整合蛋白 (Integrins) 是普遍存在的細胞黏著受器，會與其他種類細胞表面和細胞外基質的配體 (ligands) 結合。他們參與橫跨細胞膜的雙向訊息傳遞，來調控細胞的黏著、分化、遷移、生長和存活。整合蛋白alpha5beta1 是唯一明確具有前血管新生的整合蛋白，對於治療癌症是一個具有潛力的藥物目標。Rhodostomin (簡稱 Rho) 是一種去整合蛋白 (Disintegrin) 藉由阻斷血小板上的整合蛋白alphaIIbbeta3 來抑制血小板凝集。Rho 是由68個胺基酸所組成，在48至53的位置上有一段PRGDMP的序列。根據噬菌體呈現 (phage-display) 的試驗中，他們發現噬菌體含有序列為 PRGDGW、 FRGDGW、 TRGDGW、 SRGDGW、 FRGDGF、 YRGDGF 和 TRGDGF 可以選擇性地結合到整合蛋白alpha5beta1。因此，我們提出將這些序列併入到 Rho 的 RGD loop 上，來設計出具有整合蛋白alpha5beta1-專一性的去整合蛋白。本研究中，我已經在 pichia pastoris 系統中表現出十六株 Rho 的突變蛋白，包含序列為 ARGDEP、ARGDAP、ARGDDN、ARLDDL、AKGDWN-NPHKGPAT、SRGDGW、FRGDGW、TRGDGW、RRGDGW、KRGDGW、FRGDGF、TRGDGF、PRGDGF、ARGDGF、RRGDGF和KRGDGF 並均質純化。當與分子量理論值作比較時，利用質譜儀測出在 P. pastoris 表現下的 Rho 和其突變株之分子量實驗值誤差小於1 Da。而這些計算數值已經考慮所有半胱胺酸形成的雙硫鍵，暗示著在 Rho 和其突變株有六對雙硫鍵的形成。我也利用血小板凝集和細胞黏著試驗去檢視他們對於整合蛋白 alphaIIbbeta3、alphaVbeta3 和 alpha5beta1 的專一性與活性。相對於噬菌體呈現試驗的結果，我們的結果顯示出突變株 SRGDGW、 FRGDGW、 TRGDGW、 FRGDGF、 TRGDGF 和 PRGDGF無法選擇性地抑制整合蛋白alpha5beta1。而 RRGDGF 和 RRGDGW 突變株能夠抑制整合蛋白alphaIIbbeta3、alphaVbeta3 和 alpha5beta1，其IC50數值分別是 >5950、 1157 和 280.6 nM與 >5950、 1813 和 64.2 nM。這些結果指出 Rho 的 RRGDGF 和 RRGDGW 突變株可以選擇性地抑制整合蛋白alpha5beta1，建議相鄰在 RGD motif N端的 R48 殘基可能會與整合蛋白 alpha5 次單位作用。

Integrins are ubiquitous cell adhesion receptors that bind ligands on the surface of other cells and in the extracellular matrix. They are involved in bidirectional signaling across the plasma membrane, regulating cell adhesion, differentiation, migration, growth, and survival. Integrin alpha5beta1 is the only unambiguously proangiogenic integrin, which is a potential drug target for the treatment of cancer. Rhodostomin (Rho) is a disintegrin that inhibit the platelet aggregation by blocking integrin alphaIIbbeta3 of platelets. Rho consists of 68 amino acids with a PRGDMP sequence at positions of 48-53. Based on phage-display experiments, they found that the phages containing PRGDGW, FRGDGW, TRGDGW, SRGDGW, FRGDGF, YRGDGF, and TRGDGF sequences can selectively bind to integrin alpha5beta1. Therefore, we proposed to incorporate these sequences into the RGD loop of Rho to design integrin alpha5beta1-specific disintegrin. In this study I have expressed sixteen Rho mutant proteins, including ARGDEP, ARGDAP, ARGDDN, ARLDDL, AKGDWN-NPHKGPAT, SRGDGW, FRGDGW, TRGDGW, RRGDGW, KRGDGW, FRGDGF, TRGDGF, PRGDGF, ARGDGF, RRGDGF, and KRGDGF sequences in Pichia pastoris and purified them to homogeneity. The experimental molecular weights of Rho and its mutants produced in P. pastoris were deviated less than 1 Da when compared with their theoretical values. These values were calculated by assuming all cysteines formed disulfide bonds, indicating the formation of six disulfide bonds in Rho and its mutants. I also used platelet aggregation and cell