| 题名 | 大蹼铃蟾（Bombina maxima）白蛋白的皮肤表达、蛋白酶抑制及细胞调亡诱导活性 |
| 作者 | 张英霞 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 大蹼铃蟾 血清白蛋白 皮肤 血红素b 胰蛋白酶抑制剂 细胞调亡 |
| 其他题名 | Bombina maxima albumin is expressed in skin with trypsin inhibitory and apoptosis-inducing activity |
| 中文摘要 | 两栖类动物的皮肤是其得以生存的重要器官，它担负着许多生理功能，如呼吸、水份调节、温度控制、排泄、繁殖、抵抗微生物、防御天敌等。存在于两栖动物皮肤分泌物中的生物活性成分已成为研究热点。目前，己分离鉴定出许多具有各种生物活性的蛋白质及多肤。通过三步分离纯化过程：DEAE-SephadexA-50离子交换，SephadeX075凝胶过滤，和DEAE-SephadexA-50离子交换层析，我们从大蹼铃蟾（Bombinamaxima）皮肤匀浆物中分离得到纯化的大蹼铃蟾皮肤白蛋白（Bm-A-skin）。SDS-PAGE电泳表明，该蛋白为单链蛋白质，在还原状态下表观分子量为67kDa。非还原状态下至少存在三条带，分子量分别为50，55和110kDa。经N-端氨基酸序列分析，其序列均相同，故该蛋白在SDS存在下有同分异构体及多聚体形式。根据所测得的N端氨基酸序列及内肤序列设计引物，通过筛选已构建的大蹼铃蟾皮肤cDNA文库，获得了编码该蛋白的全长cDNA。经序列分析发现该蛋白由三个保守的血清白蛋白结构域组成，并且同人血清白蛋白及牛血清白蛋白的序列相似度分别为39％和38％。随后，我们从血清中也分离纯化到血清白蛋白（BmA-serum），并通过RT-PCR，从大蹼铃蟾肝脏中扩增得到其全长cDNA序列。对由cDNA序列推导的两个大蹼铃蟾白蛋白的氨基酸序列进行比较，发现二者基本相同，只有两个氨基酸的差异，即BmA-skin的Gly417，Ser569，在BmA-serum中均为Asn。造成这两个氨基酸差异的只有一个碱基的突变，即编码BmA-skinGly417，Ser569密码子的第二位碱基A在BmA-serum中变为G。另外，从肝脏获得的BmA-serum的cDNA3'非翻译区还有8个碱基的插入。经扫描光谱分析，BmA-skin含有大量的血红素b，含量为0．95moFinol蛋白，而BmA-serum中含量较少，为0.05mol／mol蛋白。经schiff试剂染色发现，BmA-skin及BmA-serum均为非糖蛋白质。两者均具有抑制trypsin水解小肚底物的活性，但对其它丝氨酸蛋白酶的活性则无抑制，如thrombin、chyomotryPSin、elastase及substilisin。利用表面等离子共振技术研究BmA-skin及BmA-serum与trysin的相互作用，分别得到它们与trrpsin结合的动力学常数，解离平衡常数KD为两者均通过由一对二硫键cys53-Cys62形成的一个暴露的活性位点环，以1：1分子摩尔比同tryrsin形成稳定的非共价结合的复合物，其反应活性位点为Arg58（P1）-His59（P1'）。利用免疫组织化学方法研究发现，BmA-skin广泛地分布于成年大蹼铃蟾上皮细胞的细胞膜及真皮的疏松结缔组织层。表明其在蛙皮肤的生理功能中发挥重要作用，如水及代谢物质交换，渗透压的维持，皮肤呼吸等。另外，我们还从非洲爪蟾（xenopus勿即is）的血清及皮肤中分离到其68扔a的血清白蛋白，经初步鉴定也具有trtPsin抑制活性，但其抑制机制与B．maxima白蛋白不同，还有待于进一步研究。通过MTT法研究发现，BmA-skin对人T淋巴细胞H9、C8166及hemin处理的红白血病细胞K562具有细胞毒活性。三种细胞经BmA-skin处理72h，CC50A片段化，细胞核形态变化及流式细胞仪分析，结果显示，BmA-skin具有选择性诱导细胞调亡的特性。而BmA-serum对三种细胞均无毒性作用，单独的hemin对三种细胞的，胜也很弱。实验结果表明，BmA-skin结合的血红素b可能对其细胞毒及诱导细胞调亡的活性具有较大的贡献。用Cy3标记的BmA-skin与Hg和C8166细胞保温后，发现其进入细胞内发挥作用，这可能是其诱导细胞调亡机制之一。 |
| 英文摘要 | Amphibian skin is a morphologically, biochemically and physiologically complex organ that Mfills a wide range of functions necessary for amphibian survival. A serious of mutually compatible functions takes place in the skin, including respiration, water regulation, anti-predator, antimicrobial defense, temperature control, reproduction, etc. Although numerous studies have focused on the bioactive components existed in amphibian skin "secretions", from which lots of peptides with diverse biological activities have been isolated and characterized, the physiological and functional complexity and the relative biochemical mechanisms of amphibian skin are incompletely understood. Following determination of trypsin inhibitory activity, a novel serine protease inhibitor (BmA-skin) was purified and characterized from frog Bombina maxima skin by a three steps protocol including DEAE-Sephadex A-50, Sephadex G-75 gel filtration and the DEAE-Sephadex A-50 ion exchange. BmA-skin is composed by a single peptide chain as demonstrated by SDS-PAGE and native-PAGE. Under reducing conditions, its apparent molecular weight is 67 kDa. However, under non-reducing conditions, there are at lest three bands (50 kDa, 55 kDa and 110 kDa) with identical N-terminal amino acids are determined by N-terminal protein sequencing. Thus, the protein may exist in isomeric and polymeric forms. