Western blotting across eosinophil subfractions 1 Introduction

A range o f approaches using synaptic vesicle and Golgi membranes as well as

yeast secretion mutants have led to a model o f vesicle docking and fusion being

proposed known as the SNARE hypothesis. Although the original SNARE hypothesis

has undergone considerable reassessment and refinement, SNARE proteins are still

thought to provide a core mechanism to specifically pair membrane compartments.

Many v- and t-SNARES have been identified in yeast, plants and animals. They have

also been localised to specific subcellular compartments including the ER, endosomes

and plasma membrane, but little is known o f their role in secretory lysosome docking

and fusion events during regulated secretion in haematopoietic cells. Antibodies to

key components o f this complex, as well as other synaptic vesicle proteins also

thought to play a role in regulation o f membrane fusion events such as Rab3,

synaptophysin and synaptobrevin are now available. Therefore it should be possible to

couple subcellular fractionation techniques with western blotting to determine if any

o f these components are present on the eosinophil granule. Identification o f granule

docking and fusion components would be an initial step in understanding the

underlying mechanism o f regulated secretion in haematopoietic cells.

3.3.2 Results

After fractionation, ISOpl o f each eosinophil subffaction was loaded onto an

7.5-13% polyacrylamide gel, then stained with coomassie blue. A typical gel across

an eosinophil subfractionation is shown in figure 3.3. The majority o f protein was

present in cytosolic fractions, although granule fractions also contained high amounts

o f a few proteins with molecular weights o f approximately, 50, 14 and 18kDa. These

corresponded to known granule content proteins, eosinophil peroxidase large subunit,

major basic protein and eosinophil derived neurotoxin (Hamann et al, 1991). Very few other bands were visible in the granule fractions. Furthermore, electron

micrographs o f eosinophil granules demonstrate the abundance o f granule content and

highlight the high ratio o f granule lumen content to membrane in these organelles

Fraction number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

f-A

Figure 3.3: Coomassie stained polyacrylamide gel of across eosinophil subfractions. An equal volume of each fraction was loaded onto each lane with protein levels of 2.5- 40pg/well.

kDa 66 Lane 2 3 35 21 14

e

25 20 > Ü CD CO O o 0 2 4 6 8 10 12 14 16 18 20 22 24

-o - EPO

-e - AP

fraction no. kDa < 66

Figure 3.4: Synaptic vesicle protein expression across hum an eosinophil subfractions, (a) Western blot showing expression of synaptic vesicle proteins in human brain. VAMP (synaptobrevin) (lane 1); SNAP25 (lane 2); syntaxinl (lane 3); Rab3 (lane 4); synapsinl (lane 5) (b) marker enzymes across human eosinophil subfractions. EPO (eosinophil peroxidase); AP (alkaline phosphatase), (c) Western blot showing expression of synapsin I across eosinophil subfractions.

In an attempt to identify granule fusion machinery components, Western blots

across the subfractionation were prepared. 150|ul/well o f each fraction was loaded

onto an 7.5-13% polyacrylamide gel, which was then transferred onto ECL-

nitrocellulose as described in chapter 2. Table 3.1 details the synaptic vesicle protein

antisera tested on eosinophil subtractions and the results obtained. These antisera all

reacted positively when tested against a human brain western blot (Fig. 3.4a). With

the exception o f synapsin I however, none o f these antisera reacted positively with the

eosinophil subtractions (Fig. 3.4c). Synapsin I localised to cytosolic fractions and to

light membrane fractions corresponding to plasma membrane and Golgi membranes

(Fig. 3.4b). No synapsin immunoreactivity however was localised to the ER,

lysosomal or granule fractions.

Antisera to protein Specificity

Rab3(a-d) Human, rat, invertebrates

SNAP-25 Human, rat

Synaptobrevin Human, pig

Syntaxin Human, pig, hamster, rat

Synapsin I Human, bovine, rat

Table 3.1: Summary o f eosmophil granule immunoblot analysis o f synaptic vesicle complex components. Sources o f antibodies are listed in chapter 2.

3.3.2 Discussion

Despite exhaustive probing for members o f the synaptic vesicle fusion

complex, none o f those tested with the exception o f synapsin I reacted against blots o f

human eosinophil subfractions. There are a number o f possible explanations for these

observations. The first is that the secretory lysosome o f haematopoietic cells utilises a

different mechanism for docking and fusion with the plasma membrane. It is possible

however that cells o f this lineage use homologues o f the known docking and fusion

machinery components, that are not recognised by available antibodies. A third

number o f granules per cell means that insufficient membrane protein has been loaded

onto the gel for detection by western blotting.

The demonstration o f synapsin I immunoreactivity in eosinophil subtractions

is intriguing. Synapsin I immunoreactivity was not observed in the granule-enriched

fractions. Synapsins are a multigene family o f neuron-specific phosphoproteins. They

interact in vitro with lipid and protein components o f synaptic vesicles and with cytoskeletal proteins including actin (Hilfiker et al., 1999). These observations have led to the hypothesis that synapsins tether synaptic vesicles to each other and to the

actin-based cytoskeleton, to maintain a pool o f vesicles in the vicinity o f the active

zone. Although synapsins are widely accepted to have neuronal specific expression,

there is recent evidence suggesting that synapsin I is also associated with the insulin

secretory granules o f MIN6 cells and it has also been detected by PCR in normal rat

islets (Matsumoto et a l, 1999). Synapsin I immunoreactivity has not previously been observed in cells o f the haematopoietic lineage, although further studies in this

laboratory demonstrated synapsin I mRNA expression by Northern blotting (C.

Paddon, personal communication). The absence o f synapsin immunoreactivity from

granule fractions, but its presence in cytosolic and light membrane fractions, suggests

that synapsins may be involved in the exocytosis o f small secretory vesicles, although

as o f yet there is no further evidence for this.

Parallel studies using guinea pig eosinophils also demonstrate the absence o f

synaptic vesicle proteins in the eosinophil (Lacy et a l, 1995) and are in agreement with the observations described here. This study also noted the absence o f the synaptic

vesicle proteins synaptotagmin and cellubrevin. During the course o f this study, the

presence o f the t-SNARE syntaxin 4, VAMP-2 and the 39kDa isoform o f the

secretory carrier membrane protein (SCAMP) have been demonstrated in neutrophils

(Brumell et ah, 1995). Syntaxin 4 was localised predominantly to the plasma membrane. VAMP-2 was present in tertiary granules and secretory vesicles, but

largely absent from primary (lysosomal) granules and secretory vesicles. SCAMP was

also absent from primary granules, but was localised to secondary and tertiary

granules and secretory vesicles. The apparent difference in synaptic vesicle marker

expression in eosinophils and neutrophils may be due to the presence o f multiple

that none o f these proteins were present on the secretory lysosome o f the neutrophil.

This suggests that either these proteins are present at undetectable levels, that

secretory lysosomes use homologues o f these proteins, or that the secretory lysosome

utilises a different control mechanism for membrane fusion during degranulation. In

attempt to address these possibilities other approaches are necessary that do not rely

3.4 Direct identification of proteins from eosinophil subfractions by mass

In document An investigation into the regulated secretory pathway of granulocytes (Page 81-87)