Protein sulfation had been extensively researched for more than 50 years, but there remains a lot of questions and basic knowledge that still needs to be investigated. The present study serves as the first to examine the tissue-specific sulfoproteomics by using
Drosophila melanogaster as the animal model. TPST in nervous system is specifically
knockdown due to the highly TPST mRNA expression in the head and brain of the D.
melanogaster. By the manipulation of RNA interference technique and GAL4-UAS system,
the TPST in the nervous system was successfully knockdowned with statistical significance.
Proteomic analysis following protein identification from mass spectrometry revealed that protein tyrosine sulfation might be involved in metabolism and oxidative stress. Moreover, the oxidative stress assay showed the surprisingly result that the neuron-specific TPST knockdown flies had remarkably longer survival rates. These results are important to comprehend the biological roles and regulatory actions of protein tyrosine sulfation in the nervous system.
23
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27
Tables
Table 1. The tyrosylprotein sulfotransferase gene expression analysis of adult D.
melanogaster (FlyAtlas).
Tissue mRNA Signal Present Call Enrichment Affy Call
Brain 264 ± 10 4 of 4 2.8 Up
Table 2. Criteria of selecting spots in 2-D gel for protein analysis.
APPL-GAL4>
UAS-TPSTRNAi UAS-TPSTRNAi/+ APPL-GAL4/+
o o o o x o o o x
o x x up-regulation
x o o down-regulation
x x o
x o x x x xO indicated the up-expression of protein spot X indicated the down-expression of protein spot
indicated the up/down-regulation of protein spot that actually changing by TPST knockdown system.
29
30
Table 3. Protein changes in neuron-specific TPST knockdown Drosophila melanogaster.
Spot # ORF Names Protein Name (Possible) Function
1 GE16567 Retinoblastoma-associated protein B domain Retinal cancer up-regulated
2 CG4105 Cytochrome P450 4e3 Oxidation reduction down-regulated
3 CG31779 ACP24A4 Serine-type endopeptidase inhibitor activity down-regulated
4 GM05777p Unknown down-regulated
7 CG9062 RE72568p Adaptor/regulatory modules in signal transduction up-regulated pre-mRNA processing and cytoskeleton assembly
8 GA20134 Alanine dehydrogenase Metabolic process down-regulated
Rossmann-fold NAD(P)(+)-binding proteins
17 GA10647 Phosphoenolpyruvate carboxykinase Gluconeogenesis up-regulated 18 CG6058 Fructose-bisphosphate aldolase Glycolysis down-regulated
20 CG11793 Superoxide dismutase [Cu-Zn] Oxidation reduction down-regulated
31
Table 4. The superoxide dismutase [Cu-Zn] gene expression analysis of adult D.
melanogaster (FlyAtlas).
Tissue mRNA Signal Present Call Enrichment Affy Call
Brain 1789 ± 20 4 of 4 1 None
Figures
Figure 1. The tyrosylprotein sulfotransferase reaction. TPSTs catalyze the transfer of sulfate
from the universal sulfate donor PAPS to the hydroxyl group a luminally oriented peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3’, 5’-ADP.32
Figure 2. Directed gene expression in D. melanogaster. To generate transgenic lines
expressing GAL4 in neuron-specific patterns, the GAL4 gene is inserted randomly into the genome, driving GAL4 expression from genomic enhancer (APPL). A GAL4-dependent target gene can then be constructed by subcloning any sequence behind GAL4 binding sites.The target gene is silent in the absence of GAL4. To activate the target gene in neuron-specific pattern, flies carrying the target (UAS-TPSTRNAi) are crossed to flies expressing GAL4 (Enhancer Trap GAL4). In the progeny of this cross, it is possible to activate UAS- TPSTRNAi in cells where GAL4 is expressed and to observe the effect of this directed misexpression on development.
