【作者】 周东文；

【导师】 滕脉坤； 牛立文；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2008， 博士

【摘要】 (Ⅰ)凝集素是自然界广泛存在的一大类非免疫来源的、无酶活性的多价的糖类结合蛋白质,能使细胞发生凝集。凝集素可从不同的角度来分类,根据来源可分为动物凝集素、植物凝集素和微生物凝集素。半乳糖凝集素galectin为S型动物凝集素,它广泛存在于各种动物体内,galectin家族成员具有高度保守的特征性的氨基酸序列,拥有一个大约130个氨基酸的核心序列,通常称之为糖识别结构域（CRD:carbohydrate recognition domain）,能特异性地结合β—半乳糖苷,且进化保守,种类繁多,功能复杂,可能参与细胞与细胞、细胞与细胞间质之间的相互作用,细胞粘附、凋亡及免疫反应等多种生物学过程。根据分子结构,目前galectin可划分为3种基本类型:1)原型galectin（Prototype:含有单一的糖识别结构域CRD）;2)嵌合型galectin（Chimera:一个CRD与一个胶原蛋白样重复结构域融合）;3)前后重复型galectin（Tandem-repeat:两个CRD串联融合）。原型galectin包括:galectin-1,2,5,6,7,10,11,13,14,15等;嵌合型galectin仅有一个成员galectin-3;前后重复型galectin包括galectin-4,6,8,9,12。在过去的一些年里,半乳糖凝集素家族的一些蛋白的晶体结构逐渐被解析出来。迄今为止,gal-1,gal-2,gal-3,gal-7,gal-9的N端结构域以及gal-10等的晶体结构已经投入PDB蛋白质数据库中。从这些已经报道的晶体结构来看,它们都采取了类似的大豆凝集素的凝胶卷拓扑结构。它们的保守CRD结构域包含由11或12条β-strand的双层β-sheet组成的β-三明治结构,其中一些蛋白含有短的3₁₀螺旋。这些已知晶体三维结构的蛋白中,糖结合部位的结构特征也比较相似,以Gal-1-β-galactoside复合体为例,蛋白与糖配体的结合主要靠His44,Asn46,Arg48,Val59,Asn61,Trp68,Glu71和Arg73等氨基酸残基直接与结合的二糖相互作用。GRP（即:HSPC159）是个新的人类galectin相关基因,最近通过测定人的造血干细胞CD34+全长的mRNA序列得到鉴定。尽管它编码的蛋白质含有一个比普通稍长的N端序列,但仍可以被划归到“原型”galectin。它的序列中不含有公认的分泌或者核定位信号肽序列。人类的GRP基因定位于染色体2P13上,包含有5个外显子,含有在galectin家族中保守的外显子和内含子连接处。GRP的序列在进化上非常古老并且在不同的物种中高度保守,非常相似的cDNA序列已经在人类、鼠、鸡、青蛙和鱼当中发现。GRP含有其它galectin最保守的64个残基中的51个,是一个galectin相关蛋白质。然而,像GRIFIN一样,GRP的序列只具有那些保守的糖识别残基中的2个,因此可能不结合半乳糖苷。GRP的生物功能目前还不清楚。在本论文中,我们构建表达并纯化了人源GRP的C端保守结构域CRD（氨基酸残基38-172）,然后我们使用悬滴气相扩散的方法长出了它的晶体。在北京和合肥同步辐射实验室收集到了最高分辨率达1.8（?）的衍射数据。晶体有两个空间群,分别是C2空间群,晶胞参数是:a=123.07,b=96.67,c=61.56（?）,β=118.72°;F222空间群,晶胞参数是:a=96.89,b=127.76,c=213.29,α=β=γ=90°。计算的Matthews coefficient分别为2.6（?）³/Da对应51.8%的溶剂含量,和2.55（?）³/Da对应52.5%的溶剂含量。然后,我们使用分子置换的方法解析了GRP-C的晶体相位并通过精修得到了1.9（?）的三维结构模型。GRP采用了和半乳糖凝集素类似的折叠方式即由两个反向平行的β-sheet组成的β-三明治结构,二级结构以β-sheet为主,此外还含有一个小的3₁₀螺旋。GRP蛋白的晶体堆积方式非常独特,它的不对称单位中含有4个分子,先两两形成一个含有一个非晶体学对称轴的同源二聚体,然后这两个二聚体垂直排列并且以凹面相对,形成中间带有一个圆孔的四体结构。糖结合试验和共结晶试验以及对晶体结构的分析表明GRP确实不具有结合糖配体的能力。它的功能作用方式与凝集素存在着根本的不同。因此我们又根据蛋白的表面电荷分布情况推测了GRP可能的在体内发挥生理功能的方式即通过亲水和疏水相互作用进行特异性识别以及通过与金属离子的结合来发挥生物学功能。（Ⅱ）Galectin-8属于前后重复型凝集素亚家族。它包含有一些异构体,每个异构体都由两个140个左右氨基酸残基长的结构域组成,每个结构域都包含有一个糖识别结构域。这两个结构域由一个长度不等的连接肽连接起来。人的galectin-8包含33kbp的基因组DNA,定位于染色体1q42.11上,包含11个外显子。基因通过可变剪接共产生14个转录产物,编码6个蛋白。像其它凝集素一样,galectin-8是一个分泌蛋白。通过分泌,galectin-8扮演细胞粘结的生理调控因子的角色。当它不固定时,作为基质蛋白发挥生理功能,等同于纤维粘连蛋白通过连接和聚集一些特定的细胞表面整合素受体来刺激细胞粘附。Galectin-8和整合素复合物的形成包含糖—蛋白相互作用并引发了整合素介导的信号级联反映例如FAK和paxillin的酪氨酸激酶的磷酸化。相反,当galectin-8作为过剩的可溶配体时,像纤维粘连蛋白一样,它能与整合素形成复合物负调节细胞粘附。这样的一个机制使得分泌的galectin-8发出的本地信号可以传到细胞粘附和迁移的特定区域。由于它对细胞粘附的双重作用和它与纤维粘连蛋白的联系,galectin-8可以被看作一种新型的细胞基质蛋白。Galectin-8的表达水平与某些人类肿瘤、前列腺癌正相关。过量表达的凝集素可以刺激那些肿瘤组织的生长和转移,这也是靠它的调节细胞粘结和生长的能力来完成的。因此,galectin-8可以调控细胞—基质的相互作用,也可以调节多种生理和病理环境下的细胞功能。在本研究中,我们表达并纯化了人源galectin-8以及它的C末端保守结构域CRD。然后运用悬滴气相扩散的方法结晶了CCRD蛋白。蛋白的结晶条件是:27.5%PEG8000,100mM Na Cacodylate buffer pH6.5,0.2M ammoniumphosphate,5%Ethylene Glycol。晶体在实验室铜靶光源100K的温度下收集了一套3（?）的常规衍射数据。晶体属于正交品系,空间群为P222,晶胞参数为a=54.15,b=73.13.c=179.42（?）。计算得到的Matthews coefficient为2.84（?）³/Da,对应的溶剂含量为56.71%。更多还原

