蛛丝蛋白MiSp全长基因克隆、表达及结构和功能研究
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摘要
圆网蜘蛛可分泌六种蛛丝蛋白纤维,分别在蜘蛛生命活动中扮演不同的角色。次壶腹腺丝(minor ampullate silk)主要用于构建蛛网核心框架和包裹猎物等,具有显著的机械性能和优异的生物亲和性,加之在水环境中不会发生超收缩(超收缩会严重影响丝蛋白纤维的性能)等,是一种优质的、难得的天然生物材料,在军工、航空航天以及生物医学工程领域有着巨大的潜在应用价值。
蜘蛛生性偏爱独居(同类残食),纺丝量也远少于家蚕,因此无法像高密度饲养家蚕一样大规模获取蛛丝纤维,严重阻碍了蜘蛛丝在现代科技生活中的大规模应用。自二十世纪九十年代以来,随着生物技术日趋成熟,人们开始探索通过生物技术的方法制备人造蜘蛛丝,以满足现代科技发展对高品质纤维材料的需求。多年来国内外多家研究机构专注于重组蛛丝纤维的仿生制备,但到目前为止尚未有一家机构能够仿生类似天然蛛丝的人造蛛丝纤维,严重限制这种新型战略资源的产业化。
长期研究表明,国内外蛛丝蛋白纤维的人工仿生之所以进展缓慢,主要是因为现阶段面临三大瓶颈问题：1)蛛丝蛋白全长编码基因克隆滞后：蛛丝蛋白编码基因大、重复度高以及GC含量显著等,常规PCR和小型cDNA文库技术很难钓取全长编码基因,经过近25年的克隆研究国内外仅报道三种蛛丝蛋白全长基因序列(其中第二条为本论文克隆)；2)全长编码基因外源表达难以实现：蛛丝蛋白全长编码基因大,编码的丝素蛋白达到300kDa左右；基因序列重复度高,容易出现基因重组和缺失等；蛛丝蛋白基因GC含量高,容易形成特殊的mRNA二级结构,导致核糖体脱落和翻译提前终止等；蛛丝蛋白中Gly和Ala使用频率过高,表达过程中给宿主压力太大；3)成丝机理研究不足：蛛丝蛋白分泌腺尺寸太小,腺体内环境(生理及化学条件)尚没明确；蛛丝蛋白由N端和C端非重复调节区域以及中间重复区Rp组成,它们相互协调共同赋予液态蛛丝蛋白以及固态蛛丝纤维的高度可控特性和优良品质,现阶段相关NT和CT的结构和功能还没有解析完全,Rp与丝性能的关系也不明确；次壶腹腺丝蛋白(minor ampullate spidroin, MiSp)中还包含特有的spacer间隔区,结构和功能还有待研究；由于腺体内环境以及蛛丝蛋白成丝机理研究的严重不足,致使人工纤维的仿生方法比较单一。
为系统性突破上述三个瓶颈问题,为产业化高品质重组蛛丝纤维奠定基础,本论文首次以MiSp为研究对象,主要内容包括以下三个方面：
部分一：克隆鉴定MiSp全长编码基因。大腹园蛛(Araneus ventricosus)是我国广泛分布的蜘蛛种属之一,丝纤维综合品质优异,国内外多家研究机构均在进行A. ventricosus丝蛋白编码基因的鉴定分析。次壶腹腺丝作为一种新型的蛛丝蛋白纤维,研究相对滞后,除了少量短的cDNA序列报道外,表达和仿生均无涉及。为了促进MiSp蛋白成丝机理的解析、全长MiSp蛋白重组表达、开发及仿生应用、为蛛丝蛋白的进化和表达调控研究增添新成员,本章基于前期构建的A.ventricosus大型fosmid基因组文库和STS/3D-PCR技术筛选鉴定MiSp全长编码基因序列。经过多轮筛选,我们得到含有A. ventricosus MiSp全长编码基因的阳性克隆,外源插入片段约33kb左右(GenBank accession no. JX513956),覆盖MiSp全长编码基因及其上游6647bp和下游14937bp非编码序列。A. ventricosus MiSp全长编码基因长10.9kb,由两个外显子(exon)和一个在蛛丝蛋白基因中首次发现的基因内含子(intron)组成。A. ventricosus MiSp intron长5628bp,以GT起始AG终止,符合intron的GT-AG法则。A. ventricosus MiSp全长转录子为5440bp,编码的1766个氨基酸可划分为N端和C端非重复区以及中间占到90%以上的重复区,其中重复区含有四种重复模块(由Gly-X、Gly-Gly-X、Gly-Gly-Gly-X和poly-Ala组成)和两个特有的DNA水平相似度为100%的间隔区(spacer,126个氨基酸残基)。A. ventricosus MiSp全长编码基因组成结构揭示了蛛丝蛋白编码基因intron-exon结构多样性,上下游调控序列的比对分析为进一步研究蛛丝蛋白编码基因的转录调控增添了新的保守性调控元件CACG。A. ventricosus MiSp全长编码基因为国内外首条MiSp全长基因序列(世界上第二种全长蛛丝蛋白编码基因序列),填补了国内外就A. ventricosus丝蛋白全长编码基因克隆的空白,为国内外丝蛋白全长基因的研究增添新成员。
部分二：研究MiSp结构和功能、解析组装机制和成丝机理。常温下,高浓度蛛丝蛋白经过丝腺内部一系列物理和化学变化,在极短的时间内迅速转变成性能显著的固态蛛丝纤维,这一复杂的纤维化过程目前尚无系统性阐述。蛛丝蛋白主要由氨基酸水平高度重复的重复区域构成(Rp,占到整个蛋白90%以上),Rp两端分别为N端(NT,约110氨基酸残基)和C端非重复区(CT,约130个氨基酸残基)。Rp区域主要与蛛丝纤维机械性能和品质相关,NT和CT则主要参与高浓度蛛丝蛋白储液的维持及纤维化过程中对蛛丝蛋白的多种调节作用。前期研究推断拖丝蛋白(major ampullate spidroin, MaSp) NT和CT在蛛丝蛋白成丝过程中以相似的方式发挥调节作用,本论文研究则证实次壶腹腺丝蛋白MiSp NT和CT在蛛丝蛋白纤维化过程中以相反的作用方式分别扮演不同的角色。A.ventricosus MiSp除了拥有蛛丝蛋白典型的三个结构域NT、CT和Rp外,还含有两个spacer区域。我们前期对Nephila clavipes主壶腹腺体的pH值(pH7.6-5.7,这是到目前为止测得的最宽pH范围)和多种离子种类及浓度进行测试,并发现腺体导管中含有碳酸酐酶(催化CO2+H2O←→HCO3-+H+反应维持pH梯度)。