E1A激活基因阻遏子（CREG）调控人血管平滑肌细胞表型转化的膜受体机制
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摘要
目的动脉粥样硬化(atherosclerosis, AS)和经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)术后再狭窄(restenosis, RS)等增生性血管疾病是目前威胁人类健康最严重的疾病之一,研究其防治策略具有重要意义。近年研究发现,成熟血管壁的血管平滑肌细胞(vascular smooth muscle cells, VSMC)具有极大可塑性。生理状态下血管壁中VSMC处于分化表型,血管损伤后,VSMC可发生表型逆转,由分化表型(收缩表型)转变为去分化表型(合成表型),并获得增殖、迁移及合成、分泌大量细胞外基质的能力。VSMC由分化表型向去分化表型的转化是AS及PCI术后RS等疾病发生中新生内膜形成和管腔狭窄的重要病理基础。因此,对VSMC表型转化机制的研究是增生性血管病防治研究的一个重要方向。
1998年,Gill等研究提示,人E1A激活基因阻遏子基因(cellular repressor of E1A-stimulated genes, CREG)可以诱导细胞的分化和成熟,并参与成熟组织中细胞分化状态的维持。我室自1999年应用mRNA差异显示技术首次从体外培养的人胸廓内动脉血管平滑肌细胞(Human internal thoracic artery smooth muscle cell,,HITASY)中克隆出了人CREG基因,并证实CREG表达与VSMC分化呈高度相关,提示CREG参与了体外培养的VSMC由增殖表型向分化表型转化。但是CREG作为分泌型蛋白质,其诱导VSMC分化和抑制VSMC增生的机制尚待阐明。Gill实验室通过Far Western分析提示[1],CREG可与细胞膜上的甘露糖6磷酸/胰岛素样生长因子II受体(mannose-6-phosphate/insulin-like growth factorⅡreceptor,M6P/IGF2R)发生相互作用,在人畸胎瘤细胞中发挥抑制增殖和/或促进分化的作用,并证实CREG与M6P/IGF2R的相互作用是通过CREG蛋白的糖基化位点介导的。为明确M6P/IGF2R在CREG调控VSMC分化中的作用,本室进行了相关的预实验研究,发现用蛋白磷酸酶PP2A的特异性抑制剂岗田酸(okadaic acid)处理VSMC后,细胞膜表面M6P/IGF2R减少,从而使外源性CREG蛋白诱导的VSMC分化受到抑制,提示膜受体蛋白M6P/IGF2R可能参与了CREG对VSMC分化的调控。本研究将着重阐明M6P/IGF2R是否介导了CREG蛋白的生物学功能,CREG与M6P/IGF2R之间是否发生直接相互作用、相互作用的结合位点及其与CREG蛋白分子中糖基化结构的关系。
方法(1)用RT-PCR技术扩增终止密码突变的人CREG开放读码框,构建含有myc和His标签的野生型CREG(wtCREG)真核表达载体pcDNA3.1 myc-His/wtCREG,以及3个糖基化位点(第160, 193和216位的Asn残基)全部突变为Ala残基的去糖基化突变型CREG(mCREG)真核表达载体pcDNA3.1 myc-His/mCREG。Lipofectamine 2000转染人293F细胞株,用G418筛选出稳定表达的细胞克隆,大量扩增后提取细胞蛋白,用Ni-NTA亲合层析方法纯化,并进行糖苷酶切和Western blot检测鉴定,得到野生型和糖基缺失突变的CREG蛋白(wtCREG和mCREG),并通过与标准品白蛋白对比,计算出纯化蛋白的浓度;(2)将纯化得到的重组wtCREG及mCREG蛋白以不同浓度,加入本室保存的人胸廓内动脉VSMC系(简称为HITASY细胞)和HITASY细胞衍生的、经逆转录病毒介导的、shCREG干扰后CREG蛋白低表达的人VSMC(简称为OB2细胞)培养液中,应用流式细胞分析方法检测细胞增殖情况,比较两种CREG蛋白对细胞增殖效应的调控作用,确定两种蛋白调控人VSMC生物学行为的最适浓度。将最适浓度的两种蛋白质分子分别加入HITASY和OB2细胞培养上清液中,应用细胞流式分析、Western blot、细胞刮伤实验、明胶酶电泳分析等方法观察两种重组人CREG蛋白对人VSMC增殖、迁移、分化等生物学行为的影响;通过不同浓度(2,4,8μg/μL)的M6P/IGF2R抗体阻断实验、免疫荧光双染色和免疫共沉淀等检测方法,分析CREG蛋白是否与M6P/IGF2R存在直接结合,进一步明确M6P/IGF2R介导两种CREG蛋白质生物学功能;(3)进一步应用RT-PCR合成M6P/IGF2R细胞外不同结构域(M6P/IGF2R-1:1~3结构域;M6P/IGF2R-2:4~6结构域;M6P/IGF2R-3:7~10结构域)cDNA片段,并构建带有His标签的PQE31- M6P/IGF2R-1,2,3载体,在大肠杆菌JM109菌株中表达,通过Ni-NTA亲合层析纯化得到人M6P/IGF2R胞外结构域蛋白小肽。进一步应用SDS-PAGE电泳和Western blot鉴定纯化蛋白的准确性,并通过与标准品白蛋白对比,计算出纯化蛋白的浓度。应用固相吸附实验,检测固化的M6P/IGF2R(R&D Systems)胞外结构域(其中11~15号结构域—IGF2R-4—从R&D公司购买)与纯化的wt/mCREG的直接结合,并通过Scatchard分析计算离解常数(KD值)。分别应用M6P和6-磷酸葡糖糖(glucose 6-phosphate,G6P)进行体外竞争性结合实验,进一步确定CREG蛋白与M6P/IGF2R相互作用是否与蛋白的糖基化有关。将与wt/mCREG蛋白高亲合的M6P/IGF2R胞外重组小肽片段分别添加到细胞培养上清中,确定wt/mCREG蛋白对VSMC生物学行为的调控作用(流式细胞分析检测细胞增殖、刮伤实验和明胶酶谱分析细胞迁移能力)是否通过其与M6P/IGF2R小肽的直接亲合作用介导。
