Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms

Abstract

Background: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal findings: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Figure 1. SEM images of M. tardigradum in the active and tun state.

Tardigrades are in the active form when they are surrounded by at least a film of water. By loosing most of their free and bound water (>95%) anhydrobiosis occurs. Tardigrades begin to contract their bodies and change their body structure into a so-called tun.

Figure 2. Comparison of different workup protocols for M. tardigradum.

Total protein extract of tardigrades in the active state was separated on a one-dimensional polyacrylamide gel. Lane 1: Rainbow molecular weight marker. Lane 2: Protein extract of whole tardigrades without any precipitation step. Lane 3: Protein extract after TCA precipitation. Lane 4: Protein extract after chloroform/methanol precipitation. Lane 5: Protein extract using clean-up kit.

Figure 3. Analysis of protein degradation in total protein extracts of tardigrades by Western blot analysis.

Actin (A) and alpha tubulin (B) were used as marker proteins for the detection of proteolysis. Lane 1A and 1B: DualVue Western blotting marker. Lane 2A and 2B: Total protein extract of HeLa cells as control. Lane 3A and 3B: Total protein extract of M. tardigradum. Notably, no protein degradation was observed during the workup procedure.

Figure 4. The experimental workflow to developing the proteome map.

Tardigrades were sonicated directly in lysis buffer. Total protein extracts were separated by two-dimensional gel electrophoresis. After silver staining protein spots were picked and in-gel digested with trypsin. MS/MS data obtained by LC-ESI-MS/MS analysis were searched against the NCBInr database, the clustered tardigrade EST database and the tardigrade protein database. Identified proteins with annotation were classified in different functional groups using the Blast2GO program. Identified proteins without annotation were analysed with the DomainSweep program to annotate protein domains.

Figure 5. Image of a preparative 2D-gel with selected analysed protein spots.

Total protein extract of 400 tardigrades in the active state corresponding to 330 µg was separated by high resolution two-dimensional gel electrophoresis. Proteins were visualised by silver staining. Three different categories are shown: Identified proteins with functional annotation are indicated in green, identified proteins without annotation are indicated in blue and not yet identified proteins are indicated in red.

Figure 6. Comparison of database performance for protein identification.

Protein spots were analysed by nanoLC-ESI-MS/MS and searched against the NCBInr database and the tardigrade protein database. The diagram illustrates the number of positive identifications in the respective database and the overlap between the two databases.

Figure 7. GO analysis of proteins identified in M. tardigradum.

A total of 271 spots representing 144 unique proteins was analysed with the Blast2GO program. The GO categories “molecular function” and “biological process” are shown as pie charts. A total of 9 different molecular function groups and 16 groups for biological processes are present in our result. The major parts of these categories (level 2) are shown in more detail (level 3) on the left and right side.

Figure 8. Detection of hsp60 and hsp70 by Western blotting.

Total protein extract of M. tardigradum in the active state was separated on a one-dimensional polyacrylamide gel. Hsp60 (A) and hsp70 (B) could be immunodetected with high sensitivity. Lane 1A and 1B: DualVue Western blotting marker. Lane 2A and 2B: Total protein extract of HeLa cells. Lane 3A and 3B: Total protein extract of tardigrades. Notably, the protein bands in the HeLa control lysate show molecular weights of 60 and 70 kDa as expected. In contrast the detected protein band for hsp60 in M. tardigradum is considerably smaller. For hsp70 multiple bands are observed in M. tardigradum at higher as well as at lower molecular weights.

Figure 9. Detection of hsp60 in six different tardigrade species by Western blotting.

Total protein extracts of tardigrades in the active state were separated on a one-dimensional polyacrylamide gel. Hsp60 (A) and actin (B) as loading control were immunodetected with high sensitivity. Lane 1: DualVue Western blotting marker. Lane 2: Total protein extract of HeLa cells. Lane 3: Total protein extract of M. tardigradum. Lane 4: Total protein extract of Paramacrobiotus richtersi. Lane 5: Total protein extract of Paramacrobiotus “richtersi group” 3. Lane 6: Total protein extract of Macrobiotus tonollii. Lane 7: Total protein extract of Paramacrobiotus “richtersi group” 2. Lane 8: Total protein extract of Paramacrobiotus “richtersi group” 1. Interestingly, the detected protein bands were ranging from 100 kDa to less than 24 kDa. Only hsp60 in the HeLa control lysate was detected at its expected position at 60 kDa.

Figure 10. Statistical analysis of significant peptides found in the three different databases which were used to search the MS/MS data.

The number of significant peptide hits is compared between the different databases. When searching against the NCBInr database most proteins were identified with only one significant peptide hit. In contrast when using the tardigrade protein database most proteins were represented by two or more significant peptides.