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains and its sequence exhibits 39% and 38% sequence identities with those of human and bovine serum albumins, respectively. Frog serum albumin (BmA-serum) was subsequently purified and its coding cDNA was further obtained by PCR-based cloning from frog liver. BmA-serum coding cDNA obtained from the frog liver is generally the same as that of BmA-skin from the skin, except two nucleotide mutations in protein coding region that resulted in Gly471 to Asn and Ser569 to Asn replacements, and an 8 base insertion in 3* untranslated region in the cDNA of BmA-serum. BmA-skin is characterized of binding of a haem b (0.95 mol/mol protein). However, BmA-senim is distinct from BmA-skin by a much lower content of haem b (0.05 mol/mol protein). Different from bovine serum albumin, both of them potently inhibited trypsin. No inliibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The equilibrium dissociation constant (KD) with rypsin determined are around 2 x 10"9M by BIAcore 3000. It formed a stable non-covalent complex with trypsin at 1:1 moler ratio through an exposed loop formed by a disulfide bond (Cys53~Cys62), which comprises the seissile bond Arg58(Pi>His59(Pi')r Immunohistochemical study revealed that BmA-skin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in frog skin, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osrnoregulation, skin respiration, etc. BmA-skin possessed cytotoxic activity on H9, C8166 human T lymphoblastoid cells, but not on K562 erythroleukemia cells. Pretreatment K562 cells with hemin to induce erythroid differentiation, it inhibited the viability of them too. After treatment cells with BmA-skin for 72 h5 fifty percentage cytotoxic concentrations (CC50) of BmA-skin on H95 C8166, and hemin-treated K562 cells were 0.96, 1.05, and 1.27 fiM, respectively. . BmA-skin induced apoptosis of the cell lines, which was demonstrated by nuclear morphological changes, DNA fragmentation, and DNA subdiploidy of apoptosis cells. BmA-serum did not affect the growth of the three cell lines. These results indicated that bound heam in BmA-skin contributed importantly to its 'cytotoxic and apoptosis-inducing activity on the cell Ikes assayed. BmA-skin and serum labeled with cy3 incubated with H9 and C8166 cells at different time points, and determined by laser scanning confocal microscopy. The results showed that more BmA-skin enter cells than BmA-senim, suggesting that entry cells of BmA-skin is a way to induce apoptosis |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6177]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 张英霞. 大蹼铃蟾（Bombina maxima）白蛋白的皮肤表达、蛋白酶抑制及细胞调亡诱导活性[D]. 北京. 中国科学院研究生院. 2005. |
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