结果( 1 )制备并纯化得到重组的人wtCREG/myc-His和mCREG/myc-His标签蛋白:1)RT-PCR扩增出终止密码突变的人CREG cDNA片段,插入PMD-18T载体中,经酶切及测序证实插入片段序列正确;用BamH I/EcoR I双酶切将CREG cDNA片段亚克隆至pcDNA3.1 myc-His真核表达载体中,构建完成pcDNA3.1 myc-His/wtCREG重组质粒,测序确定插入序列正确;将生工公司合成的氨基酸突变的CREG cDNA片段经BamH I/EcoR I酶亚克隆至pcDNA3.1 myc-His真核表达载体中,构建完成pcDNA3.1 myc-His/mCREG重组质粒,测序确定插入序列正确;2)将构建完成的pcDNA3.1 myc-His/wt/mCREG重组质粒转染至人293F工程细胞株中,通过G418抗性筛选获得高表达CREG蛋白的抗性克隆。大量扩增克隆细胞株,收集细胞后进行裂解提取细胞中蛋白质,经Ni-NTA亲合层析纯化获得重组人wtCREG/myc-His和人mCREG/myc-His标签蛋白。经糖苷酶切和Western blot检测证实分别为含有糖基结构的CREG和去糖基突变型的CREG蛋白;SDS-PAGE电泳确定重组蛋白质的分子量,与标准品白蛋白对比,计算出纯化的重组蛋白浓度分别为0.893μg/μL和0.972μg/μL,纯度为92%和93%;(2)两种重组CREG蛋白生物学效应检测及其细胞膜表面受体机制:1)最适宜效应浓度分析:将两种纯化的重组CREG蛋白稀释成不同浓度(200、400、800、1600nM),加入经去血清培养72h进行同步化的HITASY和OB2细胞培养上清中,8h后收集细胞用流式细胞仪分析细胞周期,结果显示:各组细胞G0/G1期细胞比例均有所增加,添加wtCREG蛋白组作用更加明显,而且两种CREG蛋白发挥生物学功能的最佳浓度均为400nM。上述实验结果证实:两种重组CREG蛋白对VSMC增殖均有剂量依赖性的抑制作用,并且相同浓度的糖基化的CREG蛋白对细胞增殖的抑制效应更为显著,最佳效应浓度为400nM;2)两种重组CREG蛋白添加后对HITASY和OB2细胞生物学行为的影响:①CREG蛋白对VSMC迁移的影响:刮伤实验发现,加入最佳效应浓度的wtCREG和mCREG蛋白24h后,OB2组迁移能力下降,HITASY组无明显变化;细胞外基质金属蛋白酶-2,9(Matrix metallo-proteinase 2,9,MMP2 ,9)明胶酶电泳检测和Western blot检测结果证实,两种CREG蛋白均可以使OB2细胞合成细胞外基质MMP2,9减少,而组织金属蛋白酶抑制物(Tissue Inhibitors of Metalloproteinases,TIMPs)增加;②CREG蛋白对VSMC分化的影响:加入400nM的wtCREG和mCREG蛋白12h后,OB2细胞myocardin、SMα-actin、MHC、caldesmin表达增加,LM-1、FN表达减少;③流式细胞仪分析细胞周期和BrDU染色分析证实,加入400nM的wtCREG和mCREG蛋白后,OB2组G0/G1期细胞由0.5308分别增加至0.5773和0.5572,HITASY组G0/G1期细胞由0.6297分别增加至0.7369和0.7034;3)M6P/IGF2R在重组CREG蛋白的生物学功能中的调控作用:①免疫共沉淀和免疫荧光双染色分析结果显示,CREG蛋白与M6P/IGF2R存在直接结合;②应用抗体阻断实验:将不同浓度的anti- M6P/IGF2R(2、4、8μg /mL)与两种CREG蛋白同时加入培养液中,CREG蛋白抑制VSMC增值、迁移和合成细胞外基质、促进分化的效应减弱,而且与加入anti- M6P/IGF2R浓度正相关。上述实验结果证实:两种重组CREG蛋白对体外培养的HITASY和OB2细胞向分化表型转化有明显的促进作用,同时抑制细胞的增殖、迁移和合成细胞外基质的能力,并且两种重组CREG蛋白对VSMC生物学行为的调控均可被M6P/IGF2R中和抗体阻断,CREG蛋白与M6P/IGF2R存在直接结合;(3)与两种重组CREG蛋白相互作用的M6P/IGF2R结构域分析:1)RT-PCR扩增含有不同结构域M6P/IGF2R cDNA片段,插入PMD18-T载体中,测序确定插入片段序列准确。用Sph I/Sal I双酶切将M6P/IGF2R cDNA片段亚克隆至PQE31-His原核表达载体中,构建完成重组质粒,测序确定插入序列正确;载体质粒感染大肠杆菌JM109菌株后扩增,并通过Ni-NTA亲合层析纯化获得原核表达蛋白,SDS-PAGE分析确定重组蛋白的分子量分别为:54kD(IGF2R-1),44kD(IGF2R-2),62kD(IGF2R-3)。Western blot分析证实所获得的M6P/IGF2R-1,2,3重组蛋白均为M6P/IGF2R和His标签重组蛋白,并将三种重组蛋白的浓度均调整为10nM。ELISA分析结果证实:重组wtCREG蛋白与IGF2R-3和IGF2R-4具有高结合力,KD值分别为0.1063pM和0.2106pM;重组的mCREG蛋白与M6P/IGF2R IGF2R-4存在高亲和力, KD值为6.488pM;其中wtCREG与M6P/IGF2R-3/ IGF2R-4结合作用可以被M6P竞争抑制;2)将不同浓度的M6P/IGF2R的第11~15结构域和第7~10结构域小肽片段与两种重组CREG蛋白共同加入体外培养的OB2细胞中,观察重组CREG蛋白对OB2增殖和迁移的效应变化,结果发现:wtCREG和mCREG对细胞生物学行为的调控作用均被M6P/IGF2R的第11~15结构域小肽阻断,而第7~10结构域小肽片段仅对wtCREG蛋白的生物学效应有明显的阻断作用,对mCREG的生物学作用无效。
结论CREG可以作为分泌型蛋白,通过细胞膜表面M6P/IGF2R蛋白抑制VSMC的增殖、迁移、分泌细胞外基质,并促进其由未分化表型向分化表型转化,其作用与其分子中糖基化结构无关,糖基化可以增强CREG的生物学功能,wtCREG蛋白在M6P/IGF2R作用位点主要是7~10号和11~15号结构域,而mCREG蛋白在M6P/IGF2R作用位点主要是11~15号结构域。
Object Atherosclerosis(AS)and restenosis(RS) post percutaneous coronary intervention(PCI) are most danger to human health as cellular proliferative desease. It is important to study how to prevent AS and RS post-PCI. Recent study suggested mature vascular smooth muscle cells(VSMC) had remarkable plasticity. In physiologic condition, VSMC show the differentiation phenotype. Whereas, when injury occurred, the VSMC would transform its differentiation phenotype to un-differentiation phenotype and gain the abilities to migrate, proliferate and metabolize extracellular matrix. The phynotye transition of VSMC is the pathologic mechanism of AS and RS post-PCI. So, to study the mechanism of phynotype transition is an important aspect to provent hyperplasic vascular desease.
