Protein identification and determination of linear epitopes
In the genome of ‘Candidatus Kuenenia stuttgartiensis’, four different gene clusters have been identified to encode membrane-bound complexes of ATPase (van Niftrik et al., 2010). All of these can function either as an ATP-dependent proton pump or as an ATP synthase. The gene cluster of the F-ATPase 1 (open-reading frames, ORF; kuste3787 – 3796) shows similar protein sequences as compared to an F-ATPase 1 in E. coli with an experimentally verified function of ATP synthase (van Niftrik et al., 2010). To elucidate the expression of the putative ATPases in the bacterial cells, efforts have been made to study the transcriptome (mRNA) and proteome (peptides) (van Niftrik et al., 2010). Only the F-ATPase 1 (ORF; kuste3787 – 3796) was found to be significantly expressed. Immunolocalization suggested a diffuse subcellular localization of the F-ATPase 1, with several possible loci within the anammox cell. Strongest labeling was found in the outermost membrane and in the anammoxosome membrane (van Niftrik et al., 2010). This observation is quite in contrast to the genomic prediction that rather suggests that the anammoxosome has a targeted subproteome (Medema et al., 2010). In accordance, the F-ATPase 1 would only be associated with the anammoxosome membrane.
This study utilized a recently developed technique to select and experimentally verify linear epitopes of the F-ATPase 1 (Karlsson et al., 2009). Peptides found in the MS analysis were therefore regarded as suitable substrates for antibodies (Fig. 1). One of the main advantages of using the lipid-based protein immobilization (LPI™) technology in a flow cell format is the ability to use short digestion times which enables a surface shaving of the exposed proteins. This was recently illustrated by performing a sequential digestion approach to maximize the number of protein hits in a protein profiling study (Jansson et al., 2012).
Figure 1. Schematic illustration of the differences in work-flow using in silico prediction tools vs. proteomics-based generation of experimentally verified linear epitopes for antibody generation. Using software tools, it is possible to predict sequence regions which are most likely buried in a cellular membrane and therefore not suitable for the selection of linear epitopes. When selecting epitopes predicted to be in exposed extracellular loops, there is always the risk of generating antibodies against target sequences hidden in the three-dimensional protein structure. Using a proteomic-based approach followed by bioinformatics enables the selection of experimentally verified linear epitopes (peptides) that are exposed on the surface of the protein structure. Such exposed linear epitopes are subsequently used for antibody generation, thus increasing the probability of a specific binding event.
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As the ATP-generating ATPase is one of the most conserved proteins in bacteria (Deckers-Hebestreit & Altendorf, 1996), peptides found in the MS/MS analysis may not be unique to anammox bacteria. Following analysis, the blast network service (www.expasy.org, Altschul et al., 1997) was therefore used to screen for homologue peptides originating from bacteria other than ‘Candidatus K. stuttgartiensis’. In the database search for peptides from the beta-subunit of the F-ATPase 1, there were hits from the anammox bacterial strain KSU-1 (Shimamura et al., 2007) and from uncultured Planctomycetes. Altogether, the sequence overlap between peptides from the beta-subunit of anammox bacteria and other bacteria was quite low (Karlsson et al., 2009). In the search for peptides originating from the alpha-subunit, however, there were complete sequence overlaps with many species of bacteria. To retain the structure and function of the ATPase during evolution, key peptide sequences are likely preserved. As many hits were found in the blast search, peptides arising from the alpha-subunit are probably peptides that originate from such highly conserved regions of the ATPase. The unique morphology of anammox bacteria still facilitated a positive localization of the alpha-subunit of the F-ATPase 1.
Two peptides were used as linear epitopes for raising antibodies in rabbits (Innovagen AB). AIDAMIPIGR (single letter amino acid sequence) was the target peptide in the alpha-subunit of the F-ATPase 1 (kuste3793), and QIAELGIYPAVDPLR was the target peptide in the beta-subunit (kuste3795). The generated antibodies were thus named anti-kuste3793 and anti-kuste3795, respectively.
EM immunocytochemistry and subcellular localization of the F-ATPase 1
The subcellular organization of anammox bacteria was well preserved after cryopreparation, whereby the cell structure, including the anammoxosome membrane, was clear and distinct (Fig. 2). In general, there was a low but favored labeling of gold particles associated with the anammoxosome membrane after use of the two specific antibodies. In contrast, the control antibody (anti-CRAMP) resulted in extremely few nanogold particles over cell profiles. The background using pre-immune serum was for unknown reasons rather high. Results from the immunocytochemistry are exemplified in Fig. 2.
Figure 2. EM immunocytochemistry and localization of the F-ATPase 1. (a) Schematic illustration of the protocol for immunogold labeling. Target peptides from either the alpha (kuste3793)- or beta (kuste3795)-subunits of the identified F-ATPase 1 were chosen from the MS/MS analysis as linear epitopes. Antibodies against these peptides (anti-kuste3793 and anti-kuste3795) were raised in rabbits. During the immunogold localization, the antibodies bound to their target proteins in cryo-sectioned anammox colonies. Protein A labeled with gold particles (10 nm) was used to localize the antibodies, thereby revealing the location of the F-ATPase 1. (b–c) Anammox cells incubated with the antibodies (anti-kuste3793 and anti-kuste3795). In each image, one or two 10 nm gold particles were associated with the anammoxosome membrane. Width of images is 500 nm. (d) Colony of anammox bacteria after immunogold incubation with anti-CRAMP antibody (control). The cytoplasmic membrane (white arrow, CM) and the anammoxosome membrane (black arrow, AM) are indicated. (e) Schematic illustration of the cellular organization of anammox bacteria (adapted from
van Niftrik et al., 2008b).
