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研究生:楊智雯
研究生(外文):Yang,Chih-Wen
論文名稱:探討類上皮生長因子區在GP-83和精子蛋白間的交互作用中所扮演的角色
論文名稱(外文):The Role of EGF-like domain in the interaction of GP-83 and Its binding protein
指導教授:劉鴻文劉鴻文引用關係
指導教授(外文):Liu,Hwan Wun
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:63
中文關鍵詞:類上皮生長因子功能區
外文關鍵詞:GP-83ADAM7EGF-like domain
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哺乳類精子在副睪中的成熟過程是獲得生殖能力必要的步驟。本實驗室先前的研究發現GP-83,一個由人類副睪分泌的醣蛋白在精子成熟過程中,黏附到精子膜上,並在受精作用過程中扮演重要角色。GP-83的載錄基因分為α和β兩同型序列;α-型擁有3,451個核酸,而β-型擁有2,643個核酸;其蛋白表現區(open reading frame)皆為2,262個核酸,載錄754個胺基酸。由基因序列推定的胺基酸序列包括訊息序列(signal sequence)、前功能區(pro-domain)、金屬蛋白酶(metalloprotease)、去整合素(disintegrin)、半胱胺酸豐富(cysteine-rich)、類上皮生長因子(EGF-like)、穿膜(transmembrane)和細胞內(cytoplasmic)等功能區;由於GP-83的基因序列和猴子ADAM7的基因序列有高達93.3%的相似性,因此我們將其命名為人類ADAM7。過去的研究顯示ADAMs 主要與細胞膜融合,細胞移動及精卵結合有關。成熟的精子具有上皮生長因子受器,而上皮生長因子的重組蛋白可促進精卵結合。本實驗室推論GP-83可能藉由類上皮生長因子區來和精子上的受器結合,進而黏附在精子頭部。因此本論文主要目的在於探討類上皮生長因子區在GP-83和精子蛋白間的交互作用中所扮演的角色。以原核細胞表現全長及剔除類上皮生長因子區的人類ADAM7重組蛋白。將全長重組蛋白和精子蛋白萃取液作用後,利用沉降分析將重組蛋白及結合蛋白ㄧ起分離出來。二維電泳結合質譜儀分析的結果顯示人類ADAM7重組蛋白的精子結合蛋白可能是sperm associated antigen 4 protein(SPAG4),分子量為48kDa,位於精子濃縮核周圍的一層囊膜上,而GP-83和SPAG4間的交互作用需進一步探討。
In mammals, epididymal maturation is essential for fertility development of sperm. Our previous studies shown GP-83, a glycoprotein secreted by human epididymis, was conjugated to sperm and might play a major role in fertilization. GP-83 is encoded by two isoforms of cDNA sequences, α-form of 3,451 bp and β-form of 2,643 bp. Both α-form and β-form exhibited same open reading frame of 2,262 bp encoding a peptide of 754 amino acid residues. Deduced amino acid sequence revealed signal sequence, pro-domain, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains, and 93.3% homology to monkey ADAM7. The molecule was therefore designated as human ADAM7 (h-ADAM7). ADAMs are shown involved in membrane fusion, cell migration, and fertilization. EGF receptor was found in mature sperm, and recombinant EGF might increase fertilization rate. We propose that EGF-like domain may mediate the conjugation of GP-83 to sperm. In this study, full-length and EGF-like domain-deleted recombinant proteins of GP-83 were expressed in E coli.
Sperm protein of 80 kDa and 42 kDa bound full-length GP-83, but not EGF-like domain deleted GP-83. 2D-gel identified a protein of 48 kD bound full-length GP-83. Amino acid sequence of the protein was analyzed by MALDI-TOF. Comparing to database, it might be sperm associated antigen 4 protein (SPAG4), a membrane protein localizes to microtubules of manchette around condense nucleus in sperm. Interaction between GP-83 and SPAG4 will be verified in the future studies.
