【作者】 张亮；

【导师】 刘欣；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2021， 博士

【摘要】 N-糖基化作为蛋白质重要的翻译后修饰之一,广泛参与包括增殖生长、信号传导、免疫调节和炎症反应等重要生理活动。N-聚糖的定性和定量分析是蛋白质N-糖基化研究的重要组成部分。然而,研究表明N-聚糖具有微观不均一性、结构多样性以及动态变化等特性,不利于直接进行分析研究,因此需要有效的样品前处理方法将N-糖基化样品高效的、特异性的释放分离和富集标记。研究报道,诸多具有特殊功能的蛋白质可以有效应用于聚糖的释放分离和富集标记研究。本文旨在对这些具有特殊功能的蛋白质进行改造,使其更有效的用于蛋白质N-糖基化研究,具体工作如下:肽N-糖苷酶F(Peptide N-glycosidase F,PNGase F)是糖组学研究中重要的工具酶,它可以在温和条件下,专一性地释放完整的N-聚糖,在N-聚糖的结构和功能分析等研究中起到重要作用。目前His-PNGase F的生产常利用其可溶性的重组蛋白来进行纯化,但His-PNGase F在表达过程易形成无活性的包涵体蛋白,导致其生产成本相对较高。为有效利用包涵体形式的His-PNGase F,我们通过优化诱导条件,获得占菌液蛋白总量40%以上的重组His-PNGase F包涵体蛋白,再采用尿素变性和复性的方法,从而得到大量表达的目的蛋白。此外,实验证明末端引入共融合谷氨酰胺标签(Gln-substrate peptide tag,Q tag),可有效提高可溶性PNGase F表达量,利于目的蛋白的直接纯化,也可提高PNGase F的产量。两种表达及纯化方案均可获得纯度>95%的目的蛋白,均具有较大的应用前景。酶固定化方法是提高PNGase F的使用效率的重要手段之一。本文对以上表达得到的带有两种不同分子标签的PNGase F的固定化进行研究。利用化学缩合和亲和相互作用的方法对His-PNGase F进行非定向和定向的固定化研究,得到非共价定向固定化方法相较于共价非定向化方法可提高约120倍的His-PNGase F负载量,且非共价定向固定化的His-PNGase F能够在重复使用五次后仍保留52.8%酶活性。该酶活性降低的部分原因是非共价定向固定化的PNGase F在使用过程中会发生流失。而Q tag在转氨酶的催化作用下能够介导PNGase F的共价定向固定化,共价定向固定化的PNGase F在重复使用五次后仍可具有78.6%的酶活性,显示融合Q tag的PNGase F在定向固定化中的应用优势。为了进一步提高固定化PNGase F的酶切效率,我们首次提出固定化PNGase F与微波辅助酶切相结合的方法,可有效地在5 min内实现N-聚糖的完全释放。PNGase F的定点突变和晶体结构分析验证其活性中心由D60N、E206和E118三个关键的氨基酸残基组成,其关键氨基酸位点的突变可导致水解活性丧失或改变与底物的亲和力。在前期研究的基础上,我们选择三种无水解酶活性的PNGase F突变蛋白进行分子改造和蛋白纯化,并用于聚糖的特异性识别。结果表明,R911突变型蛋白识别底物的能力相对较强,可以分别从混合样品中富集部分糖蛋白或糖肽而对非糖蛋白或非糖肽没有识别作用。然而,R911蛋白质结合底物的能力较弱,难以应用于大规模N-聚糖的分析。我们对另一种糖结合蛋白-识别糖链的F-box突变蛋白(a mutant of F-box that recognizes sugar chains,Fg)进行N-聚糖识别能力的分析,并首次验证N-聚糖的核心五糖结构对于Fg识别的必要性。融合谷胱甘肽硫转移酶标签的Fg(GST-tagged Fg,GFg)在温和的条件下能直接固定于谷胱甘肽修饰的琼脂糖微球,形成固定化的GFg-琼脂糖微球,可从免疫球蛋白G(Ig G)和辣根过氧化物酶(HRP)的酶切产物中分别鉴定到17条和11条糖肽,且在复杂的样品体系中表现出良好选择性和抗干扰性,利于糖肽的分析和检测。此外,GFg-琼脂糖微球用于蛋白质混合物的样品中,可以分离富集含有高甘露糖型聚糖的核糖核酸酶B(RNase B)和含有唾液酸型聚糖的转铁蛋白(transferrin),有效地去除非糖蛋白的干扰。实验结果表明GFg-琼脂糖微球能有效分离富集N-糖基化样品,且具有成本低廉、操作简单和生物相容性好等优点,有望高通量和自动化的应用于糖肽或糖蛋白分析。基于Fg对N-聚糖的识别能力,我们设计融合绿色荧光蛋白(GFP)和Fg蛋白质的荧光探针(GFP-Fg),对细胞膜上N-聚糖进行标记研究。共聚焦荧光成像技术和流式分析结果表明GFP-Fg可对细胞的N-聚糖实现特异性标记,且标记效率达到99.9%。这种特异性的荧光探针为细胞膜上N-聚糖的分析提供了新的研究手段。综上所述,基于PNGase F和Fg的结构和功能,两类蛋白质可有效应用于N-聚糖的分离、富集和标记研究。结果表明,定向固定化的PNGase F展现出良好的酶活性和重复使用性,为高通量的N-聚糖的研究提供了新的技术手段。同时,改造后的PNGase F和Fg蛋白质可特异性应用于N-聚糖的识别和富集研究,并且能够进一步应用于细胞表面N-聚糖的标记。两类功能蛋白的相关研究为蛋白质N-糖基化的研究提供新的手段,具有广阔的应用前景。更多还原

