冬眠不同时期达乌尔黄鼠比目鱼肌糖蛋白质组学的研究

发布时间：2018-05-30 13:11

  本文选题：冬眠 + 糖蛋白质组学　； 参考：《西北大学》2016年博士论文

【摘要】：废用性肌萎缩是临床上亟待解决的重要问题之一。尽管科研人员已经作了大量的研究,但目前仍未找到阻止和治疗废用性肌萎缩的有效措施,其主要的原因在于我们对废用性肌萎缩的分子机制仍缺乏了解。冬眠是冬眠动物应对冬季低温和食物缺乏等不利因素所特有的生存策略,其过程可以长达数月。在历经长期的冬眠期不活动后,冬眠动物的骨骼肌并未出现明显的废用性肌萎缩。因此,冬眠动物是一个天然的抗废用性肌萎缩研究模型。阐明冬眠动物骨骼肌这种特殊适应现象的发生机制是生理生态学领域亟待研究的重要课题,对航天、临床、运动及康复医学等领域亦有重要借鉴意义。蛋白的糖基化(glycosylation)是指将糖或者寡糖通过酶促反应连接到蛋白质上,这是蛋白质最重要的翻译后修饰方式之一。据估计,人类约70%的蛋白质被多种糖结构所修饰。越来越多的证据表明多种病理状况引起的肌肉萎缩与蛋白的糖基化密切相关。作为糖基化方式之一,O-乙酰葡萄糖胺(O-acetylglycosamine,O-GlcNAc)可以通过控制肌蛋白合成来调控废用性肌萎缩；蛋白的唾液酸化(sialylation)水平的降低是导致肌纤维降解、肌肉力量减弱及肌重减轻的重要原因之一；多种导致肌肉萎缩或功能障碍的肌肉疾病如有镶边空泡远端肌病(distal myopathy with rimmed vacuoles, DMRV)和先天性肌营养不良(congenital muscular dystrophy, CMD)均被证实与肌肉中蛋白糖基化的异常紧密相关。我们本次研究以达乌尔黄鼠(Spermophilus dauricus)为实验对象,选取典型的慢肌比目鱼肌(soleus, SOL)为代表,首次对冬眠不同时期黄鼠比目鱼肌糖蛋白组质学进行系统研究,从新的角度探讨冬眠动物骨骼肌的抗废用性肌萎缩机制。第一部分：采用凝集素芯片技术检测冬眠不同时期达乌尔黄鼠比目鱼肌中糖蛋白糖基化的变化情况,并使用凝集素组化技术对芯片结果进行验证。芯片的分析结果显示,有8种凝集素的荧光强度值在冬眠过程中发生了显著性变化,分别为马鞍树凝集素-Ⅱ(Maackia Amurensis Lectin Ⅱ, MAL-Ⅱ)、四棱豆凝集素-Ⅰ(Psophocarpus Tetragonolobus Lectin Ⅰ,PTL-Ⅰ)、紫藤花凝集素(Wisteria Floribunda Lectin, WFA)、加纳子凝集素-Ⅰ(Griffonia Simplicifolia Lectin I, GSL-Ⅰ)、四棱豆凝集素-Ⅱ(Psophocarpus Tetragonolobus Lectin Ⅱ, PTL-Ⅱ)、曼陀罗凝集素(Datura stramonium, DSA)、欧洲卫矛凝集素(Euonymus Europaeus Lectin,EEL)和菜豆凝集素-E+L(Phaseolus vulgaris Agglutinin E+L,PHA-E+L)。我们的结果显示：1)与冬眠前组相比,冬眠组凝集素MAL-Ⅱ所识别的糖链结构唾液酸(Sialic acid, SA)α2-3半乳糖(Galactose, Gal) (SAa2-3Gal)显著降低,而在激醒组和出眠组显著增加,恢复到冬眠前水平；2)PTL-Ⅰ所识别的αN-乙酰半乳糖胺(αGalNAc)和Gal结构与SAa2-3Gal的变化趋势相似,即在冬眠中显著下降,在激醒和出眠时显著增加；3)与冬眠组相比,激醒组和出眠组的WFA特异性识别的末端GalNAc (Terminal GalNAc)结构显著增加；4)与其它三组相比,GSL-Ⅰ和PTL-Ⅱ特异性识别αGalNAc和Gal结构在激醒组中显著性的增加；5)与冬眠前组相比,DSA识别的GlcNAc结构在冬眠组显著性增加,而在激醒组和出眠组显著下降,恢复到冬眠前水平；6)与其它三组相比,凝集素EEL识别的半乳糖α1-3(岩藻糖α1-2)Gal(岩藻糖,Fucose, Fuc)结构在冬眠组显著增加；7)与冬眠前组相比,冬眠组、激醒组和出眠组SOL的PHA-E+L特异性识别的三天线和四天线复杂型N-糖链(Tri-and tetra-antennary complex-type N-glycan)结构显著性增加。我们选取了MAL-II, PTL-I,DSA和PHA-E+L四种凝集素作了凝集素组化实验。组化的结果与凝集素芯片的结果相一致,进一步验证了凝集素芯片的实验结论。总之,这一部分的结果提示,黄鼠周期性阵间激醒过程中,SAa2-3Gal结构的显著性增加有效地补偿了其在冬眠过程中的减少,对于维持冬眠黄鼠骨骼肌正常的结构和功能,防止其废用性萎缩起到了重要作用；由于PTL-Ⅰ、WFA、GSL-Ⅰ、PTL-Ⅱ四种凝集素特异性识别的糖链结构均为O-连接的糖链,因此推测,O-连接的糖链在激醒过程中的增加可能与冬眠黄鼠特有的保护性机制有关；此外,三天线和四天线型的糖蛋白N-糖链在整个冬眠过程中均显著性增加,可能与黄鼠骨骼肌对冬眠废用的生理适应机制有关。第二部分：本文第二章的结果表明,黄鼠周期性阵间激醒过程中,SAa2-3Gal结构的显著性增加有效地补偿了其在冬眠过程中的减少,而正常的唾液酸含量对于维持肌肉的形态、结构、功能有着重要的作用。因此,本部分实验的研究目的是通过测定冬眠不同时期黄鼠比目鱼肌中β-半乳糖-α-2,3-唾液酸糖基转移酶(sialyltransferasebeta-galactoside alpha-2,3-sialyltransferases)(包括ST3Gall、ST3Gal2、 ST3Gal3、ST3Gal4、T3Gal5、ST3Gal6)与唾液酸糖苷酶(sialidases)(包括NEU1、 NEU2、NEU3、NEU4) mRNA表达的变化情况,从糖相关基因mRNA水平对SAa2-3Ga在冬眠不同时期的变化机制进行分析。我们的结果显示,与冬眠前组相比,冬眠组ST3Gall的mRNA表达量显著性降低(-55.00%；P0.01),而激醒组和出眠组ST3Gall的mRNA表达量与冬眠组相比,均显著性升高(114.30%和126.20%,P0.01),与冬眠前组相比无显著性的差异(P0.05)。相比于冬眠前组,ST3Gal2的mRNA表达量在冬眠组显著性降低(-42.70%,P0.01),而与冬眠组相比,激醒组和出眠组mRNA表达量均显著性升高(77.30%和76.50%,P0.01),冬眠前组、激醒组与出眠组的ST3Gal2的mRNA表达量无显著性的差异(P0.05)。冬眠组ST3Gal5的mRNA相对表达量相比于冬眠前组显著性降低(-60.91%,P0.01),而在激醒组和出眠组中ST3Gal5的mRNA表达量分别较冬眠组出现显著性升高(160.56%和125.48%,P0.01),与冬眠前组相比没有显著的差异(P0.05)。NEU1、NEU3和ST3Gal6的mRNA表达量在冬眠四组中没有显著的差异(冬眠前组,冬眠组,激醒组,出眠组)(P0.05)。