Chinese Journal of Biochemistry and Molecular Biology. (growth inhibitory factor,gif, 2Sepharose 4B PC12,

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 20 (4) : G Rab3a ( GIF) 1) 1) 1) 2) 1) 3,,,, ( 1), ; 2), ) G Rab3a (growth inhibitory factor,gif, 23, MT23), (pheochromocytoma) PC12 hmt23 (human MT23) Rab3a pflag2cmv22 psv2ha, PC12, pflag2cmv222hmt21 psv2ha2rab3a, hmt23 Rab3a PC12, pflag2 CMV222hMT23 psv2ha2rab3a PC12, GIF F, pflag2cmv222 hmt21 psv2ha2rab3a PC12 p GEX24T212Rab3a p GEX24T212hMT23, BL21, 2Sepharose 4B Sephacryl S2100, 95 % Rab3a hmt23, Rab3a hmt23 PC12, GIF Rab3a,, Q74 The Cooperated Effect of G2Protein Rab3a and GIF to Inhibit Neuron Growth CHEN Qiao2lin 1), KANG Qiao2hua 1), REN Hong2wei 1), WANG Zong2yuan 2), RU Bing2gen 1) 3 ( 1) Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, National Laboratory of Protein Engineering, Beijing , China ; 2) College of Animal Husbandy and Veterinary Medicine, Yangzhou University, Yangzhou , China) Abstract To study the function of cooperated role of G2protein Rab3a and the growth inhibitory factor( GIF, originally termed as MT23),pheochromocytoma cell line PC12 was used as neuron model The genes of human MT23 and Rab3a were cloned into pflag2cmv22 and psv2ha,respectively,and subsequently cotransfected into PC12 cells by a lipofectamine kit Another pair of cloned genes of psv2ha2 Rab3a and pflag2cmv222 hmt21 was also cotransfected into PC12 as control After cultivation,the obvious growing status was only observed in the cloned group containing recombinant hmt23,which demonstrated similar results to the neuron treated with GIF,but the control and the clone transfected only with pflag2cmv22 or psv2ha had no remarkable effects Expressionable recombinants were constructed by linking Rab3a and hmt23 to p GEX24T21,which were then transfected into BL21, expressed, and at last purfied by affinity chromatography, thrombin digestion and gel filtration on Sephacryl S2100 The final products with high purity were used to co2cultivate PC12, and the : , : , : 962C Tel : ; Fax : ; E2mail : edu cn,, ,, : Receivd :September 18,2003 ; Accepted :February 26,2004 Supported by the National 9th Five Years Plan Special Research Programs of China No 962C Corresponding author Tel : , Fax : ,E2mail : edu cn China Academic Journal Electronic Publishing House All rights reserved

2 4 : G Rab3a ( GIF) 535 cellular biological activities were tested by in vitro experiment The results confirm that they have specific cooperative repressng effect on PC12 cellular growth,and the corresponding inhibitory curve is similar to the neuron treated with GIF and brain extracts Key words Rab3a,GIF,neuron,transfection,inhibition (Alzheimer s disease, AD) 1 2, Alzheimer s 301 [1 ] AD, 2S2, ( growth inhibitory factor, GIF), 23 (MT23), Pharmacia AD (MTT) Sigma GIF,Alzheimer s Lipofectamine DMEM Life Technologies, Clontech, (MT21 MT22) ;, HyClone GIF Alzheimer s Biolabs DNA,, GIF hmt23 PC12, 12 h, Rab3a hmt23 PC12, MTT PC12 GIF PC12 12 h, DH5 BL21 ( PC12), p GEX24T21, pflag2cmv22 psv2ha (GST) Sephacryl S2100 [26 ] 1 3 Rab3a MT23 PC12 Rab G ( superfamily), PC12 EcoR Π Rab3 ( subfamily) BamH pbs2hmt23 pbs2hmt21 ( Rab3a Rab3b Rab3c Rab3d ; ), 200 bp, Rab3a 220, pflag2cmv22 ; ;, ; Rab3a cdna PCR BamH ΠXho,, Rab3a Rabphilin23 Rim psv2ha, Noc2, Ca 2 + [79 ], Rab3a PC12 6 ( Π), [10,11 RIM ] Lipofectamine pflag2cmv222hmt23 psv2ha2 Rab3a Rab3a PC12,Western,,, pflag2cmv222hmt21 Rab3a GIF psv2ha2rab3a, 10 %, 5 % DMEM,37,5 %CO 2 [12 ] Rab3a,4 h CdCl 2 5molΠL ; MTT PC GST PCR BamH Xho hmt23 Rab3a DNA,BamH Xho p GEX24T212hMT23 p GEX24T212Rab3a China Academic Journal Electronic Publishing House All rights reserved

