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1、精品论文Cloning and purifying of Soluble amyloid precursor protein and its modulating MAPK signal pathway5CHEN Keping1, DOU Fei2(1. Medical School Southeast University;2. College of life science, Beijing Normal University)Abstract: It is well known that the proteolytic processing of the amyloid precurso
2、r protein (APP)release -amyloid (A), which plays a central role in the pathogenesis of Alzheimers disease (AD).10Understanding APP processing is important for reducing A levels in AD therapeutic strategy. Solubleamyloid precursor protein (sAPPa) is a proteolyte of APP cleavage by -secretase. The sig
3、nificance ofthe cleavage and the physiological functions of sAPP are poorly understood. In this study, we constructed the stable cell lines expressing sAPP fusion protein and purified with tandem affinitypurification (TAP) technology. We also found sAPP could modulate the MAPK signal pathway15involv
4、ing in NCAM-induced neurite outgrowth, likes the full length APP.Key words: cell biology; secreted APP; tandem affinity purification; MAPK0IntroductionIn view of the worlds rising elderly population, Alzheimer disease (AD) threatens to precipitate a20public health crisis. AD is characterized by the
5、presence of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain. The proteolytic processing of amyloid precursor protein (APP) has long been studied because of its association with the pathology of AD (Sisodia et al.2002). APP processing by - or -secretase, followed
6、by presenilin-dependent -secretase cleavage, releases the majority of the ectodomain as the soluble fragment of APP, termed sAPP or sAPP,25respectively; as well as p3 or -amyloid (A) peptides, and the APP intracellular domain (AICD). Earlystudies have demonstrated that sAPP may protect neurons again
7、st oxygen-glucose deprivation and excitotoxicity by inhibiting calcium currents (Mattson et al. 1993). sAPP alsoplay an important role in neurite outgrowth, synaptogenesis and cell adhesion (Mattson 1997; Gakhar-Koppole et al. 2008). sAPP likely has growth promoting functions in dividing cells of ep
8、ithelial origin (Herzog et al. 2004;30Siemes et al. 2006), including embryonic and adult neural stem cells (Ohsawa et al. 1999; Caille et al.2004). In the light of the reduction in sAPP concentrations in individuals brains with AD (Lannfelt et al.1995), the research of sAPP potential function is int
9、eresting for the mechanism of the ADpathogenesis.In our previous study, we found APP can interact with NCAM-140 (Chen et al. 2011), an35immunoglobulin superfamily member which linked to human brain disorders such as Alzheimers disease (Todaro et al. 2004), bipolar disorder and schizophrenia (Arai et
10、 al. 2004; Arai et al. 2006). Whats more, the interaction could modulate the phosphorylation levels of ERKs involving in NCAM-induced neurite outgrowth. The interaction site of these protein was in the conserved ectodomain domain of the APP (Chen et al. 2011), that is the the central APP domain (CAP
11、PD)40contained in sAPP. So in this study, we would like to construct sAPP recombinant plasmid andpurify this protein, and then test whether sAPP could modulate the NCAM-induced neurite outgrowth, like the full length APP.Foundations: Specialized Research Fund for the Doctoral Program of Higher Educa
12、tion（No.20090003110015）NSFC(NO.81200877)Brief author introduction:CHEN Keping (1977,1-)Assistant Professor,neurodegenerative diseaseCorrespondance author: DOU Fei(1975,11-),male,professor,neurodegenerative disease. E-mail: douf- 6 -451Materials and methodsConstruction of the recombinant plasmidsAPP
13、cDNA was subcloned into the pCTAP-C vector using the EcoR V and Sal I restriction sites. To generate the fusion protein containing the extracellular domain of APP with streptavidin-binding peptide (SBP) and calmodulin-binding peptide (CBP) affinity tags at its COOH-terminal end50(sAPP-SBP-CBP), the
14、forward primer (GCT AGA TAT CAT GCT GCC CGG TTT GG) wasdesigned on the basis of the N-terminal sequence of APP gene, an EcoR V restriction enzyme site was added at the start of the gene (underlined). The reverse primer (GCG CGT CGA CTT TTT GAT GAT GAA C) was designed on the basis of the C-terminal a
15、mino acid sequence, introducing a Sal I restriction site (underlined). After PCR amplification and purification, the purified DNAs encoding55sAPP and pCTAP-C vector were digested with EcoRV and Sal I, respectively. The correspondingfragments were purified separately, and then Ligation of the fragmen
16、t with the vector DNA was done in reaction mixture. The ligation products were transformed into E. coli DH5, and plated on LB agarose medium containing Kanamycin Sulfate to culture overnight. A single colony was selected andgrown at 37 overnight in 3mL LB medium containing Kanamycin Sulfate. The rec