33
Figure 3. TPST mRNA expression of five neuron-specific TPST knockdown lines detected by RT-PCR. The neuron-specific TPST (black) was knockdowned compared to
flies carrying UAS-TPSTRNAi alone (light gray) and APPL-GAL4 alone (dark gray).34
Figure 4. RNAi knockdown of TPST in the fly detected by RT-PCR. The neuron-specific
TPST knockdown by APPL-GAL4. The neuron-specific TPST (black) was knockdowned compared to flies carrying UAS-TPSTRNAi alone (light gray) and APPL-GAL4 alone (dark gray), whose P<0.05. The p values were calculated by Student’s t test. All experiments were carried out with at least three independent replicates.35
Figure 5. 2-D electrophoresis of neuron-specific TPST knockdown fly and two controls.
(a)APPL-GAL4>UAS-TPSTRNAi; (b) UAS-TPSTRNAi/+; (c) APPL-GAL4/+. The arrow indicates the up-regulated protein expression. The flies ranging from 50 to 100 were homogenized by ultrasonication. The 2-D electrophoresis was performed under 13-cm Immobiline DryStrips with pH 4–7 for isoelectric focusing and then 12.5% SDS polyacrylamide gels with silver staining. Spot 20 is the protein spot of superoxide dismutase [Cu-Zn].
36
Figure 6. Oxidative Stress Assay of neuron-specific TPST knockdown flies. The 10 mM
paraquat was treated to neuron-specific TPST knockdowned flies (black), UAS-TPSTRNAi alone (light gray), and APPL-GAL4 alone (dark gray). The knockdown flies have remarkably longer survival rate compared with the two controls, whose P<0.001. The p values were calculated by Student’s t test.37
Figure 7. The mRNA of superoxide dismutase [Cu-Zn] (SOD1) remains intact in
neuron-specific TPST knockdown flies as measured by RT-PCR.38
Figure 8. Sequence alignment of human TPST1 (hTPST1), human TPST2 (hTPST2), and D. melanogaster TPST (dmTPST). The sequence alignment is performed by ClustalW
(http://www.ebi.ac.uk/clustalw/). The * indicated identity to each other and the．meant conserved substitutions.39
Figure 9. Sequence alignment of D. melanogaster TPST. Tango-PB and Tango-PC were
the two isoforms from one TPST gene. The sequence alignment is performed by ClustalW (http://www.ebi.ac.uk/clustalw/). The * indicated identity to each other and the．meant conserved substitutions.40
Appendices
Appendix 1. Some common and important post-translation modifications (Mann et al.,
2003).41
Appendix 2. General ST-catalyzed reaction with PAPS as the cosubstrate (Chapman et al.,
2004).42
Appendix 3. Sulfate activation and tyrosine O-sulfation. Inorganic sulfate enters the cell
by the action of one of several sulfate transporters. Once in the cytosol, sulfate is then activated by the action of one of two PAPS synthases (PAPSS1 or PAPSS2). These bifunctional enzymes contain a C-terminal ATP sulfurylase domain and an N-terminal adenosine phosphosulfate (APS) kinase domain. In the first step of sulfate activation, ATP and inorganic sulfate are converted to APS and pyrophosphate by ATP sulfurylase. APS is then channeled directly between the ATP sulfurylase and APS kinase active sites. In the second step catalyzed by the APS kinase domain, a second ATP is consumed to phosphorylate the 3’-hydroxyl of the ribose ring of APS to yield PAPS and ADP. PAPS is then transported into the Golgi lumen by a PAPS translocase that has been purified but not yet cloned. This transporter functions via an antiporter mechanism with PAP as the returning ligand. Once inside the Golgi lumen PAPS acts as the sulfate donor for TPSTs and all other carbohydrate sulfotransferases, and the sulfated products are either secreted or retained in the membrane of lysosomes, secretory vesicles, and/or the plasma membrane. TGN, trans-Golgi network. (Moore, 2003).43
Appendix 4. Comparison of the expression level of TPST-1 and TPST-2 in 20 human tissues. Human TPST-1 and TPST-2 mRNA levels were quantified in arbitrary units,
normalized against b-actin signal. The data represent calculated mean values derived from three experiments. The solid bars correspond to the levels detected for TPST-1, and the open bars correspond to the levels detected for TPST-2 (Mishiro et al., 2006).44
Appendix 5. Schematic representation of cell entry by HIV-1 following sulfonation of CCR5 by a tyrosylprotein sulfotransferase (Chapman et al., 2004).
