Tyrosine sulfation was discovered in 1950s by the first sulfated protein, bovine fibrinogen (Bettelheim, 1954), and then tyrosylprotein sulfotransferase was denoted as the enzyme that catalyzed this reaction in 1983 (Moore, 2003). Protein sulfation has been largely researched over the past 50 years, however, a number of bottlenecks serve as challenges in previous studies, such as the difficulty of sourcing the homogeneous enzyme, limited information of enzyme characteristics (kinetics), unstable sulfated groups on the substrate, and lack of sensitive detecting methods for the sulfate group. Tyrosine-sulfated proteins have been indentified in physiological processes, including coagulation, leukocyte adhesion, chemokine signaling, and HIV entry (Seibert and Sakmar, 2008). At present, the understandings of TPST function are confined on the specific substrate involved in biological functions as described above. In order to systematically analyze the roles of protein sulfation in physiological regulation, we designed a proteome-wide screening tool that basically relied on proteomic techniques. Drosophila melanogaster was chosen as the source of study animal, due to it can grow easily, short generation span, well-established genomic database, commercial transgenic lines, and more importantly, D. melanogaster only has a single TPST gene (Moore, 2003). The amino acid sequence of TPST in D.
melanogaster shares 58% and 56% with human TPST1, and TPST2, respectively (Figure 8).
Approximately 75% of known human disease genes have a recognizable match in the genetic code of D. melanogaster, and 50% of D. melanogaster protein sequences have mammalian analogues (Reiter et al., 2001) which makes D. melanogaster an appropriate animal model for pathological studies on TPST.
To study the biological function of protein sulfation, knockdown endogenous TPST by RNAi technique in D. melanogaster is the most direct way. At present, TPST knockout mice was developed by Moore group, and the dysfunction of TPST has been indicated to
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cause a severe defection of sperm motility which reveals that protein sulfation may be involved in the reproductive system (Hoffhines et al., 2008). Moreover, there is no information available about the relationship between protein sulfation and neurons, we are interested in focusing on neuron-specific knockdown due to the highly mRNA expression of TPST in the D. melanogaster nervous system (Table 1). The neuron-specific driver, APPL-GAL4, was used to cross with five different UAS-TPSTRNAi transgenic flies as our animal sources; only one had accomplished the neuron-specific knockdown in statistical significance compared to the controls, APPL-GAL4 alone and UAS-TPSTRNAi alone (Figure
3, 4). The mRNA was extracted from whole flies, therefore, the result of RT-PCR does not
directly indicate the knockdown in the nervous system, and instead, represents the knockdown of TPST in the whole fly. Theoretically, the two controls, APPL-GAL4 alone and UAS-TPSTRNAi alone, should have similar quantity of TPST mRNA expression. Figure 3 shows the two controls have different relative quantity of TPST, which may contribute to the distinct genetic background by different maintenance and growing conditions between the two controls.Total proteins were extracted from 1-7 day-old male flies with ultrasonication, and 200μg protein were loading onto each drystrip. pH4-7 Drystrips were chosen because the protein spots mostly tended to be distribute in the acidic region. Approximately 1200 proteins spots were visualized for each SDS-PAGE after silver staining, and different patterns of protein spots varied by different extracting methods. It has been estimated that only about 8% of the protein encoded by the genome could be analyzed in a two-dimensional SDS-PAGE of a total protein extract of D. melanogaster (Ericsson, 1999). Twenty-two protein spots showed a significant difference when compared with the controls, with 4 proteins up-regulated and 18 down-regulated. Among those protein spots, a number of spots seemed to have a pI shifting on the gel. Only 9 proteins were identified from MALDI-TOF mass spectrometry after preceding an in-gel trypsin digestion with trypsin. The protein loss
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during the process of in-gel digestion could be a reason to decrease the protein identification of mass spectrometry. More importantly, however, the visualizing method, silver staining in this study, was the major cause for the low efficiency of protein identification by mass.