17、ombinant plasmid60was extracted and purified from the amplified cells. The plasmid was confirmed by sequencing. cDNAs encoding human NCAM-140 were generous gifts from Dr. Irina Korshunova (University of Copenhagen, Copenhagen, Denmark) (Korshunova et al. 2007 )Cell Culturing, Transfection, Generatio
18、n of Stable Cell LinesCOS-7 cells were among the rare cell lines known to express absolutely no NCAMs, So we used65COS-7 cells for ERKs experiment. COS-7 and CHO cells were propagated in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated FCS. All cells (2104cells/cm2
19、) were grown in a humidified atmosphere at 37, 5% v/v CO2. All transfections were carried out usingFuGENE HD reagent according to the manufacturers protocol (Roche). Transfections were carried out in six-well tissue culture dishes for generation of stable cell lines. Briefly, CHO cells were plated a
20、t7070% confluence the day before transfection. Cells were transfected with 3g of recombinant pCTAP-sAPP-SBP-CBP plasmid. About 16 hrs post transfection, the medium was exchanged with medium supplemented with 0.5g/L G418 for selection. Exchanging the medium every day was kept for 7-10 days. Stable cl
21、ones were picked after 1020 days in culture. Expression levels of sAPP in the stable cell lines were confirmed by Western blotting.75Production of the fusion proteinThe fusion proteins with the SBP-CBP tag were purified with the tandem affinity purification (TAP) technology. The key feature of TAP w
22、as the use of two different affinity purification tags that are fused to the protein. The SBP (streptavidin-binding peptide) tag had a high affinity for the streptavidin resin, and could be effectively eluted with biotin. The CBP (calmodulin-binding peptide) tag had a high80affinity for the calmodul
23、in resin in the presence of calcium. Upon removal of calcium with a chelatingagent, recovery of the tagged protein from the resin was achieved. Performing two consecutive purification steps using SBP and CBP tags, and then having gentle washing and elution conditions allows for isolation without dis
24、ruption of the targeted protein. Purified protein was stored at 20C. Silver Staining of SDS-PAGE Gels85Immediately after the electrophoresis, sliced the stacking gel from the slab, and placed the gel in a glassdish with fixing solution (50% methanol, 12% acetic acid, 500ul/l 37% Formaldehyde) for ov
25、ernight. The next day, placed the gel in the silver solution (0.2% silver nitrate, 800ul/l 37% formaldehyde) for30 minutes, and then rinsed rapidly three times with ddw. Finally added developer (6%Na2CO3, 0.5%lNa2S2O3.5H2O, 500ul/l 37% Formaldehyde) and shook gently until the brown protein bands rea
26、ched90desired intensity and then stopped development with stop solution (50% methanol, 12% acetic acid).The silver solution and developer must be prepared freshly95100105110115120125Western blot analysisCell lysates were prepared by directly extracting cells in RIPA buffer(25 mM TrisHCl, pH 8.0, 150
27、 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) containing a protease inhibitor cocktail (Roche), followed by centrifugation at 15,000g. The samples were separated by SDS-PAGE on12% polyacrylamide gels, transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore), and blocke
28、d with 5% BSA in TBST. The blots were incubated with primary antibodies at room temperature for overnight at 4C. Horseradish peroxidase-conjugated secondary antibodies were visualized using an enhanced chemiluminescence detection system (Thermo) and exposed to Kodak X-OMAT AR films (Kodak).Antibodie
29、sThe following antibodies were used: p44/42 MAPK and Phospho-44/42 MAPK polyclonal antibody (CST); the 6E10 monoclonal antibody (Signet) identifying residues 116 of human A was mainly used for immunoblotting.2ResultsConfirmation of the stable cell linesIn order to generate the fusion proteins (sAPP-
30、SBP-CBP), we sucloned sAPP cDNA fragment into the pCTAP-C vector using the EcoR V and Sal I restriction sites. The recombinant plasmids were determined by double restriction enzyme digestion, and then confirmed by DNA sequencing. The CHO cells were transfected with the recombinant plasmids containin
31、g the sAPP cDNA fragment, and the stable cell lines were selected with G418. The results showed some cells expressed sAPP either in the cell lysate or conditioned media (Fig.1). So we got the stable cell lines (CHO/ sAPP-SBP-CBP) with high expression of the sAPP for the next purification procedure.F