In 1998, Gill’s study showed the cellular repressor of E1A-stimulated genes(CREG) could promote cell differentiation and maturity. Also, CREG might effect on the maintenance of cellular differentiation status. In 1999, we firstly cloned the CREG genes with mRNA differential display technology from human internal thoracic artery-Shenyang(HITASY-Shenyang)and confirmed that expressing of CREG was highly correlated to VSMC’s differentiation, which suggested CREG took part in switching from proliferation phenotype to differentiation phenotype. While, the mechanism of CREG, as one secreted glycoprotein, promotes cellular differentiation and inhibits its proliferation need to be defined. By Far Western assay, Gill et showed the CREG executed the biologic function after binding to the mannose-6-phosphate/insulin-like growth factorⅡ(M6P/IGF2R) depended its glycosylation sites. To confirm the role of M6P/IGF2R in the CREG biologic function, we decreased the quantity of M6P/IGF2R on the cellular membrane using okadaic acid and found the effect of CREG on promoting VSMC differentiation was inhibited, which suggested the M6P/IGF2R might mediate the process of CREG regulating VSMC differentiation.This study intend to clear if CREG directly bind to M6P/IGF2R, if the binding depends on its glycosylation sites, if the binding is on the VSMC’membrane and the mechanism of M6P/IGF2R mediating CREG biologic function.
Methods (1)The ORF(open reading frame) fragment of human CREG(wtCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector, pcDNA3.1 myc-His/wtCREG, was constructed. At the same time, change the Asn residue of the glycosylation sites (160,193,216 Asn residue) with Ala residue and construct expression vector pcDNA3.1 myc-His/mCREG. The human 293F cells were transfected with pcDNA3.1 myc-His/wt/mCREG using Lipofectamine 2000, and stably transfected cell clones were selected by G418. The recombinant secreted wt/mCREG/myc-His protein were purified by Ni-NTA affinity chromatography and characterized with glucosidase and Western blot assay. Measure the concentration compared to the standard albumn.(2)The optimal effect concentration of CREG on VSMC was studied. Add .the wt/mCREG with optimal concentration to HITASY cell (VSMC line gained from human internal thoracic artery) and OB2 cell (HITASY which do not express CREG by retrovirus jamming the shCREG). The effects of CREG on the change of proliferation, migration and differentiation of VMSC were studied by cell cycle, wound-healing, Western blot assay and gelatinase digestion analysis. To clear the role of M6P/IGF2R in the CREG biologic function, we add M6P/IGF2R anti-body (2,4,8nM) to OB2 cell with wt/mCREG at the same time to study the blocking effect. Also, double immunofluorescence staining and co-immunoprecipitation were performed to analyses if CREG peotein directly binded to M6P/IGF2R.(3)Segments of M6P/IGF2R domains were recombined by RT-PCR(IGF2R-1:1~3.domains; IGF2R-2:4~6.domains;.IGF2R-4:7~10.domains).PQE31 IGF2R-1,2,3 expression vectors with His-label were constructed and transfected to E coli JM109. The domains protein was gained by Ni-NTA affinity chromatography and purified. SDS-PAGE and Western blot assay were performed. Measure the concentration compared to the standard albumin. To analyses binding of CREG with M6P/IGF2R domains(IGF2R-4:11~15 domain bought from R&D company) and calculate the dissociation constant (KD value) using solid phase binding experiment. Then, the segment which wt/mCREG most binding to was identified.M6P and G6P blocking test was performed to study if the glycosylated structure of CREG mediated its binding to M6P/IGF2R by M6P. The in vivo competitive binding test was study by adding the segment protein which CREG most binding to into the supernatant with wt/mCREG receptively. The change of CREG biologic effects on VSMC were analyseses to identified the direct binding of CREG to IGF2R domains。 Results (1) Produce and purify the recombinant wt/mCREG protein: 1) The ORF(open reading frame) fragment of human CREG with terminator codon mutation was amplified by RT-PCR and inserted into PMD-18T vector, which was confirmed by restricted endonuclease digestion and DNA sequencing. Subclone the CREG cDNA fragment into pcDNA myc-His vector and the eukaryotic expression vector pcDNA3.1 myc-His/wtCREG was constructed which was confirmed by DNA sequencing. The designed CREG cDNA(combined by biotechnique company) with amino acid residue mutated was subcloned into pcDNA myc-His vector by the BamH I/EcoR digestion, which was confirmed by DNA sequencing.2)The recombinant expression vector, wt/mCREG, were transfected to 293F engineering cell sand selected by G418 to obtain the cell clone with CREG high expressing. The cell clone were amplified and harvested. After that, the two kind recombinant fusion proteins, wt/mCREG with myc/His label, in 293 cell lysates were purified by Ni-NTA affinity chromatography and tested by PNGaseF digestion and Western blot assay. SDS-PAGE showed the molecular mass. The concentration were 0.893μg/μl and 0.972μg/μl and the purity were 92% and 93% respectively compared to standard protein.