PARP1 from
5.1 Generation of Baculoviruses and Production of recombinant fishPARP1
To get insights in the characteristics of fishPARP1 enzyme, comparing them among each other and to draw first conclusion regarding activity differences of mammals to fish PARP1, the recombinant proteins had to be produced.
The first step in generating recombinant fishPARP1 was to isolate total RNA from different fish species. Fishes were euthanized in concordance with ethical standards and internal tissues without the gastro-intestinal tract was used for RNA isolation, following the protocol of Chomczynski and colleagues (Chomczynski and Sacchi, 1987). Internal tissues contain higher levels of PARP1 compared to scales, fins and bones. The gastro-intestinal tract was avoided because of possible contamination by the contents of stomach and intestine and degrading substances in those tissues. Concentration, contamination and quality differed in a wide range, which may have be due to insufficient phenol/chloroform extraction and mainly degradation. However, it was possible to choose a largely unscathed RNA sample for each fish species (Figure 4-1).
DNA sequence information of medaka and zebrafish PARP1 was retrieved from databases (www.ensembl.org), whereas the PARP1 sequences of the Nothobranchius species was not available at the start of the thesis. A small stretch in the middle of the sequence of N.furzeri was sequenced during my master thesis and starting from this point 3´RACE and 5´RACE was conducted.
Using the 3´RACE it was possible to amplify DNA coding the 3´-UTR region of PARP1 mRNA from the four different Nothobranchius species together with almost one third of the 3´-end of the coding sequence. This part of DNA sequence information allowed the design of gene-specific reverse primer in the 3´-UTR for further reverse transcription and PCR, thereby excluding all non-PARP1 mRNAs from amplification.
PARP1 sequence comparison of zebrafish, medaka, stickleback, salmon and two different pufferfish (Tetraodon nigroviridis and Takifugu rubripes) using GeneiousPro program allowed the design of a forward primer near the 5´-end
(bases 151 – 172) due to the high homology found in this region (middle of ZnI (bases 33 – 287)). The combination of two specific primer (14) and (46) for N.furzeri, and (14) and (47) for N.orthonotus, N.korthausae and N.rubripinnis, allowed the amplification and sequencing of the middle part of PARP1 cDNA of the Nothobranchius species, respectively.
The third and most challenging part of the PARP1 sequencing was the 5´RACE.
Different strategies were performed without success before using the 5´RACE System for Rapid Amplification of cDNA ends from LifeTechnologies. This multiple step kit allowed the amplification of the missing 5´-region of the Nothobranchius PARP1 sequences.
Employing the complete coding sequence, the PARP1 cDNAs were amplified by PCR and introduced into the cloning vector pSL1180. Transformed bacteria colonies were checked for plasmid insertion by colony screen PCR. Typically, 70 – 80 % of the analyzed bacteria were positive regarding the insertion of a DNA fragment with the correct size. Plasmids of at least three positive clones per PARP1 from each species were sequenced step by step to avoid sequencing based errors and to identify PCR caused base exchanges.
Interestingly, the PARP1 sequences of all fish species were inserted reverse into the pSL1180 vector and therefore indicating a vector – dependent condition utilized for an easy subcloning strategy.
Comparison of sequenced medaka PARP1 (olPARP1) with the published sequence revealed 8 base exchanges resulting in 1 amino acid exchange (lysine to asparagine) at position 294. This position is in a quite variable region between ZnI and ZnII, probably presenting an allelic insignificant variation.
The comparison of PARP1 sequence from all fish species and consequential conclusions will be discussed in chapter 5.3.
The generation of recombinant PARP1 was accomplished using the baculoviral expression system. This system allows the overexpression in high yields with subsequent posttranslational modifications. Therefore, the PARP1 coding sequences were subcloned into baculoviral expression vectors (pVL1392 for medaka and zebrafish, pBacPAK8 for the Nothobranchius species). Due to the
three facts, that (i) the PARP1 cDNA of all fishes was inserted reverse into the pSL1180 vector, (ii) none of the PARP1 cDNAs exhibit a restriction side for the endonucleases XmaI and XbaI, and (iii) that both baculoviral expression vectors have the desired restriction sides in the same order, an identical subcloning strategy for all PARP1 cDNAs was employed.
The vector carrying the PARP1 cDNA was amplified and tested again by colony screen PCR. Up to 90 % of the analyzed clones were typically positive in this screen. At least 2 clones from each species were stepwise sequenced to verify the correct insert and orientation.
The generation of baculoviruses carrying fish PARP1 was achieved by co-transfection of Sf9 insect cells. Positive viruses were isolated and amplified. The virus titer of all fish species was comparable (~9 x 106 pfu / ml), verifying the reliability of the system and the successful production of baculoviruses carrying fish PARP1.
The purification of recombinant proteins was conducted following the protocol of Beneke and colleagues (Beneke et al., 2000) with small modifications, described in 3.6.3. In the present study it was possible to reduce the time necessary for the purification process further from 2.5 to 1.5 days by using precast gels for SDS-PAGE and the Trans-Blot TurboTM Transfer System for Western Blotting, thus reducing the time necessary for SDS-PAGE and Western Blotting from 3.5 h to 40 min, thereby allowing to perform all purification steps before the dialysis in one day. This is a remarkable improvement, further ensuring that the activity of the enzymes is unaltered by decay due to prolonged storage in liquid phase.
