人尿嘧啶糖基化酶cDNA的克隆及其在食管鳞癌组织中的检测

尿嘧啶糖基化酶是碱基切除修复过程的起始酶,对于维护基因稳定具有重要意义。在不同组织及不同细胞周期中,该酶的表达水平存在差异。通过反转录PCR克隆了人尿嘧啶糖基化酶的cDNA编码序列,进一步以克隆所得的已知UNG基因拷贝数的重组质粒作为定量标准,通过实时荧光定量RT-PCR测定了食管癌病人手术切除组织中尿嘧啶糖基化酶的mRNA水平,探讨了尿嘧啶糖基化酶表达水平与食管癌之间的联系。     

乙型肝炎病毒表面抗原 T131N／M133T 变异株糖基化和抗原性研究

为探讨乙型肝炎病毒表面抗原（HBsAg）T131N／M133T变异株的糖基化和抗原性,本研究构建了T131N／M133T变异的HBsAg过表达质粒,将质粒转染细胞,用蛋白免疫印迹法检测 HBsAg表达和糖基化情况,用酶联免疫吸附试验（ELISA）和免疫荧光法检测 HBsAg的抗原性。结果显示,T131N／M133T变异使HBsAg产生了新的 N‐糖基化修饰,该变异质粒转染细胞内 HBsAg水平显著低于野生型,上清液中 HBsAg水平也有一定程度降低。结果提示,T131N／M133T变异使 HBsAg发生了新的 N‐糖基化修饰,该变异产生的糖基化可能影响HBsAg的胞内稳定性,对HBsAg的抗原性也有一定影响。     

N-糖基化对一种新型重组耐高温β-甘露聚糖酶(ReTMan26)稳定性的影响

【背景】对来源于嗜热枯草芽孢杆菌(TBS2)的一种新型重组耐高温β-甘露聚糖酶(ReTMan26)基因序列进行分析,该基因中含有3个N-糖基化位点(N8、N26与N255),经毕赤酵母表达时可进行N-糖基化修饰。【目的】确定N-糖基化对ReTMan26稳定性的影响。【方法】通过构建ReTMan26蛋白质三维结构模型,初步分析N-糖基化对该酶稳定性的影响。在此基础上,利用天然蛋白去糖基化试剂盒除去ReTMan26的N-多糖链,获得去除N-糖基化的耐高温β-甘露聚糖酶(ReTMan26-DG),并对纯化后的ReTMan26及ReTMan26-DG进行相应的稳定性对比检测。【结果】ReTMan26与ReTMan26-DG的最适反应pH均为6.0,但在pH1.5-9.0范围内,ReTMan26的稳定性比ReTMan26-DG有小幅提高。ReTMan26的最适反应温度为60°C,比ReTMan26-DG高5°C;ReTMan26经100°C处理10 min,剩余酶活为58.6%,而ReTMan26-DG经93°C处理10 min,剩余酶活为58.2%,100°C处理10min则完全失活。经胃蛋白酶及胰蛋白酶在37°C处理2h后,ReTMan26的剩余酶活分别为70.5%及91.2%,比ReTMan26-DG分别提高了23.7%及25.6%。【结论】N-糖基化可提高ReTMan26的pH稳定性、耐热稳定性及抗蛋白酶消化性能。     

毕赤酵母表达的嗜热蓝状菌β-葡萄糖苷酶N-糖基化修饰的作用与功能

【目的】研究N-糖基化对来源于嗜热蓝状菌β-葡萄糖苷酶(β-glucosidase,Bgl3A)的酶学性质影响。【方法】采用定点突变技术构建了3个去N-糖基化的突变体T44A、S228A、S299A,并分别在毕赤酵母GS115中表达纯化。【结果】与野生型Bgl3A相比,突变体S228A分泌蛋白产量极低,仅能微量检测到p NPG活性;突变体T44A和S299A的最适pH和最适温度没有改变,分别为4.0和75°C,但二者的T_m值和70°C下的热稳定性都明显优于野生型。以p NPG为底物时,突变体S299A和T44A的催化效率分别降低了14.5%和70.0%;以纤维二糖为底物时,T44A的催化效率基本不变,而S299A的催化效率提高了1.1倍。【结论】Bgl3A不同位点的N-糖基化修饰对酶的分泌和酶学性质的影响具有明显差异。其中,N226位的N-糖基化在维持酶的表达和功能方面至关重要,而去除N297位点的N-糖基化可以提高酶的热稳定性及对纤维二糖的催化效率。     