(2)CREG protein biologic function study and the membrane surface receptor mechanism study:1)the optimal effect concentration analysis: add different concentration (200,400,800,1600 nM) wt/mCREG to supernatant of the synchronized HITASY and OB2 cells which were cultured with free serum medium for 72h. After eight hours, the cells were harvested and cell cycle was analysesed showed the ratio of cell in GO/G1 period was increased in all of group. The effect of wtCREG is more powerful than that of mCREG..The optimal concentration of two kinds of CREG proteins was 400nM. The study showed the recombinant wt/mCREG protein depressed the VSMC proliferation depending on dose and the optimal concentration was 400nM;2)biologic function of CREG protein and the membrance receptor mechanism:①effect on VSMC migration: the wound healing experiment showed the OB2 cells migration was slower significantly after added wt/mCREG(400Nm) in supernatant. The HITASY cells migration were very slowly and no remarkable change. The gelatinase digestion and Western blot analysis showed the matrix metalloproteinase(MMP) was decreased and TIMPs was increased;②effect on differentiation: after added wt/mCREG(400nM), the expression of myocardin, SMα-actin, MHC and caldesmin were increased and that of LM-1 and FN were decreased in OB2 cells. These effects were more significant when adding wtCREG.;③effect on VSMC proliferation: Cell cycle assay and BrDU stain showed: after added the wtCREG and mCREG protein, the ratio of cell in G0/G1 phase increased to 0.5773 and 0.5572 from 0.5308 respectively in OB2 group, which increased to 0.7369 and 0.7034 respectively from 0.6297 in HITASY group;3)Role of M6P/IGF2R in CREG biologic function:①ELISA and co-immunoprecipitation showed the wt/mCREG binding to M6P/IGF2R directly.②antibody blocking test: when the anti-IGF2R was added to medium at the same time with wt/mCREG at different concentration(2μg/mL、4μg/mL、8μg/mL),the effects of CREG protein which depressing proliferation, migration, secretion and promoting differentiation were blocked, which had the positive correlation to the concentration of added anti body. The studies showed two combinant CREG promoted VSMC switch to differentiation phaenotype, at the same time, depress VSMC proliferation, migration and secreting extracellular matrix. The function of wt/mCREG can be blocked by anti-M6P/IGF2R. CREG protein binded to M6P/IGF2R directly;(3)analysis of which M6P/IGF2R domain CREG most likely bind to:1) The ORF(open reading frame) fragment of human M6P/IGF2R domains with terminator codon mutation was amplified by RT-PCR and were inserted into PMD-18T vector which were confirmed by DNA sequencing. The M6P/IGF2R cDNA were subcloned to the prokaryotic expression vector PQE31-His and construct the PQE31 IGF2R-1,2,3 recombinant plasmid which were confirmed by DNA sequencing. The recombinant expression vectors were tranfected into Eo1 JM109 and amplified. The expressed M6P/IGF2R domain proteins were gained by Ni-NTA affinity chromatography and analysesed by SDS-PAGE and Western blot. The SDS-PAGE showed the molecular mass were 54kD(IGF2R-1),44kD(IGF2R-2),62kD(IGF2R-3). Adjust the concentration to 10nM. ELISA analysis suggested the wtCREG had the most affinity to IGF2R-3 and IGF2R-4, while the mCREG to IGF2R-4.The KD value were 0.1063pM, 0.2106pM and 6.488pM respectively;The binding of CREG protein to M6P/IGF2R-3 was depressed by M6P; 3) Add the M6P/IGF2R-3 and M6P/IGF2R-4 to OB2 cell with wt/mCREG at the same time by the different concentration. The study showed M6P/IGF2R-4 blocked the effects of wt/mCREG depressing VSMC proliferation and migration, while M6P/IGF2R-3 only blocked the effects of wt/CREG was blocked by M6P/IGF2R-4.
Conclution Binding to cytomembrane M6P/IGF2R, CREG protein can down-regulate the proliferation, migration and up-regulate differentiation of VSMC promoting the cells’phaenotype transform from undifferentiation to differentiation no matter if glycosylation structures exist. But , the biologic function is more remarkable when the glycosylation structures exist. CREG protein has different affinity to M6P/IGF2R domain depend on glycosylation structures existing or not. The wtCREG showed high affinity to the 7-10 domains but the mCREG to 11-15 domains.
1998年,Gill等研究提示,人E1A激活基因阻遏子基因(cellular repressor of E1A-stimulated genes, CREG)可以诱导细胞的分化和成熟,并参与成熟组织中细胞分化状态的维持。我室自1999年应用mRNA差异显示技术首次从体外培养的人胸廓内动脉血管平滑肌细胞(Human internal thoracic artery smooth muscle cell,,HITASY)中克隆出了人CREG基因,并证实CREG表达与VSMC分化呈高度相关,提示CREG参与了体外培养的VSMC由增殖表型向分化表型转化。但是CREG作为分泌型蛋白质,其诱导VSMC分化和抑制VSMC增生的机制尚待阐明。Gill实验室通过Far Western分析提示[1],CREG可与细胞膜上的甘露糖6磷酸/胰岛素样生长因子II受体(mannose-6-phosphate/insulin-like growth factorⅡreceptor,M6P/IGF2R)发生相互作用,在人畸胎瘤细胞中发挥抑制增殖和/或促进分化的作用,并证实CREG与M6P/IGF2R的相互作用是通过CREG蛋白的糖基化位点介导的。为明确M6P/IGF2R在CREG调控VSMC分化中的作用,本室进行了相关的预实验研究,发现用蛋白磷酸酶PP2A的特异性抑制剂岗田酸(okadaic acid)处理VSMC后,细胞膜表面M6P/IGF2R减少,从而使外源性CREG蛋白诱导的VSMC分化受到抑制,提示膜受体蛋白M6P/IGF2R可能参与了CREG对VSMC分化的调控。本研究将着重阐明M6P/IGF2R是否介导了CREG蛋白的生物学功能,CREG与M6P/IGF2R之间是否发生直接相互作用、相互作用的结合位点及其与CREG蛋白分子中糖基化结构的关系。
