PARP1 from
4.3 Comparison of Amino Acid Sequences from six Fish Species
PARP1 is a highly conserved enzyme. Human and murine PAPR1 are about 92 % homolog (Beneke et al., 2002), and sequence identity between human and fruit fly PARP1 is still 31 %. The similarity of human and zebrafish PARP1 is about 70 %.
In human it was possible to identify an allelic variation of PARP1 in the primary sequence impacting significantly on activity. The exchange of only one amino acid at position 762 from alanine to valine results in a strong difference in enzymatic activity (Beneke et al., 2010; Wang et al., 2007).
The differences of the amino acid sequences from the investigated fish species are depicted in
Table 4-3. The four Nothobranchius species show as expected a strong similarity among each other, between 98.2 to 99.3 %, equivalent to only 7 – 18 amino acid exchanges. The most similar species with only 7 amino acid exchanges are the N.orthonotus and the N.furzeri. Of note, the zebrafish D.rerio is the most outlying fish in the study, showing a similar relation to the killifish and to medaka with between 180 to 188 amino acid exchanges. The similarity of medaka and the Nothobranchius species is between 86.5 to 87.1 %, corresponding to 131 – 137 amino acid exchanges.
O.latipes D.rerio N.korthausae N.rubripinnis N.orthonotus N.furzeri
O.latipes 188 133 131 137 135
D.rerio 81.6 % 186 180 185 187
N.korthausae 86.9 % 81.8 % 11 17 18
N.rubripinnis 87.1 % 82.3 % 98.9 % 14 16
N.orthonotus 86.5 % 81.9 % 98.3 % 98.6 % 7
N.furzeri 86.7 % 81.7 % 98.2 % 98.4 % 99.3 %
Table 4-3 Distances of PARP1 amino acid sequences from six fish species to each other.
Amino acid sequences were compared and the identity in percent (blue) and the amount of differences (yellow) were calculated using geneious8.
The phylogenetic tree of nine fish species illustrates the relationship of the fish regarding PARP1 (Figure 4-14). Including PARP1 cDNA sequences of two pufferfish species (Tetraodon nigroviridis and Takifugu rubripes) and stickleback (Gasterosteus aculeatus) (www.ensembl.org) into the phylogenetic tree, allows for a more accurate evaluation of the relationship between the species.
As mentioned above, N.furzeri and N.orthonotus, the two fish exhibiting the shortest life spans, are the closest related species. As expected, all Nothobranchius are very closely related. Interestingly, medaka and the Nothobranchius family have a higher degree of relation to each other than to the zebrafish, whereas the life spans and investigated enzymatic parameters show an ascending order from the annual killifish to zebrafish to medaka. This suggests that for the enzymatic activity of PARP1 the number of amino acid differences is less important than which actual amino acids are exchanged.
Figure 4-14 Bayesian phylogenetic tree from nine fish species. Phylogenetic tree based on the coding sequence of PARP1. Black color indicates fish used in this study. Distances shown at the nodes were calculated using geneious8.
In order to determine not only how many exchanges, but at which position the amino acids were different, the coding sequences of the fish PARP1s are depicted in a graph with weighted homology (3.6.7) (Figure 4-15). This is the first step toward the possibility to track the differences in the enzymatic activity back to single or short stretches of amino acids. The comparison of the Nothobranchius species (Figure 4-15, A) shows that most of the differences are located in the DNA binding domain and the automodification domain. The catalytic domain (position 525 – 1013) contains only four varying amino acids:
position 717 (N.furzeri, N.korthausae, N.rubripinnis: valine; N.orthonotus:
methionine), position 759 (N.f., N.o.: valine; N.k., N.r.: isoleucine), position 961 (N.f., N.o., N.r.: alanine; N.k.: aspartic acid) and position 977 (N.f., N.k., N.o.:
histidine; N.r.: asparagine). The sequence of the PARP signature (PS) is absolutely conserved, not only within the Nothobranchius family, but in all investigated fish species (Figure 4-15, B). Largely conserved is the N-terminal stretch of the catalytic domain (position 520 – 614) containing part of the WGR
domain. In contrast to this, more variant regions are located between DNA binding domain and automodification domain, in the BRCT domain and between zinc finger I, zinc finger II and the NLS in the DNA binding domain.
Figure 4-15 Weighted homology ofPARP1 amino acid sequences. (A) Comparison of four killifish from the Nothobranchius genus, N.korthausae, N.rubripinnis, N.orthonotus and N.furzeri.
(B) Comparison of D.rerio, O.latipes and the Nothobranchius species. Above the graphs is the schematic PARP1 protein, displaying its domains and structural features.
Although, this initial data are far from being complete, the comparison of the amino acid sequences and the analysis of the different enzymatic activity of the recombinant fishPARP1s gained in this study are a further interesting and important component towards a better understanding of the connection between PARP1-dependent DNA damage signaling and longevity.
5 Discussion
In the present study PARP1 activity and its correlation to ageing in fish was investigated. Therefore six different fish species were chosen (D.rerio, O.latipes, N.furzeri, N.korthausae, N.orthonotus and N.rubripinnis), considering phylogenetic aspects, maximum life span and morphological features. All fish display a similar size of about 4 – 7 cm, the Nothobranchius species in contrast to zebrafish and medaka show sexual dichromatism. It has been reported that D.rerio, O.latipes, N.korthausae and N.furzeri display classical biomarkers of ageing, similar to mammals, which is not a mandatory feature in fish (Ding et al., 2010; Kishi et al., 2009; Lucas-Sanchez et al., 2011; Patnaik et al., 1994;
Terzibasi et al., 2007).
Considering phylogenetic aspects, the Nothobranchius species belong to the order of Cyprinodontiformes and are very closely related to each other sharing the same genus (Nothobranchius). One of the next relatives is O.latipes, belonging to the order of Beloniformes but sharing the same superorder (Atherinomorphae). Of note, the maximum life span of medaka is at least 3-fold higher, compared to the Nothobranchius species. The zebrafish is a Cypriniformes, resident on a phylogenetically more distant branch, belonging to another superorder (Cypriniphysae).
To gain reliable information about the life spans and maximum life spans of the fish is not an easy task. On the one hand it is not possible to investigate this in the natural habitat, on the other hand life span of fish in captivity is strongly susceptible to environmental influences, as water quality, temperature and diet (Terzibasi et al., 2009; Valenzano et al., 2006a; Valenzano et al., 2006b). The variations stated for the fish in different studies, databases (e.g. www.zfin.org, www.fishbase.org and www.nothobranchius.info) and / or from hobbyists are large. Therefore, the life spans of the fishes are an estimated average of inquired data. The range of the life spans is 9 months (N.furzeri) to 60 months (O.latipes).