我们的结果提示,ST3Gall、ST3Gal2及ST3Gal5 mRNA相对表达量的改变,即在冬眠中明显下降,在阵间激醒过程中和出眠后又恢复至冬眠前水平,可能是造成黄鼠SOL的SAa2-3Gal结构在冬眠期间变化的主要因素。第三部分：本文第二章的研究表明,达乌尔黄鼠比目鱼肌中凝集素MAL-Ⅱ特异性识别的糖链结构SAa2-3Gal在冬眠过程中的显著变化可能在其抗废用性肌萎缩机制中起到重要作用。因此,本部分实验制备制备凝集素MAL-Ⅱ-磁性微粒复合物,分别从冬眠前和冬眠黄鼠比目鱼肌中提取末端为SAa2-3Gal的糖蛋白,利用LC-ESI-MS/MS对提取的糖蛋白进行鉴定,通过生物信息学对鉴定的糖蛋白进行进一步分析。从冬眠前组和冬眠组中分别鉴定出糖蛋白227个和185个。其中,只在冬眠前组鉴定出的糖蛋白为141个,只在冬眠组鉴定出的糖蛋白为99个,在两组中均鉴定出的糖蛋白数为86个。GO (Gene Ontology)功能注释分析显示：根据细胞成分注释,鉴定的糖蛋白主要是胞内蛋白,并且很多为大分子蛋白质,主要分布在细胞器中和膜结构上。根据分子功能注释,这些糖蛋白具有结合、催化以及结构分子活性等方面的功能。根据参与的生物过程注释,这些糖蛋白主要涉及细胞过程、刺激应答、代谢过程以及生物调节等生物过程。KEGG(Kyoto Encyclopedia of Genes and Genomes)通路富集分析显示,从冬眠前组和冬眠组SOL分离出的糖蛋白显著性富集的通路分别为40条和36条,其中,只在冬眠前组显著性富集的通路有14条,而只在冬眠组显著性富集的有10条。使用IntAct数据库进行蛋白质-蛋白质相互作用分析,利用Cytoscape欠件分布构建了冬眠前组和冬眠组糖蛋白的蛋白质相互作用网络图(The protein-protein interaction network, PPI network),筛选出9个度值(degree)大于30的核心蛋白质。第四部分：本部分实验选取了2个糖蛋白进行验证,分别为热休克同源蛋白(heat shock cognate 70,HSC70)和丙酮酸激酶(pyruvate kinase,PK),检测冬眠过程中其蛋白表达和SAa2-3Gal结构的变化情况。此外,检测黄鼠SOL中14-3-3 protein epsilon蛋白表达在冬眠不同时期的变化情况,分析其在抗废用性肌萎缩机制中的潜在作用。我们的结果显示,与冬眠前相比,冬眠组的HSC70蛋白的表达量显著降低了37.39%(P0.01),与冬眠组相比,激醒组的HSC70蛋白的表达量显著上升了51.39%(P0.01),出眠组显著上升了41.67%(P0.01),冬眠前组、激醒组和出眠组三组之间无显著性差异(P0.05)；PK蛋白的表达在四组之间无显著性差异(冬眠前组,冬眠组,激醒组,出眠组)(P0.05)；与冬眠前组相比,冬眠组的14-3-3 protein epsilon的表达量显著降低了42.59%(P0.01),与冬眠组相比,激醒组的14-3-3 protein epsilon的表达量显著性上升了51.61%(P0.01),出眠组显著性上升了58.06%(P0.01),冬眠前组、激醒组和出眠组三组之间无显著性差异(P0.05)；与冬眠前组相比,冬眠组糖蛋白HSC70的SAa2-3Gal结构显著减少30.08%(P0.01),与冬眠组相比,激醒组的SAa2-3Gal结构显著增加了37.66%(P0.05),而出眠组显著性增加54.50%(P0.01),冬眠前组、激醒组和出眠组三组之间无显著性差异(P0.05)；与冬眠前组相比,冬眠组糖蛋白PK末端SAa2-3Gal结构显著减少29.40%(P0.01),与冬眠组相比,激醒组的SAa2-3Gal结构显著增加了30.91%(P0.05),出眠组显著性增加34.41%(P0.05),冬眠前组、激醒组和出眠组三组之间无显著性差异(P0.05)。结果表明,冬眠不同时期黄鼠比目鱼肌中HSC70、PK、14-3-3 protein epsilon三种蛋白的变化(包括蛋白表达和SAa2-3Gal糖结构)可能与其特有的抗废用性肌萎缩机制相关。
[Abstract]:Wasting muscle atrophy is one of the most important problems to be solved in clinic. Although a lot of researchers have done a lot of research, the effective measures to prevent and treat the disused muscle atrophy are still not found at present. The main reason is that we still lack the understanding of the molecular mechanism of the disused muscle atrophy. Hibernating is a hibernating animal to deal with the low winter. The process of survival specific to adverse factors, such as mild food deficiency, can last several months. After a long hibernation inactivity, the skeletal muscles of hibernating animals do not have obvious disuse muscle atrophy. Therefore, hibernating animals are a natural model of anti waste myatrophy. The mechanism of adaptation is an important subject in the field of physiological ecology. It is also of great significance to the fields of space, clinical, sports and rehabilitation medicine. The glycosylation of protein (glycosylation) refers to the connection of sugar or oligosaccharides to the protein by enzymatic reaction, which is the most important post-translational method of protein. It is estimated that about 70% of human protein is modified by a variety of sugar structures. More and more evidence suggests that muscle atrophy caused by a variety of