45
Appendix 6. Tyrosine sulfation plays an important role in the immune response. (a)
Leukocytes roll upon, adhere to and transmigrate between endothelial cells at sites of inflammation. P-selectin and its ligand, PSGL-1, are often required for this process. (b) PSGL-1 is a mucinlike glycoprotein that appears to be an extended rod shape in vivo. The extreme amino terminus of PSGL-1 carries three tyrosine sulfation sites, shown in yellow.These sulfate esters, and specific glycans on PSGL-1, are key binding determinants for P-selectin (Kehoe and Bertozzi, 2000).
46
(a)
1 MICCIDLIYN NVLAEKRTDL INPKFEGLPS NWTELDFRHK PHCILAYFCD 51 MTEEAKAMKA TTFRQIISSF FQASTIYGNK DTMLGLLANE NFERNLKSLN 101 ISYEQYVLSV GEFDERILSA YDAGEHTALN DQSLRPPVTP LTRKQDLPAQ 151 TAMAGDKFEP VRNATNNVKQ LTAFGRITEP TDFVKQAGEE VIAKLLSIIE 201 GIKQKFLAKY PSTEARSRFR LAKSFYFYLL DQILQAEIRN KPDIDLKRLL 251 VQKISLDIFN ITLMACCVEL VLEAYKTELK FPWVLDCFSI SAFEFQKIIE 301 IVVRHGSHEG CLNRSLIKHL NSIEETCLER LAWARNSTVW DMIASFPLPL 351 PTWLRVNLDR SAGALQIFLR KVYLLGWLRI QKLCSELSLC EKTPECIWHI 401 FEHSITHETD LMKDRHLDQN IMCAIYIYIR VKRMEDPKFS DIMRAYRNQP 451 QAVNSVYREV FIGDNEDGEP KVKDIIHFYN NTYVPVIRQF AIDYLNVTPD 501 VSGRISDLQL SPHPKERTAQ PKKVTQSHSL FVSQMPKNEI QQSPNQMVYS 551 FCRSPAKDLQ AMNEKVRGGK RMLSFGDEPG LGGIVEMKRP HLSQIKAVMD 601 DRELQSAEQR PAVPAEEGVG GVGGAGGGGG GEHET
(b)
1 MWLAVLALLV LPLITLVYFE RKASQRRQLL KEFNGPTPVP ILGNANRIGK 51 NPAEILSTFF DWWYDYGKDN FLFWIGYSSH IVMTNPKQLE YILNSQQLIQ 101 KSTIYDLLHP WLGHGLLTSF GSKWHKHRKM ITPSFHFNIL QDFHEVMNEN 151 SAKFMTQLKK ASAGDTIIDF QEHANYLTLD VICDTAMGVP INAMEQRDSS 201 IVQAFRDMCY NINMRAFHPF KRSNRVFSLT PEFSAYQKTL KTLQDFTYDI 251 IEKRVYALQN GGSKEDHDPS LPRKKMAFLD TLLSSTIDGR PLTRQEIYEE 301 VSTFMFEGHD TTTSGVSFSV YLLSRHPDVQ RKLYREQCEV MGHDMNRSVS 351 FQEIAKMKYL DLFIKEAQRV YPSVPFIGRY CDKDYDINGS IVPKGTTLNL 401 ALILLGYNDR IFKDPHHFRP ERFEEEKPAP FEYLPFSAGP RNCIGQKFAL 451 LELKTVISKV VRSFEVLPAV DELVSTDGRL NTYLGLAPDE KLKREAGRHK 501 YDPILSAVLT LKSDNGLHLR LRERRS