adhesion assays to examine their specificity and activity to integrins alphaIIbbeta3, alphaVbeta3, and alpha5beta1. In contrast to the results of phage display experiment, our results showed that the SRGDGW, FRGDGW, TRGDGW, FRGDGF, TRGDGF, and PRGDGF mutants cannot selectively inhibit integrin alpha5beta1. The RRGDGF and RRGDGW mutants inhibited integrins alphaIIbbeta3,alphaVbeta3 and alpha5beta1 with the IC50 values of >5950, 1157 and 280.6 nM, as well as >5950, 1813 and 64.2 nM, respectively. These results indicate that the RRGDGF and RRGDGW mutants of Rho can selectively inhibit integrin alpha5beta1, suggesting that the R48 residue N-terminally adjacent to the RGD motif may interact with integrin alpha5 subunit.

Alder M, Lazarus RA, Dennis MS, Wagner G.: Solution structure of kistrin, a potent platelet aggregation inhibitor and GP IIb-IIIa antagonist. Science. 253: 445-8, 1991.
Altieri DC, Plescia J, Plow EF.: The structural motif glycine 190-valine 202 of the fibrinogen gamma chain interacts with CD11b/CD18 integrin (alpha M beta 2, Mac-1) and promotes leukocyte adhesion. J Biol Chem. 268: 1847-53, 1993.
Anderluh M, Cesar J, Stefanic P, Kikelj D, Janes D, Murn J, Nadrah K, Tominc M, Addicks E, Giannis A, Stegnar M, Dolenc MS.: Design and synthesis of novel platelet fibrinogen receptor antagonists with 2H-1,4-benzoxazine-3(4H)-one scaffold. A systematic study. Eur J Med Chem. 40: 25-49, 2005.
Arnaout MA, Mahalingam B, Xiong JP.: Integrin Structure, Allostery, and Bidirectional Signaling. Annu Rev Cell Dev Biol. 21: 381-410, 2005.
Berman AE, Kozlova NI, Morozevich GE.: Integrins: structure and signaling. Biochemistry. 68: 1284-99, 2003.
Bussolino F, Valdembri D, Caccavari F, Serini G.: Semaphoring vascular morphogenesis. Endothelium. 13(2):81-91, 2006.
Chang CP, Chang JC, Chang HH, Tsai WJ, Lo SJ.: Positional importance of Pro53 adjacent to the Arg49-Gly50-Asp51 sequence of rhodostomin in binding to integrin alphaIIbbeta3. Biochem J.357: 57-64, 2001.
Chang HH, Hu ST, Huang TF, Chen SH, Lee YH, Lo SJ.: Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells. Biochem Biophys Res Commun. 190: 242-9, 1993.
Cochran AG, Tong RT, Starovasnik MA, Park EJ, McDowell RS, Theaker JE, Skelton NJ.: A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins. J Am Chem Soc. 123: 625-32, 2001.
Copie V, Tomita Y, Akiyama SK, Aota S, Yamada KM, Venable RM, Pastor RW, Krueger S, Torchia DA.: Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure. J Mol Biol. 277: 663-82, 1998.
Daly R, Hearn MT.: Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit. 18: 119-38, 2005.
Dennis MS, Carter P, Lazarus RA.: Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. Proteins. 15: 312-21, 1993.
