As described in section 4.2.1, LAT3 amber mutants were generated at multiple sites within transmembrane domain 6 and 7, including potential post-translational modification (PTM) sites Y251, S262, K264 and S267, as well as neighbouring sites T252, Q263, A265, P266, L268. Mutant constructs were confirmed by DNA sequencing and western blots of transfected HEK293 cell lysates in the presence or absence of AzF (Figure 4.7). In the presence of AzF, amber stop codons were able to be translated and full length LAT3 proteins (~55kDa) were
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able to be expressed. From the western blot, the expression level and band size showed slight difference from site to site. This is probably due to the site-specific mutation to AzF which may affect the structure and conformation of LAT3 protein. Because AzF has different structure to the original residues, it may cause different modifications of protein and affect the binding affinity of antibody leading to different expression level and band size.
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Figure 4.7. LAT3 amber mutants at various sites. A. sequencing results showed site-specific mutation into amber stop codon (TAG) was successful. B. expression of LAT3 in HEK293 cell lysates transfected with LAT3 amber mutants at various sites in the presence or absence of 1 mM AzF. GAPDH is loading control.
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We transfected different HA-tagged LAT3 amber mutants, S262 and Q263, into HEK293 cells, and examined the expression of Akt after HA immunoprecipitation in the presence or absence of AzF and UV. Western blots of IP-ed sample showed migration of bands at 100 kDa in all three conditions (Figure 4.8). In the absence of AzF in IP-ed sample, we observed protein bands around 100 kDa when probing with anti-Akt antibody. This indicated that read-through may have happened during translation (Since AzF is modified from tyrosine, there are chances that the AzF-RS would charge tyrosine onto tRNA, resulting in protein expression), resulting in the expression of HA-tagged LAT3 and co-IP of Akt. For Q263amb-HA (Figure 4.8B), the read- through was less significant compared to S262amb-HA (Figure 4.8A). In the Input samples, in the absence of AzF, there was a low level of read-through for Q263, which was further enriched by immunoprecipitation with anti-HA antibody and resulted in migrated bands around 100 kDa. Moreover, in the presence of AzF, migrated protein bands show stronger expression in the absence of UV compared to UV induced samples (Figure 4.8B). It suggested the photo- crosslinking properties at Q263 could be induced by ambient light.
The UV treatment may affect the efficiency of affinity purification of HA-tagged LAT3 with the mutation of S262 and Q263. For S262, in the unbound phase (the supernatant after incubating lysate with magnetic beads overnight), the expression level of Akt was significantly higher in the absence of AzF after UV treatment compared to non-UV treatment; while, for Q263, in the unbound phase, UV treatment increased the expression level of Akt compared to non-UV treatment. The phosphorylation of S262 may be affected by the mutation, leading to the alteration of conformational change and protein folding of LAT3, thus affecting the interaction with Akt. S262 and Q263 are very close to ubiquitination site K264, and may affect the binding of ubiquitin to K264. In addition, the stability of AzF may contribute to the discrepancy of expression of the protein complex, because the bulk of AzF could be converted to AmF (Figure 4.3A) under UV induction, which is structurally close to AzF but without
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photo-crosslinking property (Shao et al., 2015). Thus, less reactive AzF was present to form protein complex leading to lower expression being detected.
Figure 4.8. HEK293 transfected with LAT3amb-HA mutants in the presence or absence of AzF or UV and probed with anti-Akt antibody to examine Akt protein expression level after immunoprecipitation with anti-HA antibody compared to input and unbound phase of IP. A, transfection with S262amb-HA. B, transfection with Q263amb-HA.
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In addition, whether the orthogonality of tRNA and AzF-RS pair remains intact after repeated expansion of the plasmid via bacterial culture and DNA purification needs to be validated. We transfected the HEK293 cells with either S267amb, tRNA (Yam), or AzF-RS, or different combinations. Wild type LAT3-HA was used as positive control. Immunoblotting showed strong LAT3 expression in positive control (wild type LAT3-HA), and showed no LAT3 expression in negative control (no Yam, AzF-RS, or AzF) (Figure 4.9). However, in the presence of Yam, AzF-RS and AzF, there was no expression of LAT3, whereas Yam alone or with Yam and AzF-RS without AzF showed LAT3 expression (Figure 4.9). This result was unexpected and problematic, as the expression of amber mutant requires the orthogonal pair of tRNA and aaRS, and the presence of the Uaa. It raised the concern that the plasmid of either Yam and/or AzF-RS might be mutated during repeated bacterial amplifications, leading to the expression of LAT3 in the absence of AzF-RS or AzF. This comprises the Uaa expression system of LAT3, and may cause confusion in data interpretation, and the identification of crosslinking.
Figure 4.9. Western blot of co-transfection with different combination of tRNA (Yam) and AzF-RS in the presence or absence of AzF in HEK293 cells, to validate the orthogonality. Wild type LAT3-HA was used as control.
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Given the inconsistency of protein expression with the orthogonal pair and AzF, we decided to use a plasmid that contains both tRNA Yam and AzF-RS (from Dr Shixin Ye) instead of the orthogonal pair, to increase the co-transfection efficiency and reduce cytotoxicity.
Since S267 is one of the reported phosphorylation sites, we transfected HA-tagged LAT3- S267amb plasmid into HEK293 cells, and cultured in the presence or absence of AzF as well as UV. Lysates were purified by IP using anti-HA tag antibody so that HA-tagged expressed LAT3 proteins were enriched. After enrichment, anti-HA tag antibodies were used to probe the expression of LAT3 after SDS-PAGE. In the presence of AzF, LAT3-S267amb was detected in cell lysates (Figure 4.10A). Although there were some proteins being detected in the absence of AzF, it may be due to the read-through of tyrosine. The bands at higher molecular weight indicated the formation of a protein complex by UV induced crosslinking. We probed blots with anti-Akt after HA enrichment (Figure 4.10B). Akt was detected in the input samples at its regular molecular weight (60 kDa), however, in the IP samples, a band was observed around 100 kDa, which represented crosslinked protein complex including Akt and HA-tagged LAT3. As AzF was incorporated into LAT3amb mutant to successfully express LAT3, its azido moiety could couple with neighbouring protein upon UV excitation. It’s also worth noting that in the IP samples of LAT3-S267amb treated with AzF but not UV radiation, there is a faint band around 100 kDa. It implies a background crosslinking has been formed. It is most likely due to the exposure to the ambient room light, which has been observed in previous studies (Grunbeck et al., 2011, Rust et al., 2014).
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Figure 4.10. UV excitation (10 min) induced crosslinking in LAT3-S267amb transfected HEK293 in the presence of AzF. A, LAT3 amber mutant was successfully expressed, probed by anti-HA tag antibody; B, a strong band of potential cross-linked complex around 100 kDa was probed by anti-Akt antibody; the bands around 50 kDa of IP samples are of IgG heavy chain.
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We also examined amber mutants at P266 and L268. Interestingly, they exhibit distinct photo- crosslinking capability to S267 (Figure 4.11). Under UV excitation, only S267amb mutant showed bands at higher molecular weight, indicating the formation of protein complex from this residue. Since serine 267 is a potential phosphorylation site of LAT3, the crosslinking from this residue suggested that S267 could potentially be the binding and/or interacting site with its substrate that affects the function of LAT3.
Figure 4.11. Western blot of UV excitation induced crosslinking in LAT3-P266amb, S267amb, and L268amb transfected HEK293 cells supplemented with AzF. After immunoprecipitation with anti-HA tag antibody, LAT3 was probed to examine the expression level of each site. A strong band presented around 100 kDa in S267, but not in P266 or L268.
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