基因重组糖蛋白CD44s的N-糖基化研究及其被LSECtin识别机理的初步探索

【作者】 韩欢欢；

【导师】 钱小红；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物分析学， 2010， 博士

【摘要】 CD44是分布极为广泛的细胞表面跨膜糖蛋白,参与细胞的许多生理过程,包括生长调控,存活,分化和迁移等。CD44表达量的变化以及功能的异常会导致机体的很多病理变化。CD44与胞外基质中的透明质酸、层粘连蛋白等基质分子之间的相互作用使得CD44能够介导异质性粘附,如肿瘤细胞与宿主细胞及宿主基质的粘附。异质性粘附在肿瘤细胞侵袭转移中起促进作用,因此有大量的研究认为CD44与肿瘤侵润转移有关。CD44s是CD44蛋白家族中分子量最小,仅含有组成性外显子的成员,广泛分布在脊椎动物发育及成熟的各种器官中。近年来,越来越多的研究开始关注糖基化对CD44s与其配体之间相互作用的调控。CD44最重要的功能是能够与透明质酸发生结合,两者间的结合参与肿瘤转移进程中的解粘附、迁移、粘附、入侵过程中的多个步骤。阻止二者之间的相互作用能够抑制原发性肿瘤的生长和转移。越来越多的研究发现CD44s的N-糖基化状态是调控其与透明质酸间结合的重要机制。新的研究发现CD44s是L-选凝素、P-选凝素和纤维蛋白原等能够介导细胞间异质性粘附的分子的内源性配体,并且这些相互作用都受到CD44s N-糖基化状态的调控。但是目前对CD44s的N-糖基化修饰,包括修饰位点、修饰类型及其糖型的微不均一性等均未见文献报道。LSECtin是一种在肝脏和淋巴结窦状细胞中高表达的C-型凝集素,其特殊的定位暗示着它在这些细胞中发挥重要的作用。目前的研究表明,LSECtin与多种病毒表面糖蛋白之间的相互作用是造成这些病毒在肝脏和淋巴结聚集、感染的原因之一。最新的研究发现LSECtin能够识别乳腺癌细胞上的CD44s的N-糖链。已有研究表明,乳腺癌细胞在发生转移时多数是转移到对侧的淋巴结内皮细胞上。由此,我们推测LSECtin作为一种特异高表达在淋巴结窦内皮细胞上的粘附分子,可能参与了乳腺癌细胞的淋巴结转移,而这个事件的分子基础可能是LSECtin对乳腺癌细胞上CD44s的特殊糖链结构的识别。对CD44s N-糖基化修饰的研究将为探索此事件的发生机制奠定基础。糖基化修饰是最普遍的蛋白质翻译后修饰形式之一。然而对蛋白质糖基化修饰的研究远远落后于对蛋白质本身的研究,这主要是由于糖基化修饰的发生没有模板指导,在多种糖基转移酶和糖苷酶的作用下,即使同一个糖基化位点上也会存在多种多样的糖基化修饰,即微不均一性,大大增加了分析的复杂性。糖链结构本身的特点决定了其没有紫外吸收,在质谱中的响应信号强度远不如肽段。因此糖基化修饰的鉴定过程常需要对糖链进行衍生化或对糖肽进行富集,反应过程复杂且步骤繁多,尤其是在糖蛋白可获得的量比较少的情况下,对其糖基化的分析尤为困难。本文首先对NS0细胞重组表达的CD44s的N-糖基化修饰做了全面、细致的研究。采用PNGase F酶切结合18O标记N-糖基化位点-液质联用检测的方法确定重组CD44s的5个潜在的N-糖基化位点均发生了糖基化,并确定了各个N-糖基化位点发生修饰的程度。用PNGase F将其N-聚糖切除,并通过泛甲基化修饰-MALDI-TOF质谱检测和2-AB标记-Q-TOF质谱检测两种方法,得到了其N-糖分布总谱及各种糖型的相对含量。利用亲水色谱结合Q-TOF质谱检测对CD44s的酶切肽段进行分析,最终得到CD44s各个N-糖基化位点上的糖型分布。其中第5位天冬酰胺上分布的糖型主要是二三天线的复合型,少部分发生了核心岩藻糖基化,部分结构末端有唾液酸修饰。第37,80和90位天冬酰胺上分布的糖型主要是二三天线的复合型,全部发生了核心岩藻糖基化,部分结构末端有唾液酸修饰。第100位天冬酰胺上分布的糖型主要是未发生岩藻糖基化的高甘露糖型。上述研究首次对CD44s的N-糖基化作了全面系统的分析,为深入理解CD44s N-糖基化的生理功能提供了基础。在对CD44s的N-糖基化修饰全面系统分析的基础上,本研究对LSECtin识别CD44s N-糖链的分子机理做了初步探索。通过亲和捕获-质谱鉴定的方法初步确定第80位天冬酰胺上的糖链是LSECtin识别的关键部位,对此结果进行了N-糖基化位点突变的验证,结果显示该位点的突变确实会导致两者之间相互作用的减弱。根据LSECtin特异识别的糖型结构的特点,对CD44s蛋白进行三种糖苷外切酶的酶切,用ELISA检测验证CD44s酶切产物与LSECtin之间的相互作用,结果显示LSECtin与CD44s之间的相互作用依赖于CD44s末端的N-乙酰葡萄糖胺,与文献所报道的LSEctin的糖型识别特点一致。蛋白质三维结构研究的报道也证明,其80位天冬酰胺处在CD44三维结构中较有利于与其他分子发生相互作用的位置。上述研究结果首次揭示了LSECtin与CD44s之间相互作用的分子结构基础,对深入理解LSECtin与乳腺癌的识别机制具有重要意义,并为研发阻断乳腺癌转移的药物提供了新的思路。有文献报道证明不同哺乳动物细胞中表达的重组蛋白的糖链结构和糖型分布会有所不同,而CHO(Chinese hamster ovary,中国仓鼠卵巢)细胞重组表达的CD44s也具有与LSECtin发生相互作用的功能。因此,本文对CHO细胞重组表达的CD44s的N-糖基化也进行了分析,并比较了NS0和CHO细胞中表达的CD44s N-糖基化的区别。我们发现NS0细胞中合成的N-糖中发生核心岩藻糖基化的糖链个数多于CHO细胞;NS0细胞中的半乳糖均是以α键连接在糖链中,而CHO细胞中则是以β键连接;两种细胞中的糖链末端都存在NeuGc的结构,CHO细胞中还存在NeuAc。两种细胞中表达CD44s上N-糖的差异,从侧面证明LSECtin与CD44s之间的结合不依赖于半乳糖、NeuGc或NeuAc。为了解决糖蛋白质组研究中糖链在质谱中检测灵敏度低的问题,本研究还发展了一种对N-聚糖进行快速标记结合质谱检测的新方法。采用酰肼活性基团对中性N-聚糖进行标记,使其在MALDI-TOF质谱中检测的灵敏度提高了10倍。对葡聚糖、去唾液酸化胎球蛋白以及CHO细胞中表达的CD44s的N-糖链质谱分析表明,该方法快速、简便,不需要后处理,特别适用于糖蛋白样品量较少时的分析。更多还原

【Abstract】 CD44 proteins are class I transmembrane glycoproteins mediating the response of cells to their cellular microenvironment. CD44 proteins participate in many cellular processes, including the regulation of growth, survival, differentiation and motility. The altered expression or dysfunction of CD44 proteins contributes to numerous pathological conditions. The adhesive qualities of the CD44 can lead to interaction between two different cells or between a cell and its pericellular matrix. CD44 mediates cell-cell and cell-matrix interactions, in large part through its affinity for hyaluronan (HA), a glycosaminoglycan of extracellular matrices (ECMs), but also potentially through its affinity for other ligands such as osteopontin, collagens. Furthermore the interaction of CD44 with these cellular proteins involved in motogenesis and proteolysis is a