32、ig.1 Expression of sAPP in the stable cell lines (parts).A, B, C and D lane represent different stable cell lines. The C stable cell lines had high expression of the sAPP either in the conditioned media or cell lysate. Western blot was performed with mouse anti-human A monoclonal antibody and detect
33、ed withHRP-conjugated goat anti-mouse IgG monoclonal antibodyPurification of the sAPP-SBP-CBP fusion proteinThe fusion protein (sAPP-SBP-CBP) had streptavidin-binding peptide (SBP) and calmodulin-binding peptide (CBP) affinity tags at its COOH-terminal, so we could finish the purification with the h
34、elp of the Streptavidin resin and MS-Grade calmodulin resin. Both tags could be eluted from their respective resins with gentle washing and small molecule elution conditions thus increasing the amount and purity of the purified protein. The results showed the fusion protein was obtained, and the fin
35、al concentration was 25mg/ml, determined with BCA protein assay kit. The amount and purity of sAPP could meet our experiments (Fig.2a; Fig.2b).130135140Fig.2 Identification of the fusion protein purification with western blot (Fig.3a) and Silver Staining (Fig.3b). A: The supernatant after the fusion
36、 proteins were incubated with the streptavidin resin; B: The eluted proteins from the streptavidin resin; C: The supernatant after the fusion proteins were incubated with the calmodulin resin; D: The eluted proteins (sAPP-SBP-CBP) from the calmodulin resin.Fig.3 sAPP modulates the MAPK signal pathwa
37、y. The COS-7cells without any transfected were the control experiment (CTRL). Western blot was performed with rabbit source polyclonal antibody (p44/42 MAPK and Phospho-p44/42 MAPK antibody) and detected with HRP-conjugated goat anti-rabbit IgG monoclonalantibody145150sAPP modulates the MAPK signal
38、pathwayNCAM (neural cell adhesion molecule) had been implicated in a variety of cellular processes, such as cell-cell adhesion, cell migration, neurite outgrowth, and synaptic plasticity (Crossin et al. 2000). The mitogenactivated protein kinases (MAPKs), extracellular regulated kinases ERK1 and ERK
39、2, were central players in NCAM signaling for inducing neurite outgrowth. ERK1 and ERK2 were molecules that played a critical role in the intracellular signaling cascade and whose phosphorylation were induced as part of the cellular response to various different stimuli. Some results have shown that
40、 the ERKs are involved in several forms of synaptic plasticity in the hippocampus and other brain areas critically involved in learning and memory processes (Thomas et al. 2004). In our previous results, the interaction of the APP and NCAM-140 may further activate MAPKs pathways to increase the acti
41、vity of ERKs. On the other hand, we also found that NCAM-140 can interact with the conserved ectodomain domain of the APP (CAPPD). In this study, we obtained the soluble extracellular domain of APP (sAPP), so we would test whether sAPP could modulate the phosphorylation levels of the ERKs involved i
42、n the MAPKs signal pathway, like the full length APP. We155160165170175180transfected the COS7cells with the vectors encoding human NCAM-140 in 6 wells, About 6 hrs post transfection, we changed the DMEM with another 2ml DMEM containing sAPP (10ul/ml). Cells were maintained for another 24 hrs. The p
43、hosphorylation levels of the ERKs were examined by immunoblotting. The results showed the phosphorylation levels of ERK1 and ERK2 were increased when sAPP existed in the DMEM (Fig.3). On the other hand, the phosphorylation levels of ERKs were also increased when the COS-7 cells were transfected with
44、 NCAM-140 and APP, which was consistent with our previous results. These results indicated sAPP was similar to the full length APP for modulating the ERKs phosphorylation.3ConclusionConsidering the importance of the sAPP in diverse cellular processes involved in cell proliferation, cell survival, ne
45、uroprotection, enhancement of memory, we constructed the sAPP recombinant plasmid and purified the fusion protein with TAP technology. On the other hand, we also found the sAPP could modulate the phosphorylation levels of the ERKs involving in the MAPK signal pathway. Early studies reported that tre
46、atment with sAPP had been shown to stimulate ERK signaling in PC-12 cells (Greenberg et al. 1995). Recently, Demars et al. (2011) found Recombinant sAPP was able to reverse the deficits in ERK phosphorylation caused by GM6001 treatment in the neural progenitor cells (NPCs). And the results also show
47、ed that sAPP was associated specifically with ERK signaling because Phosphorylation of Akt, another integral proliferation pathway known to respond to sAPP, did not change significantly from negative control (Demars et al. 2011). Here, our results showed sAPP could modulate the phosphorylation levels of the ERKs in COS-7 cells. So these results indicated sAPP