正文錄目............................................. I
圖 錄 目............................................. Ⅴ
中文摘要............................................. Ⅶ
英文摘要............................................. Ⅷ
第一章、前言......................................... 1
第一節、精子成熟與男性不孕症....................... 1
第二節、人類副睪分泌的蛋白與精子成熟............... 1
第三節、GP-83與人類ADAM7........................ 2
第四節、ADAM家族簡介 ............................ 4
壹、金屬蛋白水解酶區.............................. 5
貳、去整合素區.................................... 6
參、半胱胺酸豐富區................................ 7
肆、C端之細胞漿區................................. 7
伍、類上皮生長因子................................ 8
第五節、類上皮生長因子的生物功能.................... 8
第六節、研究動機與研究目的.......................... 8
第二章、材料與方法.................................... 11
第一節、實驗材料.................................... 11
壹、細胞株、菌株與載體............................ 11
貳、實驗藥品與試劑................................ 11
參、實驗儀器...................................... 13
第二節、實驗步驟.................................... 14
ㄧ、精子蛋白質之萃取............................ 14
二、重組蛋白的誘導表現和萃取.................... 15
三、全長人類ADAM7重組蛋白的純化............... 16
四、沉澱試驗(pull down assay).................... 17
五、蛋白質濃度測試.............................. 18
六、Tricine硫酸十二酯鈉聚丙烯醯胺凝膠電泳法...... 19
七、西方轉漬法................................. 21
八、蛋白質濃縮................................. 22
九、二維電泳分析............................... 23
十、銀染....................................... 26
十一、膠體分離(In gel digestion)................ 27
第三章、結果......................................... 31
第一節、全長及剔除類上皮生長因子功能區人類ADAM7重組蛋白製備.................................
31
第二節、全長人類ADAM7重組蛋白的純化............. 31
第三節、全長人類ADAM7重組蛋白之精子結合蛋白的辨識.........................................
33
第四節、剔除類上皮生長因子功能區人類ADAM7重組蛋白和精子蛋白結合情形.........................
38
第五節、利用二維電泳分析全長人類ADAM7重組蛋白和精子蛋白的結合...............................
38
第六節、以質譜儀分析全長人類ADAM7重組蛋白是否有精子上的結合蛋白.............................
41
第七節、以二維電泳圖觀察人類ADAM7重組蛋白之類上皮生長因子功能區對於精子結合蛋白能力之影響...
48
第四章、討論......................................... 52
第一節、重組蛋白的誘導............................. 52
第二節、利用原核細胞表現全長及剔除類上皮因子人類ADAM7重組蛋白無轉錄後修飾作用...........
52
第三節、純化全長人類ADAM7重組蛋白............... 53
第四節、利用沈澱試驗 ( pull-down assay )及二維電泳分析全長人類ADAM7是否有和精子蛋白結合的能力...
54
第五節、二維電泳分析結果與其準確性................. 54
第六節、質譜儀分析................................. 55
第七節、SPAG4簡介................................. 56
第五章、結論......................................... 57
第六章、參考文獻..................................... 58
Almeida EA, Huovila AP, Sutherland AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P, Myles DG, White JM:Mouse egg integrin α6β1 functions as a sperm receptor. Cell 81:1095-1104, 1995.
Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP: A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385:729-733. 1997.
Blaquier JA, Cameo MS, Cuasnicú PS: On the role of the epididymal factors in sperm fertility. Reprod Nutr 28:1209-1217, 1980.
Blobel CP, Wolfsberg TG, Turck CW, Myles DG, Primakoff P, White JM: A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nature 356:48-52, 1992.
Campbell ID, Bork P: Epidermal growth factor-like modules. Curr Opin Struct Biol 3:385-392, 1993.
Chen H, Pyluck AL, Janik M, Sampson NS:Peptides Corresponding to the Epidermal Growth Factor-Like Domain of Mouse Fertilin: Synthesis and Biological Activity. Biopolymers 47:299-307, 1998.
Cho C, Bunch DO, Faure JE, Goulding EH, Eddy EM, Primakoff P, Myles DG: Fertilization defects in sperm from mice lacking fertilin beta. Science 281:1857-1859, 1998.
Fambrough D, Pan D, Rubin GM, Goodman CS: The cell surface metalloprotease/disintegrin Kuzbanian is required for axonal extension in Drosophila. Proc Natl Acad Sci USA 93:13233-13238, 1996.
Focarelli R, Giuffrida A, Capparelli S, Scibona M, Fabris FM, Francavilla F, Francavilla S, Giovampaola CD, Rosati F:Specific localization in the equatorial region of gp20, a 20 kDa sialylglycoprotein of the capacitated human spermatozoon acquired during epididymal transit which is necessary to penetrate zona-free hamster eggs. Mol Hum Reprod 4:119-125, 1998.