黑曲霉Aspergillus niger 963植酸酶基因phyA2 N-糖基化突变体的构建与表达分析

为研究N-糖基化对黑曲霉Aspergillus niger963植酸酶蛋白酶学性质的影响,利用Megaprimer PCR介导基因定点突变的技术,构建了植酸酶phyA2基因两个N-糖基化突变体,即将该基因编码蛋白质N87位和N102位的天冬酰胺密码子置换为编码与其具有相似结构的谷氨酰胺密码子,两个突变体分别命名为N87Q、N102Q,经测序结果比对和图谱分析,表明在核酸水平上成功实现了点突变,构建了酵母表达载体pPIC9-N87Q,pPIC9-N102Q,转化毕赤酵母GS115,经发酵罐水平诱导表达后,获得了N-糖基化缺失突变蛋白,对突变体蛋白在60℃进行处理发现,突变体N87Q处理1h后剩余50%的酶活,N102Q处理10min后酶活完全丧失,在37℃,不同的pH缓冲体系(pH1~10)处理1h,N87Q剩余约大于70%的活性,而N102Q在pH8的环境下,没有检测到酶活。     

糖基化对Caldicellulosiruptor sp.F32中内切型木聚糖酶热稳定性的影响

[目的]探讨糖基化对来自Caldicellulosiruptor sp.F32的GH11家族木聚糖酶的影响,揭示其工业应用价值。[方法]在大肠杆菌和毕赤酵母中表达木聚糖酶Xyn A及其催化模块Tm1。通过酶学性质对比,论证糖基化对酶学特征的影响。[结果]毕赤酵母分泌的p-Xyn A和p-Tm1可被糖基化修饰,而大肠杆菌表达的e-Xyn A和e-Tm1无法被修饰。糖基化未明显影响Xyn A的催化效率和热稳定性,但可提高Tm1的催化效率约1.5倍,而且在80℃、85℃下,pTm1的半衰期相对于e-Tm1分别提高了约1.5、2倍。另外,Tm1的热稳定性和酶活均显著高于Xyn A。通过对Tm1同源建模分析,两个糖基化位点(天冬酰胺N23、N189)可能对热稳定性有促进作用。[结论]酵母对Tm1的糖基化修饰可热稳定性和催化效率提高1.5~2倍,相比于Xyn A更适合工业应用。     

非酶糖基化对α-synuclein分子构象的影响

将纯化后的α－synuclein分别与果糖和葡萄糖孵育,通过内源荧光、非酶糖基化衍生物特征荧光、圆二色光谱以及电子显微镜等技术进行检测发现：α—synuclein与还原糖共同孵育后,308nm内源荧光强度明显降低,同时在447nm产生一个非酶糖基化衍生物特征荧光．与果糖孵育的蛋白质样品其非酶糖基化特征荧光的出现速度快于葡萄糖孵育样品．内源荧光与非酶糖基化特征荧光之间存在能量传递现象,提示Tyr残基与非酶糖基化特征荧光发色团在空间距离上彼此接近．圆二色光谱测定结果显示,α－synuclein与果糖孵育后,其α－螺旋含量增加．非酶糖基化的α－synuclein在电子显微镜下表现为短纤维状．非酶糖基化可以诱导α－synuclein蛋白分子聚集,且果糖较葡萄糖更容易使α－synuclein发生非酶糖基化．以上结果提示,非酶糖基化似乎可以导致α－synuclein在细胞内的错误折叠和分子聚集．     