【Abstract】 (I) Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates among the vast array expressed in animal tissues. Most animal lectins can be classified into four distinct families: C-type lectins （including the selectins）; P-type lectins; pentraxins; and galectins. Galectins are a family of lectins that bindβ-galactosides by means of a carbohydrate recognition domain （CRD） that has many conserved sequence elements. In addition to galectins expressed in vertebrates （fish, birds, amphibians, and mammals）, galectins have also been found in invertebrates （worms and insects） and even in protists （sponge and fungus）.The galectins can be classified into three groups: the proto-type which contains one carbohydrate recognition domain （CRD）, the chimera-type which has a proline/glycine-rich repetitive sequence connected to a CRD, and the tandem-repeat-type which contains two homologous CRDs in tandem separated by a short linker. Proto-type galectins are non-covalent homodimers composed of two identical CRDs except galectin-5 which exists as a monomer. The only member of the chimera-type is galectin-3 which is predominantly found in mammals. The tandem-repeat-type includes galectin-4, -6, -8, -9, and -12. In the past years, the x-ray crystal structures of a few galectins such as gal-1, 2, 3, 7, and 10 have been reported and they are all similar and show jelly-roll topologies typical of legume lectins. Their CRDs are all composed of 11 or 12-strand antiparallelβ-sandwich. Some of them have short 3₁₀ helices. The general architectures of the carbohydrate-binding site in galectins of known three dimension structures are very similar. The structure of human Gal-1 -β-galactoside complex reveals that the amino acids His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71 and Arg73 are directly involved in interactions with the bound disaccharide.GRP （previously known as HSPC159） is a novel human galectin-related protein whose gene was originally deduced by partial sequence alignment and confirmed by a full-length sequence for an mRNA isolated from CD34+ hematopoietic stem cells. The human GRP gene （locus #29094） is located on chromosome 2p13 （NT-031752） and is composed of five exons with exon/intron junctions located in positions generally conserved across the galectin family. GRP sequence is evolutionarily ancient and highly conserved as very similar cDNA sequences have been found in human, mouse, chicken, frog and fish. GRP shares consensus amino acids at 51 of the 64 most highly conserved residues in other galectins. On the other hand, its sequence deviates significantly at five of the seven most critical residues for carbohydrate-binding.In this work, we expressed and purified the C-terminal fragment of human GRP （GRP-C; residues 38-172） containing the CRD. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG400 and 2M ammonium sulfate in 100mM Tris-HCl buffer pH7.5. Diffraction data with resolution limit of about 1.9 A were collected at the beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a=123.07, b=96.67, c=61.56 A,β=118.72°. The estimated Matthews coefficient was 2.6A³/Da, corresponding to 51.8% solvent content. Then, we determined the crystal structure of carbohydrate-recognition-domain of hGRP at 1.9A resolution. In this structure, hGRP-C adopts a fold of 10-strand anti-parallelβ-sandwich similar to that known for other galectin structures. However, the architectures of carbohydrate-binding site between hGRP-C and other known structural galectins are completely different, which suggests a novel mode in which GRP carries out its biological function in vivo.（II） Galectin-8 belongs to the family of tandem-repeat type galectins. It consists as several isoforms, each made of two domains of -140 amino-acids, both having a carbohydrate recognition domain （CRD）. These domains are joined by a ’link peptide’ of variable length. The human galectin-8 gene covers 33 kbp of genomic DNA. It is localized on chromosome 1 （1q42.11） and contains 11 exons. The gene produces by alternative splicing 14 different transcripts, altogether encoding 6 proteins. Galectin-8, like other galectins, is a secreted protein. Upon secretion galectin-8 acts as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell surface integrin receptors. Complex formation between galectin-8 and integrins involves sugar-protein interactions and triggers integrin-mediated signaling cascades such as Tyr phosphorylation of FAK and paxillin. In contrast, when present in excess as a soluble ligand, galectin-8 （like fibronectin） forms a complex with integrins that negatively regulates cell adhesion. Such a mechanism allows local signals emitted by secreted galectin-8 to specify territories available for cell adhesion and migration. Due to its dual effects on the adhesive properties of cells and its association with fibronectin, galectin-8 might be considered as a novel type of a matricellular protein. Galectin-8 levels of expression positively correlate with certain human neoplasms, prostate cancer being the best example studied thus far. The overexpressed lectin might give these neoplasms some growth and metastasis related advantages due to its ability to modulate cell adhesion and cellular growth. Hence, galectin-8 may modulate cell-matrix interactions and regulate cellular functions in a variety of physiological and pathological conditions.In this work, we have expressed and purified the C-terminal CRD of human galectin-8 （CCRD）. The CCRD protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 27.5% PEG8000 and 0.2M ammonium phosphate in 100mM Na Cacodylate buffer pH6.5 with additive 5% Ethylene Glycol. Diffraction data with resolution limit of 3.0 A were collected in house at 100 K. The crystals belong to the orthorhombic space group P222, with unit-cell parameters a=54.15, b=73.13, c=179.42 A. The estimated Matthews coefficient was 2.84A³/Da, corresponding to 56.71% solvent content.更多还原