【Abstract】 As one of the most important post-translational modifications of proteins,N-glycosylation is widely involved in various important physiological and pathological processes,including cell proliferation and growth,signal transduction,immune response and inflammation.The qualitative and quantitative analysis of N-glycans is an important component of protein N-glycosylation research.However,studies have shown that N-glycans are hard to be direct analyzed as their characteristics of micro-heterogeneity,structural diversity and dynamic changes.Therefore,effective pretreatment methods for the release,enrichment or labeling of N-glycan are needed.It has been reported that many functional proteins can be effectively used in the release,separation and enrichment of glycans.In this paper,these functional proteins were employed and modified for effective research of N-glycosylation.The work is as follows:Peptide N-glycosidase F(PNGase F)is one important enzyme in glycomics research.It can specifically release N-glycan from glycoproteins or glycopeptides under mild conditions,playing an important role in researches of N-glycan’s structure and function.Currently,the conventional method to produce His-PNGase F based on the purification of soluble protein,but His-PNGase F is prone to form inactive inclusion body proteins during the expression process,making its production cost relatively high.In order to effectively use the His-PNGase F in the form of inclusion bodies,we optimized the induction conditions to obtain high expression of the recombinant His-PNGase F in the form inclusion body,which accounts for more than 40% of the total bacterial protein,and then used denaturation and renaturation methods to obtain a large amount of target protein.In addition,results displayed that the glutamine tag(Gln-substrate peptide tag,Q tag)co-fused at the end of PNGase F can effectively improve its solubility.Both schemes to express and purify PNGase F can obtain the target protein with a purity of >95%.Enzyme immobilization method is one of the important means to improve the efficiency of PNGase F.In this paper,the immobilization of PNGase F with two different molecular tags obtained from the above expression was studied.The non-oriented and oriented immobilization of His-PNGase F was carried out using the methods of chemical condensation and affinity interaction.It was found that the non-covalent oriented immobilization method can load His-PNGase F by about 120 times compared with the non-oriented method.Non-covalent oriented immobilization of His-PNGase F can retain52.8% enzyme activity after reused for five times.The reduced enzyme activity is partly due to the loss of immobilized PNGase F.The Q tag can mediate the covalent and oriented immobilization of PNGase F under the catalysis of transaminase.The covalently oriented immobilization of PNGase F can still own 78.6% enzymatic activity after reused for five times,showing the advantages of covalently oriented immobilization of PNGase F.In order to further improve the digestion efficiency of immobilized PNGase F,we first proposed a method combining immobilized PNGase F with microwave-assisted digestion,which can effectively release of N-glycans within 5 minutes.The site-directed mutagenesis and crystal structure analysis of PNGase F verified that its active center is composed of several key amino acid residues,D60,E206 and E118.Mutations in its key amino acid can cause loss of deglycosylated activity or change the affinity with the substrate.Based on the previous research,we selected three PNGase F mutants without deglycosylated activity for molecular modification and protein purification,and then used for the specific recognition of glycans.The results show that the R911 protein has relatively strong affinity to recognize substrates,and can enrich part of glycoproteins or glycopeptides,but has no recognition effect on non-glycoproteins or non-glycopeptides.However,the ability of R911 protein to bind to substrates is too weak to be applied to large-scale N-glycan analysis.Hence,we analyzed a mutant of F-box protein that recognizes sugar chains(Fg),and verified that the intact M3N2 structure is necessary for Fg recognition.Fg fused with Glutathione S-transferases(GFg)can be directly immobilized on agarose microspheres modified with glutathione under mild conditions to form immobilized GFg-agarose microspheres.17 and 11 glycopeptides can be identified by GFg-agarose microspheres from the tryptic peptide of immunoglobulin G(Ig G)and horseradish peroxidase(HRP),respectively.GFg-agarose microspheres also show good selectivity and anti-interference in a complex sample.In addition,GFg-agarose microspheres can separate and enrich ribonuclease B(RNase B)containing high mannose glycans and transferrin containing sialic glycans in protein mixtures.Moreover,GFg-agarose microspheres own many advantages,including low cost,simple operation and good biocompatibility.Based on the ability of Fg to recognize N-glycans,we designed a fluorescent probe(GFP-Fg)fused with green fluorescent protein(GFP)and Fg to label the N-glycans on cell membranes.The results of confocal fluorescence imaging and flow cytometry showed that GFP-Fg can specifically label the N-glycans of cells,and the labeling efficiency reaches 99.9%.In summary,based on the structure and function of PNGase F and Fg protein,two types of proteins can be effectively used in the separation,enrichment and labeling of N-glycans.Results show that the oriented immobilization of PNGase F exhibits good enzyme activity and reusability,which provides a new means for the high-throughput release of N-glycans.At the same time,the modified PNGase F and Fg proteins can be specifically applied to the enrichment of N-glycans,and further applied to the labeling of N-glycans on the cell surface.The related research of two types of functional proteins provides a new method for the research of N-glycosylation,which has broad application prospects.更多还原