pathological conditions is closely related to the glycosylation of proteins. As a glycosylation method, O- acetyl glucosamine (O-acetylglycosamine, O-GlcNAc) can regulate the use of muscle protein synthesis to regulate waste use. Myoatrophy; the decrease of protein's salivary acidification (sialylation) level is one of the important causes of muscle fiber degradation, muscle strength and muscle weight reduction; many muscle diseases that cause muscle atrophy or dysfunction such as distal myopathy with rimmed vacuoles (DMRV) and congenital muscular dystrophy (DMRV). Congenital muscular dystrophy, CMD) were proved to be closely related to the abnormal protein glycosylation in the muscles. In this study, we selected the typical slow muscle soleus (soleus, SOL) as the representative of the Spermophilus dauricus (Spermophilus dauricus) for the first time, and for the first time, the myosin composition of the soleus flounder in different periods of hibernating was carried out. The mechanism of anti waste muscle atrophy in hibernating animal skeletal muscles was explored from a new perspective. The first part: using lectin chip technique to detect the changes of sugar glycosylation in the soleus muscle of dormant of dormans at different periods of hibernating, and to verify the results by using the agglutinin histochemical technique. There were significant changes in the fluorescence intensity of 8 lectins during the hibernation process, namely, saddle tree agglutinin - II (Maackia Amurensis Lectin II, MAL- II), four ribbean lectin - I (Psophocarpus Tetragonolobus Lectin I, PTL- I), wisteria lectin (Wisteria Floribunda Lectin, WFA), and Garner lectin - I Onia Simplicifolia Lectin I, GSL- I), four prismatic lectin - II (Psophocarpus Tetragonolobus Lectin II, PTL- II), Mandala agglutinin (Datura stramonium, DSA), European Euonymus agglutinin and bean agglutinin. Our results show: 1) Compared with the pre hibernating group, the sugar chain structure sialic acid (Sialic acid, SA) 2-3 semi lactose (Galactose, Gal) (SAa2-3Gal) identified by the hibernating group of agglutinin II (Galactose, Gal) (SAa2-3Gal) significantly decreased in the waking and hibernating group and recovered to the pre hibernating level; 2) PTL- I identified the alpha N- acetyl galactoamine (alpha GalNAc) and Gal structure and SAa2-3Gal. The change trend is similar, that is, in hibernation, significant decrease in waking and hibernation; 3) the terminal GalNAc (Terminal GalNAc) structure of WFA specific recognition in the waking and hibernating group increases significantly compared with the hibernating group; 4) GSL- I and PTL- II specific identification of alpha GalNAc and Gal structure are significant in the waking group compared with the other three groups. Increase in sex; 5) the GlcNAc structure identified by DSA increased significantly in hibernating group compared with the pre hibernating group, while in the waking and hibernation group, the structure of galactose alpha 1-3 (fucose alpha 1-2) Gal (fucose, Fucose, Fuc) structure