3 p GEX24T212hMT23 p GEX24T212Rab3a BL21, [8,13 ] 50 ml PBS,, Triton X %, 30 min,4 ( rπmin,15 min) 2Sepharose 4B,,20 PBS,, [ 3 mg 2 ml 50 mmolπl Tris2HCl (ph 810), 015 l 2 ],25 16 h,,1 ml PBS, 10 mmolπl NH 4 HCO 3 Sephacryl S2100,, DC Protein Assay [3 ] ; DTNB 1 6 hmt23 Rab3a PC AD (brain extracts,be), AD, 5 1 PBS ; ( rπmin,30 min,4 ),,0122 m DC Protein Assay Rab3ahMT23 PC12 PC12 96 ( Π), 37,5 %CO 2 12 h hmt23 ( 10300gΠml) Rab3a ( 10200gΠml), BE ( 240gΠml), 24 h,mtt [14 ] 2 Fig 1 Restriction analysis of plasmid pflag2cmv2hmt21, pflag2cmv2hmt23 and psv2ha2rab3a M:100 bp DNA ladder, 1 pflag2cmv2hmt23 ( EcoR ΠBamH ), 2 pflag2cmv2hmt21 ( EcoR ΠBamH ), 3 psv2ha2rab3a ( BamH ΠXho ) Fig 2 Western blotting of Rab3a and hmt23 in PC12 cells transfected with psv2ha2rab3a and pflag2cmv2hmt23 genes 1 Mouse anti2flag was used as the antibody for Western blotting in transfected PC12 cells ; 2 Rat anti2ha was used as the antibody for Western blotting in transfected PC12 cells ; 3 Mouse anti2flag and Rat anti2ha were used as the antibody for Western blotting in normal PC12 cells 2 1 Rab3a hmt23 cdna Fig 1, PC12, pflag2 200 bp 700 bp, (CMV222hMT23 psv2ha2rab3a PC12 ) pflag2cmv2hmt23 16 h, pflag2cmv2hmt21 psv2ha2rab3a ( Fig 3 ( C) ) ; pflag2cmv PC12 hmt23 (Fig 3 (A) ) psv2ha2rab3a PC psv2ha2, pflag2cmv222hmt21 psv2ha2 Rab3a pflag2hmt23 PC12, Rab3a PC12 FLAG HA ( Fig 3B) Rab3a hmt23, Western HA2Rab3a PC12 FLAG2hMT23, MTT PC12 PC12 (Fig 2), hmt23 Rab3a, China Academic Journal Electronic Publishing House All rights reserved

4 4 : G Rab3a ( GIF) 537 Fig 3 Growth results of the PC12 cells transfected with Rab3a and hmt23 genes (A) Growth of PC12 cells tranfected with pflag2hmt23 ; (B) Growth of PC12 cells cotranfected with psv2ha2rab3a and pflag2hmt21 ; (C) Growth of PC12 cells cotranfected with psv2ha2rab3a and pflag2hmt23 ; hmt23 97 % hmt23 Rab3a, Rab3a PC12, 7 kd 25 kd(fig 5) hmt21 Rab3a (Fig 4), Fig 4 Growth results of cells transfected with different genes a Transfected with pflang2hmt23 ; b Transfected with psv2ha2rab3a ; c psv2ha2rab3a cotransfected with pflang2hmt23 and psv2ha2 Rab3a ; d Cotransfected with pflag2hmt21 and psv2ha2rab3a Fig 5 15 % SDS2PAGE of purified Rab3a and hmt23 1 Protein molecular weight marker ; 2 Purified Rab3a protein ; 3 Purified hmt Rab3a hmt23 PC12 Fig 6 (A) PC12 :, 2 3 hmt23 Rab3a, ; IPTG 2Sepharose 4B hmt23 AD (BE) hmt23 Sephacryl S2100,Rab3a PC12 : China Academic Journal Electronic Publishing House All rights reserved

5 Fig 6 Growth results of the PC12 cells cultivated with Rab3a and hmt23 proteins (A) Normal growth map of PC12 cells ; (B) Growth of PC12 cells with hmt23 (100 gπml) and AD brain extracts in culture medium ; (C) Growth of PC12 cells with hmt23 (100 gπml) and Rab3a (60 gπml) in culture medium,, Fig 6 (B) (C) Rab3a hmt23 PC12 MTT PC12 Fig 7 Fig 7 Inhibition effects of different protein combinations on PC12 cells Control ; Group with hmt23 ; ΕGroup with Rab3a ; Group with hmt23 and BE (240 mgπl) ; Group with Rab3 and hmt23 (100 mgπl) 3, GIF, AD, 56 [2 ],, GIF,, AD,,, GIF AD,, GIF GIF [12 ] GIF GTP Rab3a, Rab3a G2, Rab,,, GTP2Rab3a rabphilin23 [6,7 ] Noc2 [9 ], Ca 2 +, ;, Rab3a Rabin3 [15 ] Mss4 [16 ] [17 ] China Academic Journal Electronic Publishing House All rights reserved

