2 Material and Methods
3.2 Processing of Pyramus and Thisbe
3.2.5 Processing of Pyr and Ths is not Fur1-mediated
3.2.5.1 Mutation of Furin cleavage sites is not preventing cleavage in cell culture
Based on the inconclusive results from the in vivo analysis the Ths and Pyr variants with the mutated furin cleavage sites were tested in cell culture to determine if the mutation of the Furin sites prevents the cleavage of Pyr and Ths. For this purpose double-tagged Pyr and Ths with mutated Furin cleavage sites in a sequential fashion, adding one mutation at a time, were tested for processing in cell culture (Figure 23).Figure 23: Role of Furin cutting sites for the processing of Pyr
(A) Schematic drawing of the EGFP- and MYC tagged Pyr constructs including Furin cutting sites. Mutated Furin cutting sites (MFS) are marked in red. (B) Western blot analysis of the expressed EGFP- and MYC tagged Pyr constructs in cell supernatants. Western blots were treated with the anti-GFP antibody. Note:
Mutation of Pyr FS1 results in a double band that is lost when Pyr MFS3 is introduced in addition.
EGFP FGF domain MYC Pyr
75 If Furin is indeed mediating the cleavage of Pyr and Ths the sequential mutation of the cutting sites should result in the inhibition of the processing and therefore recover the full-length proteins. However, it cannot be excluded that the mutation of the cleavage site leads to the processing at a more C-terminal Furin site, which would result in the generation of a longer protein fragment.
Figure 24: Role of Furin cutting sites for the processing of Ths
(A) Schematic drawing of the EGFP- and MYC tagged Ths constructs including Furin cutting sites. Mutated Furin cutting sites (MFS) are marked in red. (B) Western blot analysis of the expressed EGFP- and MYC tagged Ths constructs in cell lysates. Western blots were treated with the GFP antibody and the anti-MYC antibody. Note: Mutation of Ths FS1 leads to the inhibition of cleavage and the accumulation of the uncleaved protein in cell extracts.
The experiments shown in (Figure 23B) revealed that all mutated Pyr variants were still cleaved into the 90kDa EGFP-tagged N-terminal fragment that can be detected when wild type Pyr is cleaved. However, an interesting observation made in this context was the appearance of a double band of the 90kDa fragment that appears when Pyr FS1 was mutated alone or together with the FS2. This could imply that the mutation of Pyr FS1 might block cleavage resulting in the alternative cleavage at Pyr FS3. However, the additional mutation of Pyr FS3 reversed this effect and all subsequent mutations showed the N-terminal 90kDa fragment as well. Thus the mutation of Pyr FS1 possibly affects the mobility of the protein indirectly by a potential secondary protein modification or a structural change in the Pyr protein rather than blocking the processing of Pyr.
76 Taken together the mutation of the Furin cleavage sites in Pyr alone or in combination did not prevent the cleavage of the protein, proving that cleavage of Pyr is not carried out by a Furin protease. The results obtained in cell culture and the observed small differences in the activity of wild type Pyr and Pyr with mutated Furin cleavage sites in vivo (Figure 22) strongly suggest that Pyr is proteolytically processed by another currently unknown protease.
Similar to the results obtained for Pyr the inhibition of the Furin protease via the use of the inhibitor α1-PDX in cell culture (Figure 19) and in the gain-of-function analysis in vivo (Figure 22) did not lead to the clear results necessary to validate a role of Furin proteases for the processing of Ths. Therefore double-tagged Ths variants with sequentially mutated Furin cleavage sites were also tested in cell culture and their cleavage analysed in Western Blots (Figure 24).
Figure 25: Ths MFS1 is cleaved and secreted
(A) Schematic drawing of the EGFP- and MYC tagged Ths constructs including Furin cutting sites. Mutated Furin cutting sites (MFS) are marked in red. (B+C) Western blot analysis of the expressed EGFP- and MYC tagged Ths constructs (B) in the cell extract and (C) in cell supernatants. Proteins were detected using (C) anti-GFP antibody and (B) the anti-MYC antibody. A red arrow marks uncleaved Ths protein. Note:
Mutation of Ths MFS1 is not preventing cleavage and secretion of Ths.
This approach revealed that both, the inhibition of the Furin protease by α1-PDX and the mutation of the cutting sites, led to the inhibition of cleavage in Ths in cell extracts.
77 Interestingly already the mutation of the first cutting site (Ths FS1), which is located within the FGF domain, was sufficient to inhibit the proteolytic cleavage completely. As a result the full-length Ths protein could be detected with antibodies against the N-terminal EGFG-Tag and the C-N-terminal MYC-Tag in the cell extract. This result suggests that the first Furin cleavage site is indeed used for the proteolytic processing of Ths by a Furin protease. However wild type Ths could be detected neither as cleaved 55kDa fragment nor as full-length protein within the cell extracts. This result is in accordance with earlier experiments that showed that wild type Ths could only be detected as cleaved fragment in cell supernatants (2.1.2.5). The absence uncleaved Ths in cell extracts would therefore indicate that the secretion of Ths is highly efficient, leaving hardly any protein in cell extracts. Furthermore, since only processed Ths can be detected in the cell supernatant, Ths must be entirely processed during or after the secretion from the cells. If this explanation were correct the mutation of Ths FS1 would affect the secretion of Ths and thus indirectly prevent the subsequent proteolytic processing.
This hypothesis was tested by the expression of the wild type Ths and Ths carrying a mutation in the first Furin cleavage site (Ths MFS1) in cell culture and the analysis of both the cell extracts and the supernatant. In the supernatant only the cleaved 55kDa fragment was detected both in the wild type Ths and in Ths MFS1. However, in the cell extracts full-length Ths is detectable in small amounts when the Ths MFS1 was transfected and also in case of the wild type Ths (Figure 25B).
These results exclude Fur1 as the protease involved in the cleavage of Ths, thus suggesting that Fur1-mediated cleavage is exclusive to Bnl and not presenting an overall mechanism for all FGFs in Drosophila melanogaster. Furthermore, the experiment showed that wild type Ths is hardly detectable in cell extracts neither as uncleaved nor as a cleaved protein thus supporting the model that Ths is unstable in the cellular context and that is completely processed during or after its secretion.
However, the first cleavage site seems to have an effect on the stability of Ths within the cells.
3.2.6 Identification of the Pyr cleavage site