【作者】 王冬梅；

【导师】 台桂花；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2023， 硕士

【摘要】 半乳凝素-3(Galectin-3,Gal-3)是唯一的嵌合型半乳凝素,其N端(N-terminal tail,NT)富含脯氨酸/甘氨酸,C端具有保守的糖识别结构域(Carbohydrate-recognition domain,CRD)。Gal-3参与多种细胞活动,包括活化、迁移、凋亡等,且这些活动依赖于Gal-3的寡聚化。NT在Gal-3的生物功能及寡聚化中均发挥了重要的作用。NT具有较高的脯氨酸含量(24%),是正常蛋白脯氨酸含量的5倍。本实验室前期研究发现NT 1-68序列中的脯氨酸参与了Gal-3的多种细胞活动,但NT 69-121序列中的脯氨酸作用尚不清楚。因此本文研究了NT中69-121序列的15个脯氨酸在Gal-3寡聚化中的作用,具体研究成果如下:本论文将NT 69-121序列中的15个脯氨酸突变为丙氨酸,将重组质粒导入BL21(DE3)中,获得阳性单菌落,通过扩大培养、IPTG诱导、破碎、亲和层析、透析,获得了纯度较高的Gal-3和脯氨酸突变蛋白(P71A、P74A、P76A、P80A、P82A、P83A、P86A、P90A、P95A、P102A、P106A、P110A、P113A、P117A和P121A)。为明确脯氨酸突变对Gal-3功能的影响,本文通过流式细胞术、Western Blot、红细胞凝集等方法探究脯氨酸是否影响Gal-3与细胞膜的结合、T细胞活化和红细胞凝集功能,发现多个位点的脯氨酸(如P86、P102、P106、P113)参与调节Gal-3的生物功能。接下来探究脯氨酸如何影响Gal-3功能。考虑到本文将脯氨酸进行了突变,为明确突变对Gal-3空间结构的影响,通过荧光光谱法检测Gal-3色氨酸的极性环境以及通过圆二色谱法检测Gal-3的二级结构,发现脯氨酸突变未改变Gal-3的空间结构。考虑到Gal-3功能的发挥依赖Gal-3的寡聚化,本文通过纳米粒度法(Dynamic Light Scattering,DLS)检测不同糖蛋白(CD7、CD71、CD204、CD146和CD45)诱导Gal-3寡聚化,发现多个位点的脯氨酸(如P82、P83、P86、P95、P102和P113)参与糖蛋白诱导的Gal-3寡聚化。进一步研究脯氨酸如何影响Gal-3寡聚化。Gal-3的寡聚化分为糖诱导的触发过程和多种弱相互作用引起的聚集过程。本论文通过生物信息学预测了单脯氨酸突变对Gal-3无序性及pi-pi相互作用的影响,发现脯氨酸对Gal-3的无序性以及pi-pi相互作用的影响较小,推测脯氨酸对聚集过程影响较弱;通过生物膜干涉(Bio-Layer Interferometry,BLI)和等温滴定量热法(Isothermal Titration Calorimetry,ITC)探究脯氨酸突变对Gal-3与聚糖互作的影响,发现P82、P83、P113、P121等位点的脯氨酸对Gal-3与聚糖互作影响较大,推测脯氨酸对触发过程影响较大。综上所述,本论文成功制备了野生型Gal-3及15个脯氨酸突变蛋白;发现多个位点的脯氨酸参与Gal-3的生物功能;明确了脯氨酸突变对不同糖蛋白诱导Gal-3寡聚化的影响,并且主要通过影响触发过程影响了寡聚化。本文的研究结果揭示了脯氨酸在Gal-3寡聚化中的作用,为研究与Gal-3相关的疾病机制和药物开发奠定理论基础。更多还原

【Abstract】 Galectin-3(Gal-3)is the only chimeric galectin.Its N-terminal tail(NT)is rich in proline/glycine,and the C-terminal has a conserved carbohydrate-recognition domain(CRD).Gal-3 is involved in a variety of cellular activities,including activation,migration,and apoptosis,and these activities depend on the oligomerization of Gal-3.NT plays an important role in the biological function and oligomerization of Gal-3.NT had higher proline content(24%),which was 5 times higher than that of normal protein.Previous studies in our laboratory have found that proline in NT 1-68 sequence is involved in various cellular activities of Gal-3,but the role of proline in NT 69-121 sequence is still unclear.Therefore,this paper studies the role of 15 prolines in the 69-121 sequence of NT in the oligomerization of Gal-3.The specific research results are as follows:In this paper,15 prolines in the NT 69-121 sequence were mutated to alanine,and the recombinant plasmid was introduced into BL21(DE3)to obtain a positive single colony.High purity Gal-3 and proline mutant proteins(P71A,P74A,P76A,P80A,P82A,P83A,P86A,P90A,P95A,P102A,P106A,P110A,P113A,P117A and P121A)were obtained by expanding culture,IPTG induction,crushing,affinity chromatography and dialysis.In order to clarify the effect of proline mutation on the function of Gal-3,this paper explored whether proline affected the binding of Gal-3 to cell membrane,T cell activation and erythrocyte agglutination by flow cytometry,Western Blot and erythrocyte agglutination.It was found that proline at multiple sites(such as P86,P102,P106,P113)was involved in regulating the biological function of Gal-3.The next step is to explore how proline affects the function of Gal-3.Considering the mutation of proline in this paper,in order to clarify the influence of mutation on the spatial structure of Gal-3,we detected the polarity environment of Gal-3 tryptophan by fluorescence spectrometry and the secondary structure of Gal-3 by circular dichroism,and found that the spatial structure of Gal-3 was not changed by the mutation of proline.Considering the functional dependence of Gal-3 on Gal-3 oligomerization,we detected Gal-3 oligomerization induced by different glycoproteins(CD7,CD71,CD204,CD146 and CD45)by dynamic light scattering(DLS),and found that multiple sites of proline(such as P82,P83,P86,P95,P102 and P113)were involved in glycoprotein-induced Gal-3oligomerization.Further study how proline affects Gal-3 oligomerization.The oligomerization of Gal-3 is divided into a glycan-induced trigger process and an aggregation process caused by various weak interactions.In this paper,the effect of single proline mutation on the disorder of Gal-3and pi-pi interaction was predicted by bioinformatics.It was found that proline had little effect on the disorder of Gal-3 and pi-pi interaction,suggesting that proline had little effect on the aggregation process.The effect of proline mutation on the interaction between Gal-3 and glycan was investigated by Bio-Layer Interferometry(BLI)and Isothermal Titration Calorimetry(ITC).It was found that proline at P82,P83,P113 and P121 sites had a great influence on the interaction between Gal-3 and glycan,suggesting that proline had a great influence on the triggering process.In conclusion,wild type Gal-3 and 15 proline mutant proteins were successfully prepared in this paper.Multiple sites of proline were found to be involved in the biological function of Gal-3.The effect of proline mutation on Gal-3 oligomerization induced by different glycoproteins was clarified,and the oligomerization was affected mainly by influencing the triggering process.The results of this study reveal the role of proline in the oligomerization of Gal-3,which lays a theoretical foundation for the study of the disease mechanism and drug development related to Gal-3.更多还原