determinant factor in cell migration and invasion. The smallest CD44 isoform, which is known as CD44 standard (CD44s), is ubiquitously expressed in vertebrates in developing and adult organisms. The mRNA of this isoform contains no variant exons. Recently, more and more studies found the N-glycosylation of CD44s was involved in the regulation of its function. One primary function of CD44 is binding to HA. Metastasis is a multistep process that includes de-adhesion, migration, adhesion, and invasion. CD44 and its specific interaction with HA have been implicated in many of these processes. Reagents blocking the CD44/HA interaction have been found to prevent primary tumor growth and metastasis. Moreover, recent evidence suggests that glycosylation may be a major mechanism controlling the interaction between CD44 and HA. Some studies also reported that the N-glycosylation of CD44s is associated with the interaction between CD44s and L-selectin, P-selectin, fibrin (ogen). However, the glycosylation situation of CD44s including the types of the glycans, the sites occupancy of the glycosylation has never been comprehensively studied before.LSECtin is a member of the C-type lectins that is found predominantly on sinusoidal endothelial cells of the liver and lymph nodes, suggesting that it plays a specific role in these cells. It was reported that the interaction between LSECtin and glycoproteins on some kind of virus may concentrate certain viral pathogens in liver and lymph nodes and augment infectious. Recent study found that LSECtin can recognize the N-glycan of CD44s on breast cancer cells. A large number of studies show that breast cancer cells will metastasize to endothelial cells of lymph node on the opposite side. So it was spectulated that the recognition between LSECtin and CD44s may play an important role in the tumor metastasis from breast to lymph node, and the specific glycoform of CD44s is the keypoint of the recognition. In order to study the mechanism of the incident, comprehensive characterization of the N-glycosylation of CD44s is fundamental and very important.Glycosylation is one of most common post-translational modification. But the study of glycosylatin is lagged far behind the analysis of protein. It is because their biosynthesis occurs in a non-template controlled manner and mature structures arise through the activities of a series of enzymatic reactions. So the glycans on one site are mixtures of variants (named microheterogeneity), which makes the analysis for them very complicated. The characteristics of glycan structure determine that they do not have UV absorption, and have lower response in the mass spectrometry than peptides. So the derivatization of glycan or enrichment of glycopeptides before MS analysis should be carried on in the analysis of glycosylation. The process is multistep and complicated, especially in the situation of the available amount of samples is little, the analysis of its glycosylation is particularly difficult.In this thesis, the N-glycosylation of CD44s expressed in NS0 cells was characterized completely for the first time. We used the PNGase F deglycosylation in H218O to label the N-glycosylation sites and then analyzed the peptides and glycopeptides by LC-LTQ MS. As a result, all five predicted N-glycosylation sites were confirmed to be occupied by an oligosaccharide moiety. Then the N-glycans released by PNGase F digestion were analyzed by permethylation-MALDI-TOF MS and 2-AB labeled-ESI-Q-TOF MS. The profile of CD44s’N-glycans and the relative