方法(1)用RT-PCR技术扩增终止密码突变的人CREG开放读码框,构建含有myc和His标签的野生型CREG(wtCREG)真核表达载体pcDNA3.1 myc-His/wtCREG,以及3个糖基化位点(第160, 193和216位的Asn残基)全部突变为Ala残基的去糖基化突变型CREG(mCREG)真核表达载体pcDNA3.1 myc-His/mCREG。Lipofectamine 2000转染人293F细胞株,用G418筛选出稳定表达的细胞克隆,大量扩增后提取细胞蛋白,用Ni-NTA亲合层析方法纯化,并进行糖苷酶切和Western blot检测鉴定,得到野生型和糖基缺失突变的CREG蛋白(wtCREG和mCREG),并通过与标准品白蛋白对比,计算出纯化蛋白的浓度;(2)将纯化得到的重组wtCREG及mCREG蛋白以不同浓度,加入本室保存的人胸廓内动脉VSMC系(简称为HITASY细胞)和HITASY细胞衍生的、经逆转录病毒介导的、shCREG干扰后CREG蛋白低表达的人VSMC(简称为OB2细胞)培养液中,应用流式细胞分析方法检测细胞增殖情况,比较两种CREG蛋白对细胞增殖效应的调控作用,确定两种蛋白调控人VSMC生物学行为的最适浓度。将最适浓度的两种蛋白质分子分别加入HITASY和OB2细胞培养上清液中,应用细胞流式分析、Western blot、细胞刮伤实验、明胶酶电泳分析等方法观察两种重组人CREG蛋白对人VSMC增殖、迁移、分化等生物学行为的影响;通过不同浓度(2,4,8μg/μL)的M6P/IGF2R抗体阻断实验、免疫荧光双染色和免疫共沉淀等检测方法,分析CREG蛋白是否与M6P/IGF2R存在直接结合,进一步明确M6P/IGF2R介导两种CREG蛋白质生物学功能;(3)进一步应用RT-PCR合成M6P/IGF2R细胞外不同结构域(M6P/IGF2R-1:1~3结构域;M6P/IGF2R-2:4~6结构域;M6P/IGF2R-3:7~10结构域)cDNA片段,并构建带有His标签的PQE31- M6P/IGF2R-1,2,3载体,在大肠杆菌JM109菌株中表达,通过Ni-NTA亲合层析纯化得到人M6P/IGF2R胞外结构域蛋白小肽。进一步应用SDS-PAGE电泳和Western blot鉴定纯化蛋白的准确性,并通过与标准品白蛋白对比,计算出纯化蛋白的浓度。应用固相吸附实验,检测固化的M6P/IGF2R(R&D Systems)胞外结构域(其中11~15号结构域—IGF2R-4—从R&D公司购买)与纯化的wt/mCREG的直接结合,并通过Scatchard分析计算离解常数(KD值)。分别应用M6P和6-磷酸葡糖糖(glucose 6-phosphate,G6P)进行体外竞争性结合实验,进一步确定CREG蛋白与M6P/IGF2R相互作用是否与蛋白的糖基化有关。将与wt/mCREG蛋白高亲合的M6P/IGF2R胞外重组小肽片段分别添加到细胞培养上清中,确定wt/mCREG蛋白对VSMC生物学行为的调控作用(流式细胞分析检测细胞增殖、刮伤实验和明胶酶谱分析细胞迁移能力)是否通过其与M6P/IGF2R小肽的直接亲合作用介导。
结果( 1 )制备并纯化得到重组的人wtCREG/myc-His和mCREG/myc-His标签蛋白:1)RT-PCR扩增出终止密码突变的人CREG cDNA片段,插入PMD-18T载体中,经酶切及测序证实插入片段序列正确;用BamH I/EcoR I双酶切将CREG cDNA片段亚克隆至pcDNA3.1 myc-His真核表达载体中,构建完成pcDNA3.1 myc-His/wtCREG重组质粒,测序确定插入序列正确;将生工公司合成的氨基酸突变的CREG cDNA片段经BamH I/EcoR I酶亚克隆至pcDNA3.1 myc-His真核表达载体中,构建完成pcDNA3.1 myc-His/mCREG重组质粒,测序确定插入序列正确;2)将构建完成的pcDNA3.1 myc-His/wt/mCREG重组质粒转染至人293F工程细胞株中,通过G418抗性筛选获得高表达CREG蛋白的抗性克隆。大量扩增克隆细胞株,收集细胞后进行裂解提取细胞中蛋白质,经Ni-NTA亲合层析纯化获得重组人wtCREG/myc-His和人mCREG/myc-His标签蛋白。经糖苷酶切和Western blot检测证实分别为含有糖基结构的CREG和去糖基突变型的CREG蛋白;SDS-PAGE电泳确定重组蛋白质的分子量,与标准品白蛋白对比,计算出纯化的重组蛋白浓度分别为0.893μg/μL和0.972μg/μL,纯度为92%和93%;(2)两种重组CREG蛋白生物学效应检测及其细胞膜表面受体机制:1)最适宜效应浓度分析:将两种纯化的重组CREG蛋白稀释成不同浓度(200、400、800、1600nM),加入经去血清培养72h进行同步化的HITASY和OB2细胞培养上清中,8h后收集细胞用流式细胞仪分析细胞周期,结果显示:各组细胞G0/G1期细胞比例均有所增加,添加wtCREG蛋白组作用更加明显,而且两种CREG蛋白发挥生物学功能的最佳浓度均为400nM。上述实验结果证实:两种重组CREG蛋白对VSMC增殖均有剂量依赖性的抑制作用,并且相同浓度的糖基化的CREG蛋白对细胞增殖的抑制效应更为显著,最佳效应浓度为400nM;2)两种重组CREG蛋白添加后对HITASY和OB2细胞生物学行为的影响:①CREG蛋白对VSMC迁移的影响:刮伤实验发现,加入最佳效应浓度的wtCREG和mCREG蛋白24h后,OB2组迁移能力下降,HITASY组无明显变化;细胞外基质金属蛋白酶-2,9(Matrix metallo-proteinase 2,9,MMP2 ,9)明胶酶电泳检测和Western blot检测结果证实,两种CREG蛋白均可以使OB2细胞合成细胞外基质MMP2,9减少,而组织金属蛋白酶抑制物(Tissue Inhibitors of Metalloproteinases,TIMPs)增加;②CREG蛋白对VSMC分化的影响:加入400nM的wtCREG和mCREG蛋白12h后,OB2细胞myocardin、SMα-actin、MHC、caldesmin表达增加,LM-1、FN表达减少;③流式细胞仪分析细胞周期和BrDU染色分析证实,加入400nM的wtCREG和mCREG蛋白后,OB2组G0/G1期细胞由0.5308分别增加至0.5773和0.5572,HITASY组G0/G1期细胞由0.6297分别增加至0.7369和0.7034;3)M6P/IGF2R在重组CREG蛋白的生物学功能中的调控作用:①免疫共沉淀和免疫荧光双染色分析结果显示,CREG蛋白与M6P/IGF2R存在直接结合;②应用抗体阻断实验:将不同浓度的anti- M6P/IGF2R(2、4、8μg /mL)与两种CREG蛋白同时加入培养液中,CREG蛋白抑制VSMC增值、迁移和合成细胞外基质、促进分化的效应减弱,而且与加入anti- M6P/IGF2R浓度正相关。上述实验结果证实:两种重组CREG蛋白对体外培养的HITASY和OB2细胞向分化表型转化有明显的促进作用,同时抑制细胞的增殖、迁移和合成细胞外基质的能力,并且两种重组CREG蛋白对VSMC生物学行为的调控均可被M6P/IGF2R中和抗体阻断,CREG蛋白与M6P/IGF2R存在直接结合;(3)与两种重组CREG蛋白相互作用的M6P/IGF2R结构域分析:1)RT-PCR扩增含有不同结构域M6P/IGF2R cDNA片段,插入PMD18-T载体中,测序确定插入片段序列准确。用Sph I/Sal I双酶切将M6P/IGF2R cDNA片段亚克隆至PQE31-His原核表达载体中,构建完成重组质粒,测序确定插入序列正确;载体质粒感染大肠杆菌JM109菌株后扩增,并通过Ni-NTA亲合层析纯化获得原核表达蛋白,SDS-PAGE分析确定重组蛋白的分子量分别为:54kD(IGF2R-1),44kD(IGF2R-2),62kD(IGF2R-3)。Western blot分析证实所获得的M6P/IGF2R-1,2,3重组蛋白均为M6P/IGF2R和His标签重组蛋白,并将三种重组蛋白的浓度均调整为10nM。ELISA分析结果证实:重组wtCREG蛋白与IGF2R-3和IGF2R-4具有高结合力,KD值分别为0.1063pM和0.2106pM;重组的mCREG蛋白与M6P/IGF2R IGF2R-4存在高亲和力, KD值为6.488pM;其中wtCREG与M6P/IGF2R-3/ IGF2R-4结合作用可以被M6P竞争抑制;2)将不同浓度的M6P/IGF2R的第11~15结构域和第7~10结构域小肽片段与两种重组CREG蛋白共同加入体外培养的OB2细胞中,观察重组CREG蛋白对OB2增殖和迁移的效应变化,结果发现:wtCREG和mCREG对细胞生物学行为的调控作用均被M6P/IGF2R的第11~15结构域小肽阻断,而第7~10结构域小肽片段仅对wtCREG蛋白的生物学效应有明显的阻断作用,对mCREG的生物学作用无效。
结论CREG可以作为分泌型蛋白,通过细胞膜表面M6P/IGF2R蛋白抑制VSMC的增殖、迁移、分泌细胞外基质,并促进其由未分化表型向分化表型转化,其作用与其分子中糖基化结构无关,糖基化可以增强CREG的生物学功能,wtCREG蛋白在M6P/IGF2R作用位点主要是7~10号和11~15号结构域,而mCREG蛋白在M6P/IGF2R作用位点主要是11~15号结构域。
Object Atherosclerosis(AS)and restenosis(RS) post percutaneous coronary intervention(PCI) are most danger to human health as cellular proliferative desease. It is important to study how to prevent AS and RS post-PCI. Recent study suggested mature vascular smooth muscle cells(VSMC) had remarkable plasticity. In physiologic condition, VSMC show the differentiation phenotype. Whereas, when injury occurred, the VSMC would transform its differentiation phenotype to un-differentiation phenotype and gain the abilities to migrate, proliferate and metabolize extracellular matrix. The phynotye transition of VSMC is the pathologic mechanism of AS and RS post-PCI. So, to study the mechanism of phynotype transition is an important aspect to provent hyperplasic vascular desease.
In 1998, Gill’s study showed the cellular repressor of E1A-stimulated genes(CREG) could promote cell differentiation and maturity. Also, CREG might effect on the maintenance of cellular differentiation status. In 1999, we firstly cloned the CREG genes with mRNA differential display technology from human internal thoracic artery-Shenyang(HITASY-Shenyang)and confirmed that expressing of CREG was highly correlated to VSMC’s differentiation, which suggested CREG took part in switching from proliferation phenotype to differentiation phenotype. While, the mechanism of CREG, as one secreted glycoprotein, promotes cellular differentiation and inhibits its proliferation need to be defined. By Far Western assay, Gill et showed the CREG executed the biologic function after binding to the mannose-6-phosphate/insulin-like growth factorⅡ(M6P/IGF2R) depended its glycosylation sites. To confirm the role of M6P/IGF2R in the CREG biologic function, we decreased the quantity of M6P/IGF2R on the cellular membrane using okadaic acid and found the effect of CREG on promoting VSMC differentiation was inhibited, which suggested the M6P/IGF2R might mediate the process of CREG regulating VSMC differentiation.This study intend to clear if CREG directly bind to M6P/IGF2R, if the binding depends on its glycosylation sites, if the binding is on the VSMC’membrane and the mechanism of M6P/IGF2R mediating CREG biologic function.