Although silver staining methods have raised the detection limit to the nanogram range, the protein identification of excised spots was often an obstacle that cannot be overcome easily (Poland et al., 2005). In order to solve the problem, the silver staining gels are usually treated as an analytical gel; other staining methods are then used as preparative gel for mass identification, such as coomassie staining, reverse staining, and SYPRO Ruby staining (Sasse and Gallagher, 2004). We chose SYPRO Ruby and reverse staining for the preparative gel, and the mass analysis results were shown in Table 3 and Appendix 7a-i.
The neuron-specific knockdown flies down-regulated two oxidative stress proteins:
cytochrome P450 4e3 (Cyp4e3) and superoxide dismutase [Cu-Zn] (SOD1). Superoxide dismutases is an ubiquitous enzymes that functions to efficiently catalyze the dimutation of superoxide anions (Zelko et al., 2002), which is known to protect organisms from reactive oxygen metabolites (Goulielmos et al., 2003). SOD1 is widely distributed and comprised 90% of the total SOD (Noor et al., 2002), the mRNA expression of D. melanogaster is shown in Table 4. It is obvious to notice, however, that the protein spot of SOD1 (spot 20) in the gel was completely disappeared in TPST knockdown flies (Figure 5). Interestingly, TPST is only knocked down in nervous system, but it caused SOD1, expressed ubiquitously, to completely vanish in silver staining vision. Besides the actual down-regulation of the protein, the disappearance of protein spot could be contributed to the change of either isoelectric focusing point or the molecular weight of the protein, which caused a spot shift.
Further confirmation is needed for the clarification.
Based on the down-regulated Cyp4e3 and SOD1, implications can be made that the neuron-specific knockdown flies tended to suffer stress more easily, especially oxidative stress. In the oxidative stress assay, we used paraquat to increase the quantity of free radical
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in D. melanogaster. An unexpected result revealed that the mean survival time of the TPST knockdown flies was much longer than UAS-TPSTRNAi alone for 66 hours and APPL-GAL4 alone for 25 hours, (Figure 6). The longevity of the APPL-GAL4 alone might need to be clarified in advance. The incredible longevity of neuron-specific TPST knockdown flies seems to be an opposite result as we expected. Oxidative stress has been reported to be a common underlying mechanism in the pathogenesis of many neurodegenerative disorders such as Alzheimer, Huntington, and Parkinson disease (Gruenewald C et al., 2009).
Previous researches on SOD1 and neuron have indicated that overexpression of SOD1 in D.
melanogaster can reduce oxidative damage (Landis and Tower, 2005), extend lifespan (Parkes et al., 1998) (Sampayo et al., 2003), and neuron protection (Botella et al., 2008). The
overall evidence reveals that down-regulated SOD1 should decrease the survival rate of TPST knockdown flies in oxidative stress assay. Nevertheless, our result is in conflict with previous findings. The disappearance of the SOD1 (spot 20) on the two-dimensional SDS-PAGE did not result from the mRNA depletion on the gene level, which was proved by RT-PCR (Figure 7).
Moreover, there is only a single TPST gene in D. melanogaster by the analysis from BLAST. The TPST gene, however, might express two isoforms, Tango-PB and Tango-PC, with different length of amino acids. The difference between these two isoforms is that Tango-PB possessed extended C-terminal 150 amino acid residues with polyglutamine (polyQ) and polyasparagine (polyN) (Figure 9). A number of neurodegenerative diseases are characterized by the formation of intracellular protein aggregates and neurodegeneration.
The polyQ sequence can easily cause protein misfolding and the formation of inclusion body (Nagai and Popiel, 2008). In the neurons, polyQ protein inclusion are aggregated which probably induce neurotoxicity (Li et al., 2008). Therefore, the neurons are protected by
knockdowning TPST in nervous system, which decreases the inclusion proteins conducted from the aggregation of Tango-PB. Finally, it increased the survival rate of D. melanogaster.
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Further confirmation is certainly needed for this inference.
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