radiolabelled and used to probe the eosinophil cDNA library by plaque

o f 2x10^ clones. These positives were progressed through secondary hybridization to

single positive plaques. The cDNA inserts were in vivo excised, screened for VAMP2 by PCR and sequenced. Sequencing identified all o f the recovered clones as VAMP2.

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