in the hibernation group increased significantly compared to the other three groups; and the structure of the Gal (fucose, Fucose, Fuc) in the hibernating group increased significantly; 7) The three antenna identified by the hibernating group, the hibernating group, the PHA-E+L specific identified three antenna and the four antenna complex N- sugar chain (Tri-and tetra-antennary complex-type N-glycan) structure increased significantly in the hibernating group and the hibernating group. We selected the four agglutinin histochemical experiments of MAL-II, PTL-I, DSA and PHA-E+L. The results and agglutination of the histochemistry The results of the prime chip are consistent and further verified the experimental conclusions of the lectin chip. In conclusion, the results of this part suggest that the significant increase of SAa2-3Gal structure in the periodic inter matrix arousal of the rat can effectively compensate for its decrease in hibernating process, and to maintain the normal structure and function of the skeletal muscles of the hibernating rat and prevent it. Disuse atrophy has played an important role; because PTL- I, WFA, GSL- I, PTL- II, four lectins specifically identify the sugar chain structures that are O- connected, it is presumed that the increase of the sugar chain in the O- connection may be related to the specific protective mechanism of the hibernating mice; in addition, the three antenna and the four antenna type of glycoprotein N-. The sugar chain is significantly increased during the whole hibernation process, which may be related to the physiological adaptation mechanism of the skeletal muscle to hibernation. The second part: the results of the second chapter show that the significant increase of SAa2-3Gal structure effectively compensates for its decrease during the hibernation process and normal saliva during the periodic waking process of the rat. Acid content plays an important role in maintaining the shape, structure and function of muscle. Therefore, the aim of this part of this experiment is to determine the beta galactose - alpha -2,3- sialidase (sialyltransferasebeta-galactoside alpha-2,3-sialyltransferases) (including ST3Gall, ST3Gal2, ST3G) in the soleus muscle of the rat at different periods of hibernating. Al3, ST3Gal4, T3Gal5, ST3Gal6) and the changes in the expression of sialidase (sialidases) (including NEU1, NEU2, NEU3, NEU4) mRNA, from the mRNA level of sugar related genes to the mechanism of the variation of SAa2-3Ga in different periods of hibernating. 00%; P0.01), while the mRNA expression of ST3Gall in irritable and hibernating groups was significantly higher than hibernating group (114.30% and 126.20%, P0.01), and there was no significant difference compared with the hibernating group (P0.05). Compared with the hibernating group, the mRNA expression of ST3Gal2 decreased significantly in the hibernating group (-42.70%, P0.01), and compared with the hibernating group, the arousal group and out of the hibernating group were compared with