参照腺体内环境变化趋势,我们对A. ventricosus MiSp NT、CT以及spacer的功能和结构进行了系统性研究：1)随着pH从7.5降至5.0,MiSp NT逐渐二聚化且二聚体稳定性逐渐增强,连接蛛丝蛋白单体形成牢固的蛛丝蛋白多聚体,NaCl可维持单体稳定性延缓NT二聚化；pH7.5至5.0,MiSp NT二级结构维持α-helix构象不变,高温可诱导NT由α-helix向random coil可逆转变。NMR高级结构显示A. ventricosus MiSp NT单体由5个α-helix构成,Arg64和Glu115形成分子内盐键,位于α-helix1和4上的两个Cys形成分子内二硫键。与Euprosthenops australis MaSp1NT不同,A. ventricosus MiSp NT中没有参与MaSp1NT第三步二聚化的保守Glu84,但Asp109高度保守,揭示了MiSp不一样的二聚化机制；2)我们首次发现随着pH值的降低,A. ventricosus MiSp CT始终维持二聚体构象但二聚体稳定性逐渐降低(与NT相反),与MiSp NT不同,pH7.5-6.5时CT的高温变性过程可逆,二级结构由α-helix向random coil转变,pH降至5.5时高温可诱导MiSp CT由α-helix向β-sheet不可逆转变。pH≤5.5时CT结构发生改变(解折叠)形成β-sheet淀粉状(amyloid)纳米纤维(通过透射电镜和Congo染色证实),作为重复区Rp快速纤维化的晶核,NaCl对纳米纤维的形成具有抑制作用,NT不具备形成β-sheet amyloid纳米纤维的能力(pH7.5-5.0). NMR高级结构证实A. ventricosus MiSp CT单体与MiSp NT相似,由5个α-helix构成,α-helix2和4通过Arg43和Glu87形成盐键,单体之间主要依靠疏水作用二聚化,其中α-helix5申向对应单体内部形成发夹结构。导管末端pH值低以及HC03-浓度上升,因此导管末端的pC02较高,我们在分子水平证实CS2(CO2类似物,CO2难以操控以及改变pH值)会与CT内部区域保守的疏水氨基酸结合改变蛋白高级结构,促进去折叠纤维化,NT则不受CS2的影响。碳酸酐酶、NT稳定性增强以及CT稳定降低的起始部位均发生在腺体的相同位置,证实CO2和质子依懒型的蛛丝蛋白成丝新机制；3)不同于NT和CT, A. ventricosus MiSp spacer结构域不受pH值影响,维持α-helix构象不变,Tm值均为50℃左右,高温可诱导spacer蛋白由α-helix向random coil转变；A. ventricosus MiSp spacer主要为蛋白四聚体(存在少量单体构象),可拉近蛛丝蛋白分子形成复杂的蛛丝蛋白网链,增加丝纤维力学性能；NaCl可稳定spacer单体构象,但不能完全抑制蛋白四聚体的生成；A. ventricosus MiSp spacer可快速自组装并纤维化,形成肉眼可见的短小丝纤维。通过对A. ventricosus MiSp NT、CT和spacer结构域系统性的结构和功能研究,建立了蛛丝蛋白全新的成丝机理模型,为高品质蛛丝蛋白纤维的仿生提供了新的制备工艺。
部分三：实现全长蛛丝蛋白的外源定向拼接。完整的蛛丝蛋白是制备高品质蛛丝纤维的保障,目前蛛丝蛋白的外源表达均是部分基因产物或是重复模块的嵌合体,与天然蛛丝蛋白存在较大差异,仿生的单丝纤维性能远不及天然蛛丝。在过去十几年科学家们多次尝试不同的表达系统制备蛛丝蛋白,但效果均不理想。为了寻求适合全长蛛丝蛋白的制备方法,本论文首次以A. ventricosus MiSp全长编码基因为研究对象,分别尝试不同的外源表达方法制备全长蛛丝蛋白。A.ventricosus MiSp全长编码基因大、Gla/Ala使用频率高等,很难在大肠杆菌中全长表达。本论文首先1)以Rosetta2(DE3)为宿主表达A. ventricosus MiSp全长编码基因,SDS-PAGE显示MiSp全长编码基因难以表达或者表达量及其微小,而且还伴随大量翻译不完全的MiSp蛋白分子。为了寻求简单高效的全长蛛丝蛋白的制备方法,本论文2)借助Rb intein的反式剪接技术,在国内外首次尝试体外定向拼接全长A. ventricosus MiSp重组蛋白。我们将MiSp全长编码基因分成两部分,并分别与Rbn和Rbc融合表达,表达产物分别为AvMiSpNTRbn和RbcAvMiSpCT。AvMiSpNTRbn和RbcAvMiSpCT按等比例混合后,Rbn和Rbc在体外低浓度DTT诱导下相互识别并定向剪接,AvMiSpNT和AvMiSpCT随着剪接反应通过肽键连接成为完整的MiSp蛋白。Intein介导的A. ventricosus MiSp全长蛋白拼接是一种全新的合成方法,可以有效的实现全长蛛丝蛋白(高分子量)的外源制备,为后续高品质重组蛛丝纤维的仿生和应用提供技术保障。
本论文针对当前蛛丝蛋白重组制备和仿生领域的三大瓶颈问题开展系统性研究,在国内外首次克隆MiSp全长编码基因、建立全新的蛛丝蛋白CO2和质子依赖型成丝机制以及全长蛛丝蛋白外源制备新策略,为仿生新型高品质MiSp蛋白纤维奠定坚实的基础,也为其它蛛丝蛋白纤维的制备提供强有力的技术支持。
Orb-weaving spider utilize up to six different protein-based silks, each secreted from a specialized gland, and each evolved to fulfill a certain task. The minor ampullate silk, used for auxiliary spiral and prey wrapping, displays intriguing mechanical properties and excellent biocompatibility. Particularly, minor ampullate silk does not supercontract when hydrated, which thereby avoid an obvious drawback if implantation is aimed for. Minor ampullate silk is therefore considered to be a strategic resource that can be developed for applications in high-technology fields including military, aerospace and biomedical areas.