6 4 : G Rab3a ( GIF) 539, Rab3a GIF Rab3a, GIF2Rab3a GIF [7,15 ], (NGF), [18,19 PC12 ] Rab3a GIF PC12, [20 ] GIF, 11 PC12, Rab3a BE GIF GIF,, Zn ( Zn 2001, 28(6) : Zn ) [7 ] Rab3a rabphilin23, RIM Noc2 [9,15 ] N, GIF Rab3a Zn, Zn, GIF Rab3a?? ( References) 1 Nicolini M, Zatta P F, Corain B Alzheimer s Disease and Related disorders Oxford : Pergamon Press, Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M The growth inhibitory factor that is deficient in the Alzheimer s disease brain is a 68 amino acid metallothionein2like protein Neuron, 1991, 7 (2) : Erickson J C, Sewell A K, Jensen L T, Wing D R, Palmiter R D Enhanced neurotrophic activity in Alzheimer s disease cortex is not associated with down2regulation of metallothionein2 ( GIF) Res, 1994, 649(122) : Brain 4 Sewell A K, Jensen L T, Eerickson J C, Palmiter R D, Winge D R Bioactivity of metallothionein23 correlates with its noveldomain sequence rather than metal binding properties Biochemistry, 1995, 34 (14) : Uchida Y, Ihara Y The N2terminal portion of growth inhibitory factor is sufficient for biological activity J Biol Chem, 1995, 270 (7) : Yamada M, Hayashi S, Hozumi I, Inuzuka T, Tsuji S, Takahashi H Subcellular localization of growth inhibitory factor in rat brain : light and electron microscopic immunohistochemical studies Brain Res, 1996, 735(2) : Geppert M, Goda Y, Stevens C F, Sudhof T C The small GTP2binding protein Rab3A regulates a late step in synaptic vesicle fusion Nature, 1997, 387(6635) : Chou J H, Jahn R Binding of Rab3A to synaptic vesicles J Biol Chem, 2000, 275(13) : Wang Y, Okamoto M, Schmitz F, Hofmann K, Sudhof T C Rim is a putative Rab3 effector in regulating synaptic2vesicle fusion Nature, 1997, 388(6642) : Haynes L P, Evans G J O, Morgan A, Burgoyne R D A direct inhibitory role for the Rab3 specific effector, Noc2, in Ca 2 + 2regulated exocytosis in neuroendocrine cells J Biol Chem,2001,276 (13) : Iezzi M Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion, Eur J Biochem,1999,259 :317 12,,, ( GIF) G Rab3a ( Kang Qiao2hua, Chen Qiao2lin, Ren Hong2wei, Ru Bing2gen Growth inhibitory factor ( GIF) directly interacts with G2protein Rab3a Prog Biochem Biophys) 13,,,, G2 Rab3a cdna ( Kang Qiao2hua, Chen Qiao2lin, Ren Hong2wei, Ru Bing2gen Cloning and expression of G2protein Rab3a cdna Chin J Biochem Mol Biol), 2002, 18(1) : Pauwels P J, Van Assouw H P, De Ryck M, Leysen J E, Dom R, Van Gool D Towards an improved survival of rat rain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer s type Brain Res, 1993,610 (1) : Brondyk W H, McKiernan C J, Fortner K A, Stabila P, Holz R W, Macara I G Interaction cloning of Rabin3, a novel protein that associates with the Ras2like GTPase Rab3A Mol Cell Biol, 1995, 15 (3) : Burton J L, Burns M E, Gatti E, Augustine G J, De Camilli P Specific interactions of Mss4 with members of the Rab GTPase subfamily EMBO J, 1994, 13(23) : Park J B, Farnsworth C C, Glomset J A Ca 2 + Πcalmodulin causes Rab3A to dissociate from synaptic membranes J Biol Chem, 1997, 272(33) : Pittman R N, Wany S, DiBenedeuo A J, Mills J C A system for characterizing cellular and molecular events in programmed neuronal cell death J Neurosci, 1993,13(9) : Bernd P, Greene L A Association of 135 I2nerve growth factor with PC12 pheochromocytoma cells, Evidence for internalization via high2affinity receptors only and for long2term regulation by nerve growth factor of both high and low2affinity receptors J Biol Chem, 1984, 259 : , PC12 (Ji Qing2zhou, Ru Bing2gen Growth inhibitory factor can inhibit the PC12 cell in vitro Chin Sci Bull),2001, 46 (19) : China Academic Journal Electronic Publishing House All rights reserved