Methods (1)The ORF(open reading frame) fragment of human CREG(wtCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector, pcDNA3.1 myc-His/wtCREG, was constructed. At the same time, change the Asn residue of the glycosylation sites (160,193,216 Asn residue) with Ala residue and construct expression vector pcDNA3.1 myc-His/mCREG. The human 293F cells were transfected with pcDNA3.1 myc-His/wt/mCREG using Lipofectamine 2000, and stably transfected cell clones were selected by G418. The recombinant secreted wt/mCREG/myc-His protein were purified by Ni-NTA affinity chromatography and characterized with glucosidase and Western blot assay. Measure the concentration compared to the standard albumn.(2)The optimal effect concentration of CREG on VSMC was studied. Add .the wt/mCREG with optimal concentration to HITASY cell (VSMC line gained from human internal thoracic artery) and OB2 cell (HITASY which do not express CREG by retrovirus jamming the shCREG). The effects of CREG on the change of proliferation, migration and differentiation of VMSC were studied by cell cycle, wound-healing, Western blot assay and gelatinase digestion analysis. To clear the role of M6P/IGF2R in the CREG biologic function, we add M6P/IGF2R anti-body (2,4,8nM) to OB2 cell with wt/mCREG at the same time to study the blocking effect. Also, double immunofluorescence staining and co-immunoprecipitation were performed to analyses if CREG peotein directly binded to M6P/IGF2R.(3)Segments of M6P/IGF2R domains were recombined by RT-PCR(IGF2R-1:1~3.domains; IGF2R-2:4~6.domains;.IGF2R-4:7~10.domains).PQE31 IGF2R-1,2,3 expression vectors with His-label were constructed and transfected to E coli JM109. The domains protein was gained by Ni-NTA affinity chromatography and purified. SDS-PAGE and Western blot assay were performed. Measure the concentration compared to the standard albumin. To analyses binding of CREG with M6P/IGF2R domains(IGF2R-4:11~15 domain bought from R&D company) and calculate the dissociation constant (KD value) using solid phase binding experiment. Then, the segment which wt/mCREG most binding to was identified.M6P and G6P blocking test was performed to study if the glycosylated structure of CREG mediated its binding to M6P/IGF2R by M6P. The in vivo competitive binding test was study by adding the segment protein which CREG most binding to into the supernatant with wt/mCREG receptively. The change of CREG biologic effects on VSMC were analyseses to identified the direct binding of CREG to IGF2R domains。 Results (1) Produce and purify the recombinant wt/mCREG protein: 1) The ORF(open reading frame) fragment of human CREG with terminator codon mutation was amplified by RT-PCR and inserted into PMD-18T vector, which was confirmed by restricted endonuclease digestion and DNA sequencing. Subclone the CREG cDNA fragment into pcDNA myc-His vector and the eukaryotic expression vector pcDNA3.1 myc-His/wtCREG was constructed which was confirmed by DNA sequencing. The designed CREG cDNA(combined by biotechnique company) with amino acid residue mutated was subcloned into pcDNA myc-His vector by the BamH I/EcoR digestion, which was confirmed by DNA sequencing.2)The recombinant expression vector, wt/mCREG, were transfected to 293F engineering cell sand selected by G418 to obtain the cell clone with CREG high expressing. The cell clone were amplified and harvested. After that, the two kind recombinant fusion proteins, wt/mCREG with myc/His label, in 293 cell lysates were purified by Ni-NTA affinity chromatography and tested by PNGaseF digestion and Western blot assay. SDS-PAGE showed the molecular mass. The concentration were 0.893μg/μl and 0.972μg/μl and the purity were 92% and 93% respectively compared to standard protein.(2)CREG protein biologic function study and the membrane surface receptor mechanism study:1)the optimal effect concentration analysis: add different concentration (200,400,800,1600 nM) wt/mCREG to supernatant of the synchronized HITASY and OB2 cells which were cultured with free serum medium for 72h. After eight hours, the cells were harvested and cell cycle was analysesed showed the ratio of cell in GO/G1 period was increased in all of group. The effect of wtCREG is more powerful than that of mCREG..The optimal concentration of two kinds of CREG proteins was 400nM. The study showed the recombinant wt/mCREG protein depressed the VSMC proliferation depending on dose and the optimal concentration was 400nM;2)biologic function of CREG protein and the membrance receptor mechanism:①effect on VSMC migration: the wound healing