Gilpin BJ, Loechel F, Mattei MG, Engvall E, Albrechtsen R, Wewer UM: A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo. J Biol Chem 273:157-166, 1998.
Higashiyama S, Nanba D.ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk. Biochim Biophys Acta. 1751(1):110-7, 2005
Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW.Epidermal growth factor receptor: mechanisms of activation and signalling.Exp Cell ResExp 284(1):31-53, 2003.
Kennedy C, Sebire K, de Kretser DM, O'Bryan MK. Human sperm associated antigen 4 (SPAG4) is a potential cancer marker. Cell Tissue Res. 315(2):279-83, 2004
Lax Y, Rubinstein S, Breitbart H:Epidermal growth factor induces acrosomal exocytosis in bovine sperm. FEBS Lett. 339(3):234-8, 1994.
Liu HW, Lin YC, Chao CF, Chang SY, Sun GH: GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation. Mol Hum Reprod 6: 422-428, 2000.
Martin Ruiz C, Duquenne C, Treton D, Lefevre A, Finaz C:SOB3, a human protein involved in zona pellucida binding: physiological and biochemical analysis, purification. Mol Reprod Dev 49: 286-297, 1998.
Mooney JK Jr, Horan AH, Lattimer JK: Motility of spermatozoa in the human epididymis. J Urol 108: 443-445, 1972.
Mori S, Tanaka M, Nanba D, Nishiwaki E, Ishiguro H, Higashiyama S, Matsuura N: PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J Biol Chem 278:46029-34, 2003.
Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Becherer JD: Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385:33-36, 1997.
Nishimura H, Cho C, Branciforte DR, Myles DG, Primakoff P: Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta. Dev Biol 233:204-213, 2001.
Rawlings ND, Barrett AJ: Evolutionary families of metalloproteases. Methods Enzymol 248:183-228, 1995.
Rooke J, Pan D, Xu T, Rubin GM: KUZ, a conserved metalloprotease-disintegrin protein with two roles in Drosophila neurogenesis. Science 273:1227-1231, 1996.
Shao X, Tarnasky HA, Lee JP, Oko R, van der Hoorn FA: Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. Dev Biol. 211(1):109-23, 1999.
Stone AL, Kroeger M, Sang QX: Structure-function analysis of the ADAM family of disintegrin-like and metalloproteinase- containing proteins. J Protein Chem 18:447-465,1999.
Sun GH, Lin YC, Guo YW, Chang SY, Liu HW: Purification of GP-83, a glycoprotein secreted by human epididymis and conjugated to mature spermatozoa. Mol Hum Reprod 6:429-434, 2000.
Talbot P, Chacon RS: Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. Fertil Steril 37:240-248, 1982.
Tanaka M, Nanba D, Mori S, Shiba F, Ishiguro H, Yoshino K, Matsuura N, Higashiyama S: ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands. J Biol Chem. 279(40):41950-9, 2004.
Wolfsberg TG, Bazan JF, Blobel CP, Myles DG, Primakoff P, White JM: The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications. Proc Nat’l Acad Sci USA 90:10783-87, 1993.
Wolfsberg TG, Primakoff P, Myles DG, White JM: ADAM, a novel family of membrane proteins containing a disintegrin and metalloprotease domain: mutlipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 131:275-278, 1995.
Wolfsberg TG, White JM: ADAMs in fertilization and development. Dev Biol 180:389-401, 1996.
Wong GE, Zhu X, Prater CE, Oh E, Evans JP: Analysis of fertilin alpha (ADAM1)-mediated sperm-egg cell adhesion during fertilization and identification of an adhesion-mediating sequence in the disintegrin-like domain. J Biol Chem 276:24937-24945, 2001.
Yang YS, Chen SU, Hwang JL, Ho HN, Lin HR, Lee TY: Analysis of human in vitro fertilization failure. J Formos Med Assoc 92:122-127, 1993.
Yeung CH, Cooper TG, Nieschlag E: Human epididymal secreted protein CD52 on ejaculated spermatozoa: correlations with semen characteristics and the effect of its antibody. Mol Hum Reprod 3:1045-1051, 1997.
Zhang H, Martin-DeLeon PA: Mouse epididymal Spam1 (PH-20) is released in vivo and in vitro, and Spam1 is differentially regulated in testis and epididymis. Biol Reprod 65(5):1586-93, 2001.
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