Daniel L. Allen and Colin S. Burns Chemistry Department East Carolina University DLA0430@ecu.edu A Literature and Experiment Based Evaluation of Synthesis Techniques for Prothymosin-α Derived Peptides General Protein Information Prothymosin-α (ProTa) is a protein made up of 110 amino acids and located primarily in the nucleus of cells. The precise function of this protein is unclear, however evidence strongly suggests it is associated with several cellular activities and, in particular, cell proliferation. Prior research indicates that ProTa has the following attributes: •Chromatin Remodeling Activity1 •Immunomodulatory Activity2 •Growth-Promoting Effect3 •Zinc-Binding Property4 •Enhancement of Estrogen Receptor Transcriptional Activity5 •Protection of Cellular Apoptosis6 •Oxidative Stress Defense Gene Regulation6 •Enhancement of T Lymphocyte Function7 Because of ProTa’s high level of expression in a wide variety of cell types and the fact that it is relatively conserved (especially in vertebrates), as can be seen in the alignments of Figure 1 to the right, it is expected to play an essential role in organisms. Recent research has also revealed that this protein possesses significant anti-HIV activity and efforts are underway to understand how it suppresses viral replication. The use of synthetic peptides derived from the central region of ProTa has also been shown to be effective at inhibiting HIV replication. These small ProTa derived peptides can also be used as a means to test the effects of variations in sequence identity in order to increase the desired functional response in cells (anti-HIV activity for example). There is further evidence that ProTa may be phosphorylated in vivo, and we hypothesize that this phosphorylation may be significant with regards to its anti-HIV activity. Since the phosphorylation state of a protein can be used to control its activity (i.e. functionally turn it on or off), we would also like to know if phosphorylation increases or decreases the protein’s anti-HIV properties. The protein itself can undergo many known posttranslational modifications and these too are shown in Figure 1 to the right and include the following: •Initiator Methionine Removal8 •N-Acetylation9 •Phosphorylation of Serine Residues10, 11 •Phosphorylation of Threonine Residues12, 13 •Caspase Cleavage at Three Binding Sites14 In addition to its wide range of functions and post-translational modifications, ProTa has also been found to exist at multiple sites in the human genome. Interestingly, the duplication events did not produce repeated genes ordered sequentially, but rather created copies of the gene throughout the genome as can be seen in Figure 2 to the right. Similar patterns exist in other species which indicates a temporally spaced series of duplication events, especially in vertebrates. For example, Homo sapiens (humans) have 19 copies15, Pan troglodytes (chimpanzee) 2216, Mus musculus (mouse) 2617, Bos taurus (cow) 1318, Danio rerio (zebra fish) 219, and Gallus gallus (chicken) 120. As can be seen by comparing the human karyogram in Figure 2 to the chimpanzee’s in Figure 3, a broad level of evolutionary conservation exists with regard to ProTa gene location. This is most apparent when viewing chromosomes 6, 7, 11, 13, 14, 17 and 20, which have gene placements in identical locations. The pattern of placement throughout the genome indicates that the gene may be part of a transposon and/or that the various duplicates may be expressed in response to varied signals which correspond to the different functions that ProTa possesses. Further research would be necessary to evaluate these hypotheses. Evaluation of Synthesis Techniques Figure 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 1 ------M SDA ------MSDA ------MSDA ------MSDA ----------------SDA ------MSDA ------MSDT ------MSDT ------MSDT ------MADT ------MADT -----MESVA MSTRKRKQEA 11 AVDTSSEITT AVDTSSEITT AVDTSSEITT AVDTSSEITT ---------AVDTSSEITT AVDTSSEITT SVDASVEKTT AVDASVEKTP AVDASVEKTT KVDTSKEVSA KVDK-KEISA AHKDEVTHVE EDEEELQALP 21 KDLKEK-KEV