The concentrations of the purified protein samples are ranging from 28 ng / µl to 82 ng / µl. This relatively wide variation may be due to different amounts of PARP1 produced in Sf9 cells or to a small extent due to the ability of the different enzymes to bind to the DNA column. Further reasons for protein loss may be one of the multiple steps during the purification process. However, all achieved protein concentrations were high enough for use in the activity assays and the purity of the enzyme samples are up to 95 %.
5.2 Characterization of recombinant PARP1 from six Fish Species
The use of recombinant enzymes allowed the measurement of the enzymatic activity in a cell-free environment and therefore excluding the influences of external cell-dependent factors. In order to determine the optimal conditions for recombinant fishPARP1, its temperature dependency was analyzed. It was shown that the optimal reaction temperature was between 27 and 30°C. In contrast, human PARP1 exhibit an optimal reaction temperature of 30 – 37°C.
Considering that mammals are homoeothermic organisms with a body temperature of about 36 – 37°C, whereas fish are poikilothermic animals the differences are completely plausible and adapted to the settings of the proteins, respectively. The habitat temperatures and therefore in consequence to that the body temperature of the fishes are diverse, ranging from about 6 – ~40°C (medaka and zebrafish) and ~15 – 35°C (killifish). The chosen reaction temperature of 30°C is in the higher optimum range of fish and in the lower range for humans/mammals, but perfectly providing a temperature which ensures comparability of the measured parameters.
The main reason for variances in the read-out of the PARP1 temperature dependency between independent experiments is the variation in the self-cast polyacrylamide gel quality. The ratio of stacking and separating gel may be varying as well as to some extent the structure and the concentration of the gel because of its handmade character. However, the trend within a species is always clearly the same.
Using the well-established technique of activity assays followed by slot blots it was possible to calculate the maximum reaction velocity Vmax and the Michaelis-Menten constant KM, and resulting from those parameters kcat (turnover number) and kcat / KM (enzyme efficiency) were determined, in addition.
The first noticeable fact is the correlation of the overall PAR production and life span. The total PAR amount of medaka (~ 950 fmol) is more than 3 fold higher than the PAR production of the killifish (~ 300 fmol), roughly reflecting the difference in the life spans. In contrast, the experiments with mammalian
PARP1 conducted by Beneke and colleagues showed no such clear correlation.
The overall PAR production of human PARP1 (V762 variant: ~ 600 fmol and A762 variant: ~ 300 fmol) is indeed higher than the produced amount of PAR from rat, which is about 220 fmol, but the PAR production of human to rat PARP1 is 2.7 fold (V762) and 1.4 fold (A762) higher, whereas the differences in life span of human to rat differ more than 20 fold.
The differences in the enzymatic parameters KM and Vmax between the Nothobranchius species is not significant. Nonetheless, for both parameters the tendency of the differences in correlation to life span is the same: The higher the substrate affinity, the longer lived is the species, and the maximum reaction velocity is increasing from short- to long-lived killifish.
The KM ratio of PARP1 from the fish species O.latipes, D.rerio, N.korthausae, N.rubripinnis, N.orthonotus and N.furzeri is 1 : 1.1 : 2.26 : 2.35 : 2.34 : 2.84, ranging from 58.1 µM NAD+ (O.latipes) to 165.8 µM NAD+ (N.furzeri), thus showing a strong correlation between substrate affinity and maximum life span.
However, the type of the correlation – linear or exponential decay – remains to be determined. Considering the “biological background” of the measured substrate affinity, there must be a biological limit for the minimum and maximum. Therefore, a correlation with exponential decay is probably the more likely scenario. Fitting rat PARP1 (KM: 59.1 µM; maximum life span: 48 months) into the graph results in a perfect extension for both linear and exponential decay correlation, whereas the addition of human PARP1 (KM: 33.9 µM (V762) and
38.2 µM (A762); maximum life span: ~1320 months) only supports the exponential decay variant. Assuming that mammalian PARP1 activity acts in the same way as fish PARP1 an exponential correlation of substrate affinity and maximum life span can be proposed. A further alternative explanation may be that the direct correlation of KM and maximum life span is only true for short lived animals, with a life span up to a few years. To be able to answer those questions, it would be necessary to analyze PARP1 from further fish and mammals with life spans ranging from 10 to 120 years, as for example cichlids
(depending on species, life span: ~ 10-15 years), carp (life span: ~ 40 years) and sturgeon (life span: ~ 100 years).
The maximum reaction velocity Vmax of fish PARP1 show the same strong correlation to the maximum life span as the substrate affinity. The values ranging from 806.9 (nfPARP1) to 2448.3 (olPARP1) pmol PAR / min * µg fishPARP1, presenting a 3 fold difference from long- to short-lived fish species.