hibernating group. The expression of mRNA in the sleeping group increased significantly (77.30% and 76.50%, P0.01). Before hibernating group, there was no significant difference in the mRNA expression of ST3Gal2 in the waking and sleeping group (P0.05). The mRNA relative expression of ST3Gal5 in hibernating group was significantly lower than that in the pre hibernating group (-60.91%, P0.01), while the mRNA expression of ST3Gal5 in the waking and hibernation group was respectively respectively. There was a significant increase in the hibernating group (160.56% and 125.48%, P0.01), and there was no significant difference between the hibernating group (P0.05).NEU1, the mRNA expression of NEU3 and ST3Gal6 was not significantly different in the hibernating group (hibernating group, hibernating group, waking group, and dormancy group) (P0.05). Our results suggest that ST3Gall, ST3Gal2 and ST3Gal5 mRNA are expressed in relative terms. The change of the quantity, that is, in hibernation, a significant decrease in the wake-up process and after the hibernation to the pre hibernation level, may be the main factor causing the changes in the SAa2-3Gal structure of the SOL in the rat. The third part: the study in the second chapter of this article shows that the sugar of the lectin MAL- II in the soleus muscle of the squirrel's soleus is specific. The significant changes of chain structure SAa2-3Gal in hibernating process may play an important role in the mechanism of anti waste muscle atrophy. Therefore, the preparation of lectin MAL- II magnetic particle complex is prepared by this experiment, and the glycoprotein of SAa2-3Gal is extracted from the soleus muscle of hibernating and hibernating squirrel, respectively, and extracted by LC-ESI-MS/MS The glycoprotein was identified by bioinformatics. 227 glycoproteins and 185 glycoproteins were identified from the pre hibernation group and the hibernation group. Among them, only 141 glycoproteins were identified before hibernating group, and only 99 glycoproteins identified in hibernation group were identified, and the number of glycoproteins identified in the two groups was identified. The annotation analysis for 86.GO (Gene Ontology) functional annotation shows that the glycoproteins identified according to the cell composition are mainly intracellular proteins, and many are macromolecular proteins, mainly distributed in the organelles and membrane structures. According to the molecular functions, these glycoproteins have functions of binding, catalysis and structural molecular activity. .KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that the glycoproteins isolated from pre hibernating group and hibernating group SOL were 40 and 36 respectively, according to the participating biologic process annotation. There are 14 significant enrichment pathways in the pre hibernating group, but only 10 in the hibernating group, and the protein protein interaction analysis using the IntAct database, and the network diagram of the egg white interaction of