quantity were obtained. The glycopeptides and peptides of CD44s were separated by HILIC-UPLC system at the peptide level and detected online by a Q-TOF mass spectrometer to identify the information of site heterogeneity. The glycoforms on Asn5 are bi-, and triantennary complex type, little with fucose and NeuGc. The glycoforms on Asn37, Asn80 and Asn90 are also bi-, and triantennary complex type, all of them are core fucoslated, some structure carrying sialic acid. On Asn100, there are high mannose type structures having from five to seven mannose residues and no core fucosylation. A comprehensive study on the glycosylation status of CD44s was performed for the first time, which will facilitate future understanding about the roles of oligosaccharides from CD44s in related biological process.On the basis of completely characterization of CD44s’glycosylation, we made preliminary studies on the structure bases of the recognition between LSECtin and CD44s. We used the immbolized LSECtin to capture the specific glycopeptides of CD44s which was then detected by the LTQ-MS. The results showed that the Asn80 is the very important site recognized by LSECtin. The result was first validated by the mutation of N-glycosylation sites. When Asn80 was mutated to Ser, the interaction between the LSECtin and CD44s decreased clearly. It was reported that LSECtin can bind to GlcNAc-1-2Man with very high affinity. Then we treated the CD44s with three exoglycosidases and detected the interaction with LSECtin by ELISA. The results showed that the GlcNAc at the terminal of the glycan play the important role in the interaction between LSECtin and CD44s. The analysis of three dimentaional structure of CD44s reported by reference also indicated that the Asn80 is in a palce that can interact with other molecular easily. These findings have the important research significance in the understanding the mechanism of the recognition between LSECtin and CD44s. And it may provide new perspective to develope the drug to repress the metastasis of breast cancer.The N-glycosylatin of the CD44s expressed in CHO cells was also analyzed by the same way above. All five consensus sequences for N-glycosylation were found to be glycosylated. The profile of CD44s’N-glycans are detected by permethylation- MALDI-TOF MS and exoglycosidase treatment. The glycan forms of CD44s expressed in NS0 and CHO cells are compared. The glycans in NS0 cells are more fucosylated than in CHO cells. There are Gal (α1-3) Gal in NS0 cells andβform in CHO cell just like in human cells. They all have NeuGc in glycans, which is NeuAc in human cells. The differences of N-glycans between the two cells confirmed our deduction that recognition between the LSECtin and CD44s was not affected by the amount of fucose, linkage form of Gal, type of sialic acid.To solve the widespread problem in glycoproteomics studies, such as lower response of the glycan in mass spectrometry, a rapid and convenient derivatization method of oligosaccharides was developed. The hydrazones were generated by the reaction of the reducing ends of oligosaccharides and the hydrazide of CPH. The derivatives can be directly analyzed by MALDI-TOF-MS without any treatment. An approximately 10-fold increase in detection sensitivity was observed. The new developed method was applied to the analysis of N-glycans on CD44s expressed in CHO cells and satisfactory results were obtained. The method can be used to analyze the N-glycans of glycoprotein especially when only small amount of sample is available.更多还原