47
(c)
1 MFAEICGLPK VDSGKCLAYF KLWTYDSKKN KCVIFIYGGC QGNENSFKSK 51 EEFCGLPAAA NGNCLALFSR WSYDAQYNVC FNFIYGGCQG NENSFESQEE 101 CINKCVE
(d)
1 MLYMIWYTVC AFAVPMSKNR KTKKKKK
48
(e)
1 MLTHKTCQAR KKMQVSFVIR DAEEKQHRNG VNALQLDANN GKLYSAGRDA 51 IIRVWNTRTD SSEKYIQSME HHNDWVNDIV LCCNGRNLIS ASCDTTVKVW 101 NAQKGFCMST LRTHRDYVQA LAYAKDREQV ASAGLDKAIF LWDVNTLTAL 151 TASNNTVTTS SLTGSKDSIY SLAMNPSGTV IVSGSTENIL RIWDPRTCMR 201 RMKLRGHTEN VRCLVVSPDG NQVVSGSSDG TIKVWNLGQQ RCVQTIHVHK 251 EGVWSLLMSE NFQYIISGSR DRNIIVTEMR NPSNKTLVCE EQAPVLSLGY 301 NIDKTGVWAT TWNSDIRCWK LPMYDRCTLN SSGGMDAQWT QGGTEVACIK 351 GGATIKECAV LNDKRYIITK DSQDQVVVYD VLRVVKKEQL GAVDFEAEVK 401 KRNKQVYIPN WFTVDLKTGM PTIVLGQEEV DCFSAWVSIE AGLPECVDPT 451 TEIKINYGKL LLEALLEYWT PPHSIPPNEM EPDMHGNGYF QVPKHTPVIF 501 SEVGGRTVCR LLVRDAAGDS ESTLLHETAP QWVTDVVIEK NIPKFLKIPF 551 FLQPHPQMTK PERTKKDRLV ANEFIQCRKV CEHVLEKVLN AETTPSGGNA 601 NNSLQNSQSD ANSEGSQLPA EERIELWCND VVVDPNMDLR TVRHFIWKQS 651 TDLTFQYKTK QNFNYDGK
49
(f)
1 MWRVIQQRAT IAHPLVRQRH SRVIAIRRED QSVWERRAPF GPTHVQKLVK 51 QNVKVIVQPS NRRAYPMQAY MQAGAHIQED ISEASVIFGV KQVPIDALIP 101 GKTYCFFSHT IKAQESNMPL LDAVLEKKIR LIDYERIIDE RGARQVAFGK 151 YAGVAGMVNI LHGIGLRLLA LGHHTPFMHI GPAHNYRNSS MARQAIRDCG 201 YEISLGMMPK SIGPLTFVFT GSGNVSQGAQ EVFSELPIEY VPPEMLRKVA 251 EHGNQNKLYG CEVSRSDHLE RRDGGGFDAK EYDEYPERYI STLSTKIAPY 301 ASVIVNGIYW AVGSPKLISI PDAKNLLRPA NTPWLPVSKG SPALPHRMLA 351 ICDISADPGG SIEFMNECTT IDTPFCLYDA DRNKDTKSFK GPGVLVCSID 401 NMPTQLPRES TDLFGELLAP HVHDIIKSDA KKPLSEEQFS YPIQSAIIAS 451 NGELTEGFQY IQELRESQSN RSRHKMEGSS ESHKKVLVLG AGMVSAPLVE 501 WLHREKDVNI TVCSQVKDEA DRLANQYAGV DSVYLDVNES TGHLQELCGK 551 ADVVVSLLPY SLHGMVARYC VAEGTHMVTA SYLNDEISGL HDEAKAKGVT 601 IMNEVGLDPG IDHLLALECI HEVQEKGAVV ESFVSYCGGL PAPEHSNNAL 651 RYKFSWSPRG VLLNTLSAAK YLSRGQIVEI SGGGELMSTP RSLDFLPGFA 701 LEGFPNRDST KYGSLYGLGR DVHTLLRGTI RYKGFSESIK PMQLLGLIDP 751 EPHSMLHPSG PDVTWRQLVI HLLGMSDSSI FYENLKQKLN ERIGDVDCIE 801 SLGLLEETPV VKLNTPLDTL SHYLSKRLAF EREERDLVVL RHEVGIRWPD 851 GRREERGINF VVYGQPQGHS AMAMTVGKPA AIAAKMILDG EIQERGVLLP 901 FTPDIYRPML QRLRSEGLTA TETSRWLN