experiment showed the OB2 cells migration was slower significantly after added wt/mCREG(400Nm) in supernatant. The HITASY cells migration were very slowly and no remarkable change. The gelatinase digestion and Western blot analysis showed the matrix metalloproteinase(MMP) was decreased and TIMPs was increased;②effect on differentiation: after added wt/mCREG(400nM), the expression of myocardin, SMα-actin, MHC and caldesmin were increased and that of LM-1 and FN were decreased in OB2 cells. These effects were more significant when adding wtCREG.;③effect on VSMC proliferation: Cell cycle assay and BrDU stain showed: after added the wtCREG and mCREG protein, the ratio of cell in G0/G1 phase increased to 0.5773 and 0.5572 from 0.5308 respectively in OB2 group, which increased to 0.7369 and 0.7034 respectively from 0.6297 in HITASY group;3)Role of M6P/IGF2R in CREG biologic function:①ELISA and co-immunoprecipitation showed the wt/mCREG binding to M6P/IGF2R directly.②antibody blocking test: when the anti-IGF2R was added to medium at the same time with wt/mCREG at different concentration(2μg/mL、4μg/mL、8μg/mL),the effects of CREG protein which depressing proliferation, migration, secretion and promoting differentiation were blocked, which had the positive correlation to the concentration of added anti body. The studies showed two combinant CREG promoted VSMC switch to differentiation phaenotype, at the same time, depress VSMC proliferation, migration and secreting extracellular matrix. The function of wt/mCREG can be blocked by anti-M6P/IGF2R. CREG protein binded to M6P/IGF2R directly;(3)analysis of which M6P/IGF2R domain CREG most likely bind to:1) The ORF(open reading frame) fragment of human M6P/IGF2R domains with terminator codon mutation was amplified by RT-PCR and were inserted into PMD-18T vector which were confirmed by DNA sequencing. The M6P/IGF2R cDNA were subcloned to the prokaryotic expression vector PQE31-His and construct the PQE31 IGF2R-1,2,3 recombinant plasmid which were confirmed by DNA sequencing. The recombinant expression vectors were tranfected into Eo1 JM109 and amplified. The expressed M6P/IGF2R domain proteins were gained by Ni-NTA affinity chromatography and analysesed by SDS-PAGE and Western blot. The SDS-PAGE showed the molecular mass were 54kD(IGF2R-1),44kD(IGF2R-2),62kD(IGF2R-3). Adjust the concentration to 10nM. ELISA analysis suggested the wtCREG had the most affinity to IGF2R-3 and IGF2R-4, while the mCREG to IGF2R-4.The KD value were 0.1063pM, 0.2106pM and 6.488pM respectively;The binding of CREG protein to M6P/IGF2R-3 was depressed by M6P; 3) Add the M6P/IGF2R-3 and M6P/IGF2R-4 to OB2 cell with wt/mCREG at the same time by the different concentration. The study showed M6P/IGF2R-4 blocked the effects of wt/mCREG depressing VSMC proliferation and migration, while M6P/IGF2R-3 only blocked the effects of wt/CREG was blocked by M6P/IGF2R-4.
Conclution Binding to cytomembrane M6P/IGF2R, CREG protein can down-regulate the proliferation, migration and up-regulate differentiation of VSMC promoting the cells’phaenotype transform from undifferentiation to differentiation no matter if glycosylation structures exist. But , the biologic function is more remarkable when the glycosylation structures exist. CREG protein has different affinity to M6P/IGF2R domain depend on glycosylation structures existing or not. The wtCREG showed high affinity to the 7-10 domains but the mCREG to 11-15 domains.
引文
1. Veal E, Eisenstein M, Tseng A, et al. A cellular repressor of E1A- stimulated genes that inhibits activation by E2F[J]. Mol Cell Biol. 1998; 18(7): 5032~5041.
2. Di Bacco A, Gill G. The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
3. Veal E, Groisman R, Eisenstein M, et al. The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells[J]. Oncogene. 2000; 19(11): 2120~2128.
4. Kunita R, Otomo A, Ikeda JE. Identification and characterization of novel members of the CREG family, putative secreted glycoproteins expressed specifically in brain[J]. Genomics. 2002; 80(5):456~460.
5 .韩雅玲,胡叶,刘海伟,等. E1A激活基因阻遏子促进人血管平滑肌细胞分化和迁移[J].生物化学和生物物理进展. 2005; 32(6): 517~522.
6 . Mats Hellstr?m, Mattias Kalén, Per Lindahl, et al. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse[J]. Development. 1999; 126 (14) : 3047~3055.
7.韩雅玲,康建,张剑,等.对去血清后HITASY细胞分子表达及表型分析[J].生物化学和生物物理进展. 2003; 30(6): 868~873.
8. Michael Sacher, Alessandra Di Bacco, Vladimir V. Lunin et al.The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation[J]. Proc Natl Acad Sci U S A. 2005; 102(51):18326~18331.
9 . Alessandra Di Bacco, Grace Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-likegrowth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
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2. Di Bacco A, Gill G. The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
3. Veal E, Groisman R, Eisenstein M, et al. The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells[J]. Oncogene. 2000; 19(11): 2120~2128.
4. Kunita R, Otomo A, Ikeda JE. Identification and characterization of novel members of the CREG family, putative secreted glycoproteins expressed specifically in brain[J]. Genomics. 2002; 80(5):456~460.
5 .韩雅玲,胡叶,刘海伟,等. E1A激活基因阻遏子促进人血管平滑肌细胞分化和迁移[J].生物化学和生物物理进展. 2005; 32(6): 517~522.