Vmax of PARP1 from rat is 1121.9 pmol PAR / min * µg rPARP1, and from the two different human PARP1 alleles Vmax is 3313 pmol PAR / min * µg hPARP1 (V762) and 1563.7 pmol PAR / min * µg hPARP1 (A762) (Beneke et al., 2010).
The addition of the rat PARP1 in the graph life span versus Vmax confirms the linear correlation. The maximum reaction velocities of both allelic variations of human PARP1 do not fit into this picture.
Interestingly, the differences in the ratio of Vmax of fish PARP1 is almost the same ratio as of KM values from nfPARP1 and olPARP1, thereby indicating a similar contribution of KM and Vmax to the overall PARP1 activity. In contrast to this are the PARP1s of rat and human, where the Vmax ratio is 1 : 2.95 (V762) and 1 : 1.39 (A762), and the KM ratio is 1 : 1.74 (V762) and 1 : 1.55 (A762), respectively.
The turnover number kcat is defined as number of substrate molecule each enzyme site converts to product per unit time. kcat is calculated using Vmax and the total concentration of the enzymes. Because the total concentration of recombinant PARP1 protein was identical for all fish species the turnover number is reflecting same ratio to each other as the maximum reaction velocity.
The enzyme efficiency kcat / KM on the other hand reflects the combination of both parameters, KM and Vmax, and therefore gives a characteristic value of the overall PARP1 activity. Beneke and colleagues could show that the enzyme efficiency displays the same 5-fold difference between rat and human recombinant PARP1 as measured almost 20 years ago in permeabilized mononuclear leukocytes by Grube and Bürkle (Beneke et al., 2010; Grube and Burkle, 1992). Using the enzyme efficiency the difference between the
Nothobranchius species and the “long-lived” species O.latipes and D.rerio is most obvious.
This is different to mammalian PARP1, where the enzyme activity seems to be mainly dependent on Vmax, whereas the differences in KM are less prominent.
Fish PARP1 seems to be regulated equally by KM and Vmax. Still, it remains to be investigated if this feature is dependent on the differences between “short-lived” to “long-“short-lived” or mammal versus fish.
5.3 Comparison of Amino Acid Sequences from six Fish Species
The generated bayesian phylogenetic tree based on the coding sequences of PARP1 reflects largely the results gained by Dorn and colleagues (Dorn et al., 2014), who described a classification of the south African killifish. They stated that N.furzeri and N.orthonotus belong to the same subgroup (“southern clade”), whereas N.korthausae belongs to the “coastal clade”. In this study N.rubripinnis was not included. The relationship of medaka, zebrafish and the Nothobranchius genus to each other based on the PARP1 coding sequence provided in this thesis is in accordance with the generally accepted phylogenetic analysis.
The comparison of the PARP1 amino acid sequences of the examined fish species showed, that it is crucial for the enzymatic activity, which amino acids are different, and not total variation. The enzymatic activity (KM and Vmax, as well as the enzyme efficiency kcat / KM) of recombinant PARP1 from O.latipes is higher than D.rerio PARP1, which is higher than the activity of Nothobranchius PARP1, and thus correlating with their life spans. The analysis on the other hand shows that O.latipes is quite closely related to the genus of Nothobranchius and the zebrafish is the outsider in the scenario. The activity of
the PARP1 protein seems thus to be largely independent from the level of relation, but depends probably on exchanges of specific key residues.
The PARP signature, localized in the C-terminal region of the protein (aa:
859 – 908) is completely conserved between the investigated fish species.
PARPs also can be characterized by the NAD+ binding core with a central 6-stranded β-sheet using 3 motifs, located in the first, second and fifth β-sheet.
All so far investigated eukaryotic PARP1s display the evolutionary conserved triad H-Y-E, which is formed from the above mentioned 3 motifs together (Hottiger et al., 2010). PARP1 of all six fish species possess the H-Y-E triad and further exhibit the same motifs (R/H-G-T/S motif of β-strand 1: HGS, S-X-S/Y-X-X motif of β-strand 2: YFA and S-X-S/Y-X-X-S-X-S/Y-X-X-E motif of β-strand 5: YNE) as human and mouse PARP1, thus being in line and confirming the published data.
The human population contains in the PARP1 gene the interesting polymorphism V762A, which results in a drastic reduction in PARP1 activity and is connected with the susceptibility to various types of cancer (Beneke et al., 2010; Li et al., 2007; Lockett et al., 2004; Wang et al., 2007). The residue comprising higher activity, valine, is conserved in the investigated fish, whereas the area surrounding this amino acid position (aa: 758 -768) show 6 amino acid exchanges, being a first potential area for activity regulation, as one objective of this study was to identify possible stretches of amino acids responsible for differences in activity. A promising position may be residue 763: here, N.furzeri and N.orthonotus, the both short-lived fish contain a valine, the other “longer-lived” Nothobranchius species, as zebrafish and medaka exhibit isoleucine.
Both amino acids are hydrophobic and quite similar, but the same is true for valine and alanine, the amino acids in the human PARP1 allele variation.
Another potential approach could be to determine the amino acid residues, conserved between the Nothobranchius species and the zebrafish, but show a variation in comparison to PARP1 from O.latipes, the most long-lived species.