the pre hibernating group and the hibernating group of the hibernating group and the hibernating group of the hibernating group (The protein-protein inte). Raction network, PPI network), screening out 9 degree values (degree) more than 30 of the core protein. Fourth part: this part of the experiment selected 2 glycoproteins to verify, respectively, heat shock homologous protein (heat shock cognate 70, HSC70) and pyruvate kinase (pyruvate kinase, PK), detection of hibernation in the process of protein expression and knot In addition, the changes in the expression of 14-3-3 protein epsilon protein in the SOL of the rat were detected at different periods of hibernation, and the potential role of the protein in the mechanism of anti waste muscle atrophy was analyzed. Our results showed that the expression of HSC70 protein in hibernating group was significantly reduced by 37.39% (P0.01) compared with hibernating group, compared with hibernating group. The expression of HSC70 protein increased by 51.39% (P0.01) and 41.67% (P0.01) in the hibernating group. There was no significant difference between the three groups in the waking and hibernation group (P0.05), and there was no significant difference between the four groups (hibernating group, hibernating group, waking group, hibernating group) (P0.05), and pre hibernating group. The expression of 14-3-3 protein epsilon in hibernating group was significantly reduced by 42.59% (P0.01). Compared with the hibernating group, the 14-3-3 protein epsilon expression increased by 51.61% (P0.01) and 58.06% (P0.01) in the hibernating group. There was no significant difference between the waking and hibernating groups (P0.05) between the hibernating group and the hibernating group (P0.05). Compared with the pre hibernating group, the SAa2-3Gal structure of the hibernating group glycoprotein HSC70 was significantly reduced by 30.08% (P0.01). Compared with the hibernating group, the SAa2-3Gal structure of the waking group increased by 37.66% (P0.05), and the saliency group increased by 54.50% (P0.01). There was no significant difference between the pre hibernating group and the waking and hibernating group (P0.05). Compared with the hibernating group, the winter group had no significant difference (P0.05). The structure of PK terminal SAa2-3Gal in the sleeping group decreased by 29.40% (P0.01). Compared with the hibernating group, the structure of SAa2-3Gal increased by 30.91% (P0.05) and 34.41% (P0.05) in the hibernating group. There was no significant difference between the waking group and the three groups (P0.05) before hibernating group (P0.05). The results showed that the soleus muscle of the hibernation at different stages of the dormancy Changes in the three proteins of HSC70, PK, 14-3-3 protein epsilon (including protein expression and SAa2-3Gal sugar structure) may be related to its unique mechanism of anti waste muscle atrophy.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q445
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本文编号：1955417