Dresner-Pollak R, Rosenblatt M.: Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: a potential approach to the therapy of osteoporosis. J Cell Biochem. 56: 323-30, 1994.
D'Souza SE, Ginsberg MH, Plow EF.: Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci. 16: 246-50, 1991.
Dunon D, Piali L, Imhof BA.: To stick or not to stick: the new leukocyte homing paradigm. Curr Opin Cell Biol. 8(5):714-23, 1996. Review.
Faull RJ, Du X, Ginsberg MH.: Receptors on platelets. Methods Enzymol. 245: 183-94, 1994.
Goh KL, Yang JT, Hynes RO.: Mesodermal defects and cranial neural crest apoptosis in alpha5 integrin-null embryos. Development. 124: 4309-19, 1997.
Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Neiwiarowski S.: Disintegrins: A family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Bio. Med. 195: 168-71, 1990.
Guo RT, Chou LJ, Chen YC, Chen CY, Pari K, Jen CJ, Lo SJ, Huang SL, Lee CY, Chang TW, Chaung WJ.: Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin. Proteins. 43: 499-508, 2001.
Hautanen A, Gailit J, Mann DM, Ruoslahti E.: Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor. J Biol Chem. 264: 1437-42, 1989.
Higgins DR, Cregg JM, Editors.: Methods in Molecular Biology: Pichia Protocols, Totowa, NJ: Humana Press, 1998.
Hong SY, Sohn YD, Chung KH, Kim DS.: Structural and functional significance of disulfide bonds in saxatilin, a 7.7 kDa disintegrin. Biochem Biophys Res Commun. 293: 530-6, 2002.
Hsu SL, Cheng CC, Shi YR, Chiang CW.: Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells. Cancer Lett. 26;167(2):193-204, 2001.
Huang TF, Sheu JR, Teng CM, Chen SW, Liu CS.: Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. J Biochem. 109: 328-34, 1991.
Huang TF, Wu YJ, Ouyang C.: Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. Biochim Biophys Acta. 925: 248-57, 1987.
Huang TF, Yeh CH, Wu WB.: Viper venom components affecting angiogenesis. Haemostasis. 31: 192-206, 2001.
Humphries MJ.: Cell-substrate adhesion assays. Current Protocols in Cell Biology. 9.1.1-9.1.11, 1998.
Humphries MJ.: Integrin structure. Biochem Soc Trans. 28: 311-39, 2000.
Humphries MJ, Mould AP.: Structure. An anthropomorphic integrin. Science. 294: 316-7, 2001.
Hynes RO.: Integrins: a family of cell surface receptors. Cell. 48: 549-54, 1987.
Hynes RO.: Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25, 1992.
Hynes RO.: A reevaluation of integrins as regulators of angiogenesis. Nat Med. 8(9):918-21, 2002. Review.
Kim S, Bell K, Mousa SA, Varner JA.: Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. Am J Pathol. 156: 1345-62, 2000.
Koivunen E, Gay DA, Ruoslahti E.: Selection of peptides binding to the alpha 5 beta 1 integrin from phage display library. J Biol Chem. 268(27):20205-10, 1993.
Koivunen E, Wang B, Ruoslahti E.: Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library. J Cell Biol. 124: 373-80, 1994.
Koivunen E, Wang B, Ruoslahti E.: Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology. 13: 265-70, 1995.
Kulkarni GV, Chen B, Malone JP, Narayanan AS, George A.: Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1. Arch Oral Biol. 45: 475-84, 2000.
Kuntz ID.: Structure-based strategies for drug design and discovery. Science. 257: 1078-82, 1992.
Kuwada SK, Kuang J, Li X.: Integrin alpha5/beta1 expression mediates HER-2 down-regulation in colon cancer cells. J Biol Chem. 280(31):28828, 2005
Lee JO, Rieu P, Arnaout MA, Liddington R.: Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 80: 631-8, 1995.