6 . Mats Hellstr?m, Mattias Kalén, Per Lindahl, et al. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse[J]. Development. 1999; 126 (14) : 3047~3055.
7.韩雅玲,康建,张剑,等.对去血清后HITASY细胞分子表达及表型分析[J].生物化学和生物物理进展. 2003; 30(6): 868~873.
8. Michael Sacher, Alessandra Di Bacco, Vladimir V. Lunin et al.The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation[J]. Proc Natl Acad Sci U S A. 2005; 102(51):18326~18331.
9 . Alessandra Di Bacco, Grace Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-likegrowth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
10. Kobata A. Structures and functions of the sugar chains of glycoproteins[J]. Eur J Biochem. 1992; 209(2): 483~501.
11 . Lloyd RC, BG. Davis, J B Jones. Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity[J]. Bioorg Med Chem. 2000; 8(7): 1537~1544.
12. Kwon KS, MH Yu. Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation[J]. Biochim Biophys Acta. 1997; 1335(3): 265~272.
13. Suzuki S, M Furuhashi, N Suganuma. Additional N-glycosylation at Asn(13) rescues the human LHbeta-subunit from disulfide-linked aggregation[J]. Mol Cell Endocrinol. 2000; 160(1~2): 157~163.
14. Sasaki K, Nishi T, Yasumura S, et a1. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains.United States Patent:52l8092.
15. Coloma M J, Trinh R K, Martinez A R, et al. Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1-6) dextran antibody[J]. J Immunol. 1999; 162(4): 2162~2170.
16. Davies J, Jiang L Y, Pan L z, et al. Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII[J]. Biotechnol Bioeng. 2001; 74(4): 288~294.
17. Tams JW, J Vind, KG Welinder. Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions[J]. Biochim Biophys Acta. 1999; 1432(2): 214~221.
18. Linden HM, K Kaushansky. The glycan domain of thrombopoietin enhances its secretion[J]. Biochemistry. 2000; 39(11): 3044~3051.
19. Egrie JC, JK Browne. Development and characterization of novel erythropoiesis stimulating protein (NESP) [J]. Br J Cancer. 2001; 84 (Suppl 1):3~10.
20. Mistry PK, EP Wraight, TM Cox. Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease[J]. Lancet. 1996; 348(9041): 1555~1559.
21. Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351~2356.
22. Rudd PM, et al. A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands[J]. Proteomics. 2001; 1(2): 285~294.
23 . Rudd PM, et al. Glycosylation and the immune system[J]. Science. 2001; 291(5512): 2370~2376.
24. Spellman MW. Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest[J]. Anal Chem. 1990; 62(17): 1714~1722.
25. Fryksdale BG., et al. Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis[J]. Electrophoresis. 2002; 23(14): 2184~2193.
26. Hardy MR, RR Townsend. Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection[J]. Proc Natl Acad Sci U S A. 1988; 85(10):3289~3293.
27. Karty JA, et al. Artifacts and unassigned masses encountered in peptide mass mapping[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2002; 782(1~2): 363~383
28. Settineri CA, et al. Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis[J]. Biomed Environ Mass Spectrom. 1990; 19(11): 665~676.
29. Greis KD, et al. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry[J]. Anal Biochem. 1996; 234(1): 38~49.
30. Hanisch FG., M Jovanovic, J Peter-Katalinic. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments[J]. Anal Biochem. 2001; 290(1): 47~59.
31. Apweiler R, H Hermjakob, N Sharon. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database[J]. Biochim Biophys Acta. 1999; 1473(1): 4~8.
32. Devenyi T , Gergely J .实用蛋白质化学技术.上海:上海科技出版社, 1982 , 52.
33. David R. Glycoprotein Detection in Polyacryamide Gel with Thymol and Sulfuric Acid[J]. Anal Biochem. 1979; 99(2): 474～476
34.孙秀琴,中村健,伊滕洋一,等.曼氏裂头蚴蛋白分解酶的糖链研究[J].中国寄生虫病防治杂志. 2001;14 (4) :285～287
35.孙建中,王克夷.植物凝集素的超级家族[J].生物化学与生物物理进展. 1994; 21 (2) :104～109.
36.张维杰.复合多糖生化研究技术.上海:上海科技出版社, 1987 , 160.
37. Mechref Y, MV Novotny. Structural investigations of glycoconjugates at high sensitivity[J]. Chem Rev. 2002; 102(2): 321～337.
38. Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351～2356.
39. Van Langenhove A, VN Reinhold. Determination of polysaccharide linkage and branching by reductive depolymerization. Gas-liquid chromatography and gas-liquid chromatography-mass spectrometry reference data[J]. Carbohydr Res. 1985; 143.
40. Barber M, Bordoli RS, Sedgwick RD, et al. Fast atom bombardment of solids as an ion source in mass spectrometry [J]. Nature. 1981; 293:270.
41. Aberth W, Straub KM, Burlingame AL. Secondary ion mass spectrometry with cesium ion ... trix for analysis of bioorganic compounds[J]. Anal Chem. 1982; 54: 20～29.
42. Olivares JA, Nhung T, Nguyen NT , et al .On-line mass spectrometric detection for capillary zone electrophoresis[J]. Anal Chem. 1987; 59:12～30.
43. Mechref YM, V Novotny. Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins[J]. Anal Chem. 1998; 70(3): 455～463.
44. Colangelo JR, Orlando. On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains[J]. Anal Chem. 1999; 71(7): 1479～1482.
45. Geyer H, Schmitt S, Wuhrer M, et al. Structural analysis of glycoconjugates by on-target enzymatic digestion andMALDI-TOF-MS[J]. Anal Chem. 1999; 71(2): 476～482.
46. Hirabayashi J, Hayama K, Kaji H, et a1. Afinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans[J]. J Biochem(Tokyo). 2002; 132(1):103~114.
47. Mawuenyega KG, Kaji H, Yamuchi Y, et a1. Large-scale identification of Caenorhabditis elegans proteins by multi-dimensional liquid chromatography-tandem mass spec-trometry[J]. J Proteome Res. 2003; 2(1): 23~35.
48. Yang Z, Hancock WS. Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin afinity column [J].J Chromatogr A. 2004; 1 053(1~2): 79~88.
49. Hirabayashi J. Lectin-based structural glycomics:glycoproteomics and glycan profiling[J]. Glycoconj J. 2004; 21(1~2): 35~40.
50. Dwek MV, Ross HA, Leathem AJ. Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer[J]. Proteomics. 200l; 1(6):756~762.
51. Schulenberg B, Beechem J M, Patton W F.Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology:a protein diferential display approach[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 793(1): 127~139.
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