Anti-KCNE3 Antibody Generation

In order to study KCNE3 expression at the protein level, and given the current lack of a reliable and specific anti-KCNE3 antibody, a novel anti-KCNE3 antibody was generated. Anti-KCNE3 antisera was raised in rabbits, against a 23 residues peptide corresponding to the KCNE3 cytoplasmic carboxy-terminus (Figure 5.4A) (see methods for further details).

To assess the specificity of the immunopurified antibody, HA-tagged KCNE3 cDNA was heterologously expressed in COS7 cells. As shown in figure 5.4B, Western blot analyis of KCNE3-transfected cells shows that anti-KCNE3 and anti-HA antibodies specifically detected several bands between 19 and 25 kDa (Figure 5.4B and D). These multiple bands, which appear around the expected molecular weight of the KCNE3 protein, have been previously suggested to represent differentially glycosylated KCNE3 species (Gage and Kobertz 2004).

69

Figure 5.4. Specificity of the newly generated anti-KCNE3 antibody. A, the 103 amino acid sequence of the KCNE3 protein is depicted. The anti-KCNE3 antibody was raised against a 23 amino acid peptide representing the whole C-terminal domain of KCNE3 (depicted as filled green box). The N-terminal presents 3 putative N-glycosylation sites indicated by blue diamonds and the transmembrane domain is shown by the filled grey box. B, Western blot analysis from lysates of COS-7 cells overexpressing N-terminally HA-tagged version of KCNE3. The anti-HA antibody detected a 3 band pattern (between 19 and 25 kDa, filled arrowhead) (lane T), which were absent from the non-transfected COS-7 cells (lane NT). C, a faint but similar band pattern was detected in a membrane fraction preparation of wild type colonic tissue, and it was absent from kcne3^-/- tissues.

Asterisk indicates a KCNE3-immunoreactive, high-molecular weight band, which might represent SDS-resistant KCNE aggregates. D, after deglycosylation by PNGase, the newly generated KCNE3 antibody recognized a single band (~14 kDa arrowhead) in membrane preparation of wild type colon (WT lane). No specific signal was detected in kcne3^-/- tissue (KO lane). As a positive control, total lysate of COS-7 cells overexpressing HA-KCNE3 was used (T lane), where different glycosylation states of HA-KCNE3 were still observed. The presence of several bands in the latter lane might be owed to incomplete deglycosylation caused by the N-terminal HA tag. Please note that when blot with anti-KCNE3 antibody, independently of the genotype an unspecific band at 30 kDa was homogenously labeled in all tissues and cell lysates.

As it has been previously reported, KCNE3 undergoes posttranslational modifications and contains three putative glycosylation sites on its N-terminal (Gage and Kobertz 2004). To test whether the posttranslational added sugar chains accounted for the migration shifts and the multiple band pattern observed in our Western blot in figure 5.4B-D, lysates from transfected cells were treated with Peptide N-glycosidase F (PNGaseF) prior to loading. Under these conditions, only three specific prominent bands at approx. 14, 17, and 19 kDa were detected with anti-KCNE3 antibody (Figure 5.4D last lane). These bands correspond to the expected molecular weight of non-glycosylated HA-tagged KCNE3 (14 kDa) and to incomplete non-glycosylated forms (aprox. 17 and 19 kDa). Interestingly, these last incompletely deglycosylated bands were commonly seen in HA-KCNE3 transfected cell lysates, but it never appeared when

70

the untagged KCNE3 protein was probed in native tissue after PNGaseF treatment (Figure 5.4D, first lane; Figure 5.5). Chandrasekhar et al in 2006 have also reported a very similar incomplete PNGaseF-mediated deglycosylation of HA-tagged KCNE1. I therefore assume that the N-terminal position of the HA tag of KCNE3 might have provided steric interference with the PNGaseF mediated deglycosylation reaction, thereby preventing efficient removal of one of its sugar chains.

As it was shown in figure 5.3, KCNE3 mRNA was abundant in the gastrointestinal tract, particularly in colon. To further characterize the specificity of our anti-KCNE3 antibody in native tissue, we prepared membrane-bound protein fractions from wild type and kcne3^-/- colon. Similar to what it was previously observed in Western blot analysis of HA-tagged KCNE3 transfected cells (Figure 5.4B and D), lysates from wild type colon which were not treated with the deglycosylating enzyme PNGaseF also exhibited a similar band pattern between 19-25 kDa (Figure 5.4C). Besides, in these samples an additional KCNE3-immunoreactive band was detected around 40 kDa (marked with asterisk (*) in Figure 5.4C). This additional band observed in wild type colon lysates might represent SDS-resistant KCNE3 aggregates that run at higher molecular weights, a common phenomenon that had been previously reported for several other membrane-associated proteins (Sagne et al. 1996). However, Chandrasekhar et al in 2006 when blotting against KCNE1 protein also described the same additional band at 40 kDa, and claimed that it was instead due to posttranslational KCNE modifications.

On the other hand, PNGaseF treatment resulted in a shift of the apparent molecular mass of the glycosylated species, which migrated now as a single visible band at 14 kDa (Figure 5.4D, first lane).

Taken together, these data lead us to conclude that first, KCNE3 undergoes N-linked glycosylation in heterologous expression and in native tissue. As expected, the presence of complex oligosaccharides increased the molecular weight of the KCNE3 protein, producing several KCNE3 immunoreactive bands in Western blot analysis. Similar to KCNE1 (Chandrasekhar et al. 2006), deglycosylation with PNGaseF was necessary to detect a single KCNE3-specific band at the expected molecular weight of 14 kDa in wild type tissues, which was absent in kcne3^-/- controls (Figure 5.4D). In particular, our newly generated anti-KCNE3 antibody was able to recognize both glycosylated and non-glycosylated forms of the KCNE3 protein, in either heterologous expression or

71

native tissues, thereby proving an extremely useful tool for further investigations (Figure 5.4C and D).