Li R, Hoess RH, Bennett JS, DeGrado WF.: Use of phage display to probe the evolution of binding specificity and affinity in integrins. Protein Eng. 16: 65-72, 2003.
Locardi E, Mullen DG, Mattern RH, Goodman M.: Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3. J Pept Sci. 5: 491-506, 1999.
Lueking A, Holz C, Gotthold C, Lehrach H, Cahill D.: A system for dual protein expression in Pichia pastoris and Escherichia coli. Protein Expr Purif. 20: 372-8, 2000.
Lu X, Rahman S, Kakkar VV, Authi KS.: Substitutions of proline 42 to alanine and methionine 46 to asparagine around the RGD domain of the neurotoxin dendroaspin alter its preferential antagonism to that resembling the disintegrin elegantin. J Biol Chem. 271: 289-94, 1996.
Luciana Marinelli, Axel Meyer, Dominik Heckmann, Antonio Lavecchia, Ettore Novellino, and Horst Kessler: Ligand Binding Analysis for Human 51 Integrin: Strategies for Designing New 51 Integrin Antagonists. J. Med. Chem. 48: 4204-4207, 2005.

Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM.: Heterologous protein production using the Pichia pastoris expression system. Yeast. 22: 249-70, 2005.
Marcinkiewicz C.: Functional characteristic of snake venom disintegrins: potential therapeutic implication. Curr Pharm. 11: 815-27, 2005.
Marcinkiewicz C, Calvete JJ, Vijay-Kumar S, Marcinkiewicz MM, Raida M, Schick P, Lobb RR, Niewiarowski S.: Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits alpha 5 beta 1 integrin. Biochemistry. 38: 13302-9, 1999.
Marcinkiewicz C, Vijay-Kumar S, McLane MA, Niewiarowski S.: Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site. Blood. 90(4):1565-75, 1997.
Martin KH, Slack JK, Boerner SA, Martin CC, Parsons JT.: Integrin connections map: to infinity and beyond. Science. 296: 1652-3, 2002.
Marugan JJ, Manthey C, Anaclerio B, Lafrance L, Lu T, Markotan T, Leonard KA, Crysler C, Eisennagel S, Dasgupta M, Tomczuk B.: Design, synthesis, and biological evaluation of novel potent and selective alphavbeta3/alphavbeta5 integrin dual inhibitors with improved bioavailability. Selection of the molecular core. J Med Chem. 48: 926-34, 2005.
McLane MA, Marcinkiewicz C, Vijay-Kumar S, Wierzbicka-Patynowski I, Niewiarowski S.: Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med. 219: 109-19, 1998.
McLane MA, Sanchez EE, Wong A, Paquette-Straub C, Perez JC.: Disintegrins. Curr Drug Targets Cardiovasc Haematol Disord. 4: 327-55, 2004.
Minoux H, Chipot C, Brown D, Maigret B.: Structural analysis of the KGD sequence loop of barbourin, an alphaIIbbeta3-specific disintegrin. J Comput Aided Mol Des. 14: 317-27, 2000.
Monsalve RI, Lu G, King TP.: Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast. Protein Expr Purif. 16(3):410-6, 1999.
Morris GM, Goodsell DS, Huey R, Olson AJ.: Distributed automated docking of flexible ligands to proteins: parallel applications of AutoDock 2.4. J Comput Aided Mol Des. 10: 293-304, 1996.
Mould AP, Askari JA, Aota S, Yamada KM, Irie A, Takada Y, Mardon HJ, Humphries MJ.: Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit. J Biol Chem. 11;272(28):17283-92, 1997.
Mousa SA.: Anti-integrin as novel drug-discovery targets: potential therapeutic and diagnostic implications. Curr Opin Chem Biol. 6: 534-41, 2002.
Nadrah K, Dolenc MS.: Dual antagonists of integrins. Curr Med Chem. 12: 1449-66, 2005.