拟南芥细胞分裂素糖基转移酶UGT76C2的N端亮氨酸替换对酶活性的影响

UGT76C2是负责细胞分裂素N-糖基化修饰的糖基转移酶,该基因对于维持植物体内细胞分裂素动态平衡有重要作用。为了进一步研究UGT76C2酶蛋白结构与催化活性的关系,本文采用定点突变方法,将UGT76C2的N端第31位的保守亮氨酸替换为组氨酸。结果发现,突变型UGT76C2在离体实验中完全丧失了对细胞分裂素的糖基化修饰活性,该突变基因的过表达转基因植物出现与UGT76C2突变体类似的表型,转基因植物体内的两类主要细胞分裂素的N-糖苷含量显著降低。实验结果证明了UGT76C2 N端亮氨酸残基对于糖基化修饰活性的重要性。     

山东省甲型H1N1流感病毒病原学及基因特性研究

在2009~2010年监测年度开展甲型H1N1流感病毒学监测并进行病原学分离鉴定,以及对血凝素基因(HA)和神经氨酸酶基因(NA)特性分析,研究其基因变异情况。采集了17 126份发热患者的鼻、咽拭子标本,采用逆转录实时荧光定量RT-PCR(Real-Time RT-PCR)进行核酸检测,其中甲型H1N1流感病毒核酸检测阳性4004份,总阳性率为23.38%。对阳性标本开展病毒分离,并对分离的甲型H1N1流感病毒的HA、NA基因序列进行测序。利用DNAStar软件对序列进行同源性分析发现与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株HA、NA基因同源性分别为96.9%~99.3%和99.1%~99.6%;利用Mega 4.0软件进行基因进化分析和氨基酸进化分析发现,与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株有21个血凝素基因的氨基酸发生替换,其中11个氨基酸位点位于抗原决定簇区,一个糖基化位点发生改变;有16个神经氨酸酶基因的氨基酸发生了替换,一个糖基化位点发生改变;未发生神经氨酸酶蛋白275位H→Y的替换。结果显示山东省甲型H1N1流感暴发流行株HA基因和NA基因均具有高度同源性,HA蛋白和NA蛋白均存在不同程度的氨基酸替换,部分流行株抗原决定簇和糖基化位点发生改变,所有病毒均对达菲类药物敏感。     

提高糖基化的酶蛋白可结晶性研究

糖基化修饰是一类重要的翻译后修饰,对蛋白质的表达调控、折叠、分泌和功能等方面发挥着关键作用。酶是由活细胞产生的具有高度特异性和高效催化性的生物催化剂,酶的糖基化修饰对其生物催化特性和稳定性具有重要影响。研究糖基化修饰对酶蛋白的影响机制需要获取糖基化酶蛋白的结构,X-射线晶体衍射学是获得结构信息的重要技术手段,在糖基化酶蛋白的晶体衍射研究中,复杂、多样、不均一的糖基化修饰限制了该类酶的晶体生长,这是影响糖基化的酶蛋白结构解析的关键瓶颈问题。因此,如何提高糖基化的酶蛋白可结晶性是当前蛋白质结构研究的热点和难点。糖苷酶的去糖基处理、糖基转移酶抑制剂的引入和异源表达体系优化等手段都是当前研究领域提高糖基化的酶蛋白可结晶性的重要策略,这些手段可以在避免损害糖基化的酶蛋白稳定性和催化活性的同时提高其均一性。     

Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions

Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, and the largest degree of UNG2 stimulation occurred when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.     

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.     

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover

Background

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.

Methodology/Principal Findings

We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.

Conclusions/Significance

This work identifies UNG2 and SMUG1 as novel targets for CRL4^DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4^DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4^DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignment of full-length human uracil DNA glycosylase UNG2

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.     

Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the K(d) of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The k(cat) of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, k(cat) on double-stranded DNA substrate was reduced 4-12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase.     

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.     

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.