50
(g)
1 LVGPPKSYNF RLVCRSLSVQ YGDAKVLTPA VKQYVEKCVD LCQPERVHIC 51 DGSAGESKLL QGLMLKQGTI LPLPKYDNCW LARTNPADVA RVESKTFICT 101 ERREQTVPVT PKATPGTLGN WISEADLQAA IGERFPGCMK GRTMYVIPFS 151 MGPVGSPLSK IGIEITDSPY VVESMKIMTR AGNPVLDVLR EGDGQFVKCL 201 HSVGTPKSGV QVQSSWPCDP ERTIILHKPA DNEIVSYGSG YGGNSLLGKK 251 CFALRIGSTI AKREGWLAEH MLILGITNPE GKKIYVAAAF PSACGKTNLA 301 MMTPSLPGYK VECVGDDIAW MKFDSKGVLR AINPENGFFG VAPGTSRATN 351 PIAMDTIFRN TVFTNVASTS DGGVYWEGME SAQLKDLTVT DWLGKLWSKE 401 SGKPAAHPNS RFCTPATQCP IIDPAWEDSA GVPISAILFG GRRPAGVPLV 451 YEARDWTHGV FIGAAMRSES TAAAEHKGKV IMHDPFAMRP FFGYNFGDYL 501 SHWLSMEKRG TVPKVFHVNW FRKSSEGKFL WPGFGDNSRV LDWIFRRVQG 551 EKCFEDSPIG RLPSKGALNV SGIEGNIDLS QLFDLPKAFW QQEALEIERY 601 FEEQVGGHLP RPVAEQLTEL KARVANM
(h)
1 MTLITSEGII AEVSVTLLSY KNIIVENYTR ISKMTTYFNY PSKELQDELR 51 EIAQKIVAPG KGILAADESG PTMGKRLQDI GVENTEDNRR AYRQLLFSTD 101 PKLAENISGV ILFHETLYQK ADDGTPFAEI LKKKGIILGI KVDKGVVPLF 151 GSEDEVTTQG LDDLAARCAQ YKKDGCDFAK WRCVLKIGKN TPSYQSILEN 201 ANVLARYASI CQSQRIVPIV EPEVLPDGDH DLDRAQKVTE TVLAAVYKAL 251 SDHHVYLEGT LLKPNMVTAG QSAKKNTPEE IALATVQALR RTVPAAVTGV 301 TFLSGGQSEE EATVNLSAIN NVPLIRPWAL TFSYGRALQA SVLRAWAGKK 351 ENIAAGQNEL LKRAKANGEA ACGNYTAGSV KGFAGKDTLH VDDHRY
51
(i)
1 MVVKAVCVIN GDAKGTVFFE QESSGTPVKV SGEVCGLAKG LHGFHVHEFG 51 DNTNGCMSSG PHFNPYGKEH GAPVDENRHL GDLGNIEATG DCPTKVNITD 101 SKITLFGADS IIGRTVVVHA DADDLGQGGH ELSKSTGNAG ARIGCGVIGI 151 AKV
Appendix 7. MS analysis of (a) GE16567; (b) CG4105; (c) CG31779; (d) GM05777p; (e) CG9062; (f) GA20134; (g) GA10647; (h) CG6058; (i) CG11793. The probability based
Mowse score of the mapped fragment peptides. Ions score is -10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores > 50 indicate identity or extensive homology (p<0.05). The mapped fragment peptides of (A)-(I) are colored in red.
52