Pfaff M, McLane MA, Beviglia L, Niewiarowski S, Timpl R.: Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition. Cell Adhes Commun. 2: 491-501, 1994.
Pierschbacher MD, Ruoslahti E.: Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem. 262: 17294-8, 1987.
Plow EF, Haas TA, Zhang L, Loftus J, Smith JW.: Ligand binding to integrins. J Biol Chem. 275: 21785-8, 2000.
Rahman S, Aitken A, Flynn G, Formstone C, Savidge GF.: Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation. Biochem J. 335: 247-57, 1998.
Ruouslahti E.: RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol. 12: 697-715, 1996.
Scarborough RM, Rose JW, Hsu MA, Phillips DR, Fried VA, Campbell AM, Nannizzi L, Charo IF.: Barbourin: A GPIIb-IIIa specific integrin antagonist from the venom of Sistrurus m. barbouri. J Biol Chem. 266: 9359-62, 1991.
Scarborough RM, Rose JW, Naughton MA, Phillips DR, Nannizzi L, Arfsten A, Campbell AM, Charo IF.: Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem. 268: 1058-65, 1993.
Schwartz MA, Schaller MD, Ginsberg MH.: Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 11:549-99, 1995. Review.
Shimaoka M, Springer TA.: Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov. 2: 703-16, 2003.
Shimaoka M, Takagi J, Springer TA.: Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct. 31: 485-516, 2002.
Siddiqi AR, Persson B, Zaidi ZH, Jornvall H.: Characterization of two platelet aggregation inhibitor-like polypeptides from viper venom. Peptides. 13: 1033-7, 1992.
Smith JW, Le Calvez H, Parra-Gessert L, Preece NE, Jia X, Assa-Munt N.: Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3. J Biol Chem. 277: 10298-305, 2002.
Springer TA, Wang JH.: The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Adv Protein Chem. 68: 29-63, 2004.
Takagi J, Petre BM, Walz T, Springer TA.: Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 110: 599-11, 2002.
Tamkun JW, DeSimone DW, Fonda D, Patel RS, Buck C, Horwitz AF, Hynes RO.: Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 46: 271-82, 1986.
Trikha M, De Clerck YA, Markland FS.: Contortrostatin, a snake venom disintegrin, inhibits beta 1 integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis. Cancer Res. 54(18):4993-8, 1994.
Varner JA, Cheresh DA.: Integrins and cancer. Curr Opin Cell Biol. 8(5):724-30, 1996. Review.
White CE, Kempi NM, Komives EA.: Expression of highly disulfide-bonded proteins in Pichia pastoris. Structure. 2: 1003-5, 1994.
Xiao T, Takagi J, Coller BS, Wang JH, Springer TA.: Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature. 432: 59-67, 2004.
Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, Arnaout MA.: Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science. 294: 339-45, 2001.
Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, Arnaout MA.: Crystal structure of the extracellular segment of integrin alphaV beta3 in complex with an Arg-Gly-Asp ligand. Science. 296: 151-5, 2002.
郭瑞庭 馬來蝮蛇蛇毒基因的選殖及蛇毒蛋白之功能及三度空間結構之研究。國立成功大學生物化學研究所碩士論文，1998。
陳彥青 馬來蝮蛇蛇毒蛋白及其突變株的結構與動力學之研究。國立成功大學生物化學研究所碩士論文，2001。
許家豪 含RGD序列蛋白在辨識組合蛋白上的結構與功能關係之研究。國立成功大學生物化學研究所碩士論文，2003。
劉祐禎 利用馬來蝮蛇蛇毒蛋白突變株研究在辨識組合蛋白 avb3 和 a5b1 的結構決定要素。國立成功大學生物化學研究所碩士論文，2004。
陳秋月 利用 Rhodostomin 研究 Integrin 的辨識序列和研發製備胺基酸選擇性同位素標示蛋白的方法。國立成功大學基礎醫學研究所博士論文，2005。