Due to the cannibalism of most spiders and small amounts of silks, large-scale production of spider silk is not possible by housing spiders at high densities, as for silkworms, which limits their utilization. Since1990scientists have tried to move the application of spider silks forward by biotechnological means, in order to exploit the outstanding fibers in modern technology. However, biomimetic spider silk with similar properties to native silk has not yet been developed, delaying the industrialization of this novel strategic resource.
Experience from the previous work shows mainly three bottlenecks in producing native-like spider silks artificially:1) Full-length gene cloning of spider silk proteins (spidroins) is limited. Genes coding for spidroins are extraordinarily large and repetitive with high GC content, and complete gene characterization by PCR and cDNA library is not practically feasible. From1990to2014, only three spidroin full-length genes have been sequenced (the second one is from this dissertation);2) Recombinant production of complete spidroins is not yet achieved. Spidroins are up to300kDa in size and pronouncedly modular (repetitive) with high GC content in the coding genes, which generally cause problems such as genetic instability and unwanted mRNA secondary structures. Also, the high frequency of Gly and Ala makes the spidroins difficult to express in heterologous hosts;3) Silk formation mechanism is still not unraveled. The extremely small inner diameter (micron-sized) complicates the measurements of the physiological and chemical microenvironments of silk secreting glands. Furthermore, spidroins are highly repetitive in sequence but capped by non-repetitive N-and C-terminal domains (NT and CT), and it is to date not known in detail what roles the terminal domains play in the silk formation process. Additionally, the structure and function of the unique spacer domains in minor ampullate spidroin (MiSp) are entirely unknown. Compared to the microenvironments. of silk glands and native silk formation mechanisms, the spinning technologies used so far are primitive.
In order to remove the three bottlenecks above for generating high-performance biomimetic spider silks, in this dissertation Araneus ventricosus MiSp was studied firstly in order to lay the foundations for preparation of artificial spider silks with superior properties:
Part one:full-length MiSp gene characterization. A. ventricosus, one of the spider species widely distributed in China, the silks of which exhibit striking performance, has been used