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The frequency of labeling was estimated by measurements of nanogold particle distributions over anammox cells using predetermined areas (ten images, each with an area of 14.3 μm2). The labeling frequency (described by the number of labeled cells vs. total number of analyzed cells) was highest for anti-kuste3793 (23%), somewhat lower for anti-kuste3795 (13%), and almost negligible for the control antibody (anti-CRAMP; 1%). Due to the low labeling frequency of the anti-CRAMP antibody, the total number of data points for this control was low.
In another set of experiments, a higher magnification of 50 000× was used to observe gold labeling of the anammoxosome membrane. A zone of acceptance of 25 nm to either side of the anammoxosome membrane was used to indicate a positive labeling of the membrane (Mayhew & Lucocq, 2008; van Niftrik et al., 2010). Incubations of the antibodies targeted against the alpha- and beta-subunit of the F-ATPase 1 (anti-kuste3793 and anti-kuste3795) gave rise to a repeated gold particle distribution at or in the immediate proximity of visible anammoxosomes (Fig. 2 and 3). Immunogold labeling against the alpha-subunit of the F-ATPase 1 (anti-kuste3793) showed a high specificity. The relative frequency of immunogold labeling within the zone of acceptance was 93% and 87%, respectively, for the two experimental series. For the beta-subunit of the F-ATPase 1 (anti-kuste3795), the relative frequency was lower (64% and 69%, respectively). Frequency distributions of measured distance of gold particles from the anammoxosome membrane are shown in Fig. 3. Statistical evaluation by box-and-whiskers plots clearly visualized the high specific labeling of the anammoxosome membrane-associated F-ATPase 1 using the anti-kuste3793 and anti-kuste3795 antibodies, as compared to the two negative controls (Fig. 4).
Figure 3. Frequency distributions of measured distance of gold particles from the anammoxosome membrane. The zone of acceptance (± 25 nm) is indicated by filled bars. (a) Anti-kuste3793. (b) Anti-kuste3795. (c) Control. Left panels illustrate experimental series with pre-immune serum as control. Right panels illustrate experimental series with anti-CRAMP antibody as control.
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Figure 4. Measured distance of the immunogold labeling from the anammoxosome membrane (open circles) for experimental series using pre-immune serum (a) and anti-CRAMP antibody (b) as control. Statistical evaluation of the immunogold labeling is illustrated by box-and-whisker plots. The line within the box denotes the median distance of the positive immunogold labeling from the anammoxosome membrane, the box represents the standard error, and the whiskers indicate the standard deviation for the mean distance measured. Values more than three times the height of the box are statistically considered as distant outliers (stars).
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The specific association to the anammoxosome membrane was somewhat less clear for the anti-kuste3795 antibody as compared to the labeling with anti-kuste3793. Affinity data indicate similar binding properties and specificities of both antibodies (Figs S1 and S2). It could be speculated that the anti-kuste3795 antibody detects a portion of the protein that at instances might be relocated (smeared) during cryosectioning. However, further investigations are needed to clarify this eventual difference between the two antibodies.
The specific, however low, degree of labeling suggests a low expression level (i.e. low number of ATPase copies). From studies using cultures of pure anammox bacteria, it has been inferred that the anammox bacteria grow very slowly, dividing only once in every 2 weeks (Strous et al., 2006). Recently, one of the central enzymes of the anammox reaction, the enzyme complex of the HZS, was isolated (Kartal et al., 2011). Hydrazine synthesis by the catalytic part of the HZS (open-reading frames kuste2859–2861) was investigated using a coupled assay with another enzyme, kustc1061, which converted the generated hydrazine to nitrogen gas. Using and 14NO as substrates for the HZS, the generation of 29N2 was measured using gas chromatography–mass spectrometry (GC-MS). Supporting the idea of slow growth rates, the activity of the HZS was observed to be low under the experimental conditions applied (Kartal et al., 2011, 2013). The slow growth infers a low catabolism that would restrict the number of protons generated inside the anammoxosome per unit time. The expression of ATP synthase is tightly regulated, as synthesis of such a major protein complex is energetically costly for the cell (Kramarova et al., 2008). For example, in brown adipose tissue mitochondria, where the energy is released as heat through an uncoupling protein (UCP1), the proton gradient is uncoupled from the ATP synthase complex. The copy number of ATP synthase in brown adipose tissue mitochondria is therefore kept low (Kramarova et al., 2008). Further, the bacteria clusters used in this study were sampled from a waste-water treatment facility, exposing the bacteria to conditions different from those in laboratory monocultures. Also, when performing immunogold EM, it is likely that the number of exposed ATP synthases available for immunogold staining is limited in a thin two-dimensional section arising from a three-dimensional anammoxosome membrane. The curved membrane of the anammoxosome, arising from a maximization of membrane area available for the catabolic processes (van Niftrik et al., 2008a), might also affect the steric availability of the target for the antibody. These aspects could, at least partly, explain the relatively low labeling observed in the EM images.
In conclusion, the results from immunogold labeling allowed for a highly specific localization of the alpha- and beta-subunits of the identified F-ATPase 1 to the anammoxosome membrane. The use of the experimentally verified linear epitopes (peptides) found in the proteomics analysis, proved successful in generating highly specific antibodies for the F-ATPase 1 under investigation. The high sequence similarity of the F-ATPase 1 to an F-ATPase 1 with a verified function as an ATP synthase, together with the presence of catabolic enzymes inside the anammoxosome (van Niftrik et al., 2008b; Karlsson et al., 2009) and experimental evidence of a proton gradient across the anammoxosome membrane (van der Star et al., 2010), confirm that the anammoxosome membrane is energized and a site for ATP synthesis. An intracellular membranous compartment containing a membrane-bound ATP synthase has previously not been encountered in any other bacteria.