KDLKEK-KEV KDLKEK-KEV KDLKEK-KEV ----EK-KEV KDLKXX-KEV KDLKEK-KEV KDLKSKDKEL KDLKAKEKEV KDLKAKEKEV KDLKEK--KQ KDLKEK--KL SKIEELKRKS SDESEEEEEY 31 VEEAEN GRDA VEEAENGRDV VEEAENGREA VEEAENGRDA VEEAENGREA VEEAENGREA VEEAENGRDA VEETENGKDK VEEAENGKDK VEEAENGKDK VEEAENGKDA VEEAENGKDA VDAKDVADHE QSSDAEGDDY 41 PANGNAENEE PANGNA-NEE PANGNA-NEE PANGNAQNEE PANGNA-NEE PANGNA-NEE PANGNAQNEE PANGNAENEE PTNGNAENEE PTNGKAENEE PANGNA--EN PANGKKAKEA AKNGN--DEP DDSEEGSEEE 51 NGEQEADNEV NGEQEADNEV NGEQEADNEV NGEQEADNEV NGEQEA---NGEQEADNEV NGEQEADNEV NGEDGADNENGEQE--NEG NGEPEVDNEG EENGDQENEV EENGDVED-V VVKKIRKEAG EEEEYDEDEG 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 81 EEDGDEDEEA EEDGDEDEEA EEDGDEDEEA EEDGDEDEEA ---------EEDGDEDEEA EEDGDEDEEA DDEGDEDDEA DD--DEDDEV DD--DDDDEV DDD--EDEEA EDD-EDDDEV DEDGTEDDAS KAEDQEDDDE 91 ESATGKRAAE ESATGKRAAE EAATGKRAAE EAPTGKRVAE -APTGKRAAE EAATGKRAAE EAPTGKRVAE DGATGKRAAE EGATGKRAAE EGVTGKRAAE EGGTGKRAAE EGRAGKRAAE QDTKGTNGTS DGAGEEYDEE 101 DD-----EDD DD-----EDD DD-----EDD DD-----EDD DD-----EDD DD-----EDD DD-----EDD DD----DEDD DDE---DDDD DDE---DDDD DD----DDDE DDEDDDDDDE KCC------DDEGTNDTAD 111 D----VDTKK D----VDTKK D----VDTKK D----VDTKK D----VDTKK D----VDTKK D----VETKK D----VDAKK D----VETKK DD---VEIKK DD---VDPKK ED---VGTKK -----VCVIT KSGSAQAAKK 121 QK----TDED QK----TDED QK----TDED QK----TEED QK----TDED QK----TDED QKK---TDED QK----TDDD QK----TDED QK----TDED QK----TDVQK----TD-D S--------GKGGDVPQED 131 D-------D-------D-------D-------D-------D-------D-------D-------D-------D---------------D---------------DVSEVEDED Species Key Variations from Human Sequence 1. Homo sapiens (Human) (0 changes)9 2. Pongo abelii (Sumatran orangutan) (2 changes)21 3. Bos taurus (Bovine) (3 changes)22 4. Mus Musculus (Mouse) (4 changes)23 5. Capra hircus (Goat) (4 changes)24 6. Sus scrofa (Pig) (5 changes)25 7. Rattus norvegicus (Rat) (7 changes)26 8. Rana esculenta (Edible frog) (25 changes)27 9. Xenopus tropicalis (Western clawed frog) (26 changes)28 10. Xenopus laevis (African clawed frog) (32 changes)29 11. Danio rerio (Zebrafish) (38 changes)30 12. Salmo salar (Atlantic salmon) (46 changes)31 13. Schistosoma mansoni (Blood fluke) (83 changes)32 14. Penicillium marneffei (strain ATCC 18224 / CBS 334.59 / QM 7333) (117 changes)33 61 DEEEEEGGEE DEEEEEGGEE DEEEEEGGEE DEEEEEGGEE ---------DEEEEEGGEE DEEEEEGGEE -EEEEVD--E DEEEEVD--E DEEDEVD--E DEEDDDV-AE DEEEDVG-EE DTDEDEKAHS GEESKENAPA 71 EEEEEEGDGE EEEEEEGDGE EEEEEEGDGE EEEEEEGDGE ---------EEEEEEGDGE EEEEEEGDGE EDEEDEGEGD EDEEDEVEGD EDEEDEVEGE EDEEDDGEGD EDEEEDVEGD PESRSAVAEL PKKRKTAHDD The goal of the experimental portion of this project was to develop useful techniques for synthesizing small ProTa derived peptides to be used to evaluate the protein’s anti-HIV activity. Specifically, three such peptides were created which addressed the issues associated with the creation of phosphorylated peptides in vitro as well as glutamimide formation as an unwanted side reaction during synthesis. The first two peptides dealt with the difficulty of creating peptides with bulky phosphorylated residues. A ten residue amino acid chain corresponding to residues 8190 in the human ProTa sequence (EAESATGKRA) was first created with a phosphoserine at a 0.1 mmol scale. All reagents were in 0.4 mmol excess except for the phosphoserine which was used in 0.3 mmol excess to reduce wasted reagent. The difficulty of the phosphoserine coupling was circumvented by allowing the coupling times of the residue and the subsequent residue to be increased to 2 hours (from the usual 1 hour). The resultant peptide was purified via HPLC and then analyzed by mass spectrometry. It was found to be highly successful with o a small percentage of the product containing deletions. What’s more, the vast majority of the deletions that did occur were in residues other than the phosphoserine meaning that the conditions and extended coupling time used for that residue were adequate. Post-Translational Modifications to Human Sequence 7 Initiator methionine removed8 8-36 Thymosin α1 8-139 Prothymosin α 8 N-acetylation9, Phosphoserine for approximately 2% of prothymosin during entire cell cycle10, 11 14, 19, 20 Phosphothreonine created in the cytosol by prothymosin α phosphorylating kinase which is composed of two protein subunits of mass 64 and 60 kDa12 127 Phosphothreonine13 47 Deletion in prothymosin α isoform 2 101-117 3 caspase binding sites (DDED, DEDD, DDVD)14 96-97, 119-122 Proposed nuclear localization site14 This figure shows the various post-translational modifications of Human ProTa as well as the level of conservation of sequence between various species (see keys above). Note that the numbers associated with the modifications listed correspond to their position in the graphic and not the number of the amino acid(s) affected in the human sequence. Figure 2 This figure shows a human karyogram with the various locations of ProTa replicates marked by arrows. The most highly expressed replicate is marked by a rectangle.15 The second phosphorylated peptide synthesized utilized the same sequence and conditions except that a phosphothreonine was used and not a phosphoserine. In the case of this peptide, both the conditions used and results obtained were the same meaning that extended coupling times again produced proper incorporation of the phosphorylated residue. Figure 3 This figure shows a chimpanzee karyogram with the various locations of ProTa replicates marked by arrows. The most highly expressed replicate is marked by a rectangle.16 Conclusion The experimental goals of this project centered on developing high-fidelity synthesis techniques for both the highly charged phosphorylated as well as other glutamimide prone ProTa derived peptides. These were both elucidated with a high success rate attributable to altered coupling times and conditions during the synthesis process. The literature evaluation focused on mapping the points of post-translational modifications, comparing the human sequence to various phylogenetic lineages, assessing and describing the various functions attributed to the peptide as well as where these functions are expressed, and assessing the expression of the protein throughout the cell cycle. As research continues, elucidation of the evolution of the protein and the ramifications this has towards the overall functions will be explored. Additional testing of the ProTa derived peptides for anti-HIV activity will undoubtedly continue. The final peptide synthesized was derived from residues 66-75 of human ProTa (EEEGDGEEED) and was created to model techniques to evade cyclization and glutamimide formation during synthesis of larger peptide sequences. It utilized a benzyl ester protecting group (Obzl) on the glutamate residues to prevent both synthetic as well as analytical problems attributable to negative charges. Additionally, this peptide used a nonconventional dimethoxybenzyl protected glycine at position 71 as a way to prevent the previously mentioned glutamimide formation. As before, a 0.4 mmol surplus of reagents was used for all residues except for the Fmoc-(Dmb)Gly-OH where a 0.3 mmol excess was used. The products were again purified and analyzed and found to contain a much smaller percentage of cyclized peptides compared to the non-Dmb protected product. References “Function of Prothymosin α in Chromatin Decondensation and Expression of Thymosin β-4 Linked to Angiogenesis and Synaptic Plasticity.” Jaime Gomez-Marquez Annals of the New York Academy of Sciences, Vol: 1112. Pages: 201-209. 2007. Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela, Santiago, Galicia, Spain. 2 “Novel Function of Prothymosin Alpha as a Potent Inhibitor of Human Immunodeficiency Virus Type 1 Gene Expression in Primary Macrophages.” Mosoian, Arevik, et. al. J. Virol. 2006 80: 9200-9206. 3 “Prothymosin α promotes cell proliferation in NIH3T3 cells.” Wu C.-L.; et. al. LifeSciences Volume 61, Number 21, 17 October 1997 , pp. 2091-2101(11). 4 “TI: Divalent metal cation binding properties of human prothymosin α.” Nina V. Chichkova, et. al. European Journal of Biochemistry. Vol: 267.15. Pages 4745-4752. 2000. Belozersky Institute of Physico-Chemical Biology and Center of Molecular Medicine, Moscow State University, Russia. 5“Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity.” Martini, Paolo G. V et. al. Mol. Cell. Biol. 2000 20: 6224-6232. 6 "New Functions of a Well-Known Protein: Prothymosin α Is Involved in Protecting Cells from Apoptosis and Oxidative Stress." Evstafieva, A. G., et al. Molecular Biology 39.5 (Sep. 2005): 631-645. Biomedical Reference Collection: Comprehensive. EBSCO. 7 “Enhancement of Human T Lymphocyte Function by Prothymosin α: Incremed Production of Interleukin-2 and Expression of Interleukin-2 Receptors in Normal Human Peripheral Blood t Lymphocytes.” Constantin N. Baxevanis, et. al. Immunopharmacology and Immunotoxicology, 1990, Vol. 12, No. 4, Pages 595-617. 8Identification of N-terminal acetylation of recombinant human prothymosin alpha in Escherichia coli. Wu J et. al. Biophys Acta. 2006 Aug;1760(8):1241-7. Epub 2006 Apr 19. [PubMed: 16737780] 9"The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA." Eschenfeldt W.H., Berger S.L. Proc. Natl. Acad. Sci. U.S.A. 83:9403-9407(1986) [PubMed: 3467312] 10Phosphorylation of human and bovine prothymosin alpha in vivo. Sburlati A.R., et. al. Biochemistry 32:4587-4596(1993) [PubMed: 8485135] 11Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Olsen J.V., et. al. Cell 127:635-648(2006) [PubMed: 17081983] 12A 180-kDa protein kinase seems to be responsible for the phosphorylation of prothymosin alpha observed in proliferating cells. Pérez-Estévez A, et. al. J Biol Chem. 1997 Apr 18;272(16):10506-13 [PubMed: 9099694] 13A quantitative atlas of mitotic phosphorylation. Dephoure N., et. al. Natl. Acad. Sci. U.S.A. 105:10762-10767(2008) [PubMed: 18669648] 14The thymosins. Prothymosin alpha, parathymosin, and beta-thymosins: structure and function. Hannappel E, Huff T. Vitam Horm. 2003;66:257-96. Review. [PubMed: 12852257] 15http://www.ensembl.org/Multi/blastview/BLA_9I9mYNTM6 16http://www.ensembl.org/Multi/blastview/BLA_fnr5Fq8S9 17http://www.ensembl.org/Multi/blastview/BLA_NRZ5NXXQM 18http://www.ensembl.org/Multi/blastview/BLA_dhOq7d6bV 19http://www.ensembl.org/Multi/blastview/BLA_c6kDCmCZY 20http://www.ensembl.org/Multi/blastview/BLA_nIrLon2Jk 21The German cDNA consortium. Submitted (NOV-2004) to the EMBL/GenBank/DDBJ databases 22"The amino acid sequence of bovine thymus prothymosin alpha." Panneerselvam C., et. al. Biochem. Biophys. 265:454-457(1988) [PubMed: 2901823] 23"Nucleotide sequence of the murine prothymosin alpha cDNA and its deduced primary and secondary protein structure." Schmidt G., et. al. Biophys. Acta 1088:442-444(1991) [PubMed: 2015308] 24"Isolation and partial sequence of goat spleen prothymosin alpha." Frillingos S., et. al. Cell. Biochem. 108:85-94(1991) [PubMed: 1770947] 25"Evaluation and characterization of a porcine small intestine cDNA library: analysis of 839 clones." Winteroe A.K., et. al. Genome 7:509-517(1996) [PubMed: 8672129] 26"Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs." Frangou-Lazaridis M., et. al. Biochem. Biophys. 263:305-310(1988) [PubMed: 3377505] 27"First evidence of prothymosin in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta." Aniello F., et. al. 110:213-217(2002) [PubMed: 11744386] 28NIH - Xenopus Gene Collection (XGC) project Submitted (FEB-2005) to the EMBL/GenBank/DDBJ databases 29NIH - Xenopus Gene Collection (XGC) project Submitted (JUN-2003) to the EMBL/GenBank/DDBJ databases 30"A zebrafish prothymosin alpha-like protein selectively enhances proliferation of embryonic hematopoietic progenitor cells." Marty S.D., et. al. Submitted (APR-2001) to the EMBL/GenBank/DDBJ databases 31"Salmo salar full-length cDNAs." cGRASP (B.F. Koop & W.S. Davidson) Leong J., et. al. Submitted (JAN-2009) to the EMBL/GenBank/DDBJ 32"The genome of the blood fluke Schistosoma mansoni." Berriman M., et. al. Submitted (APR-2009) to the EMBL/GenBank/DDBJ databases 33"Genome sequence of Penicillium marneffei strain ATCC 18224." Fedorova N.D., et. al. Submitted (OCT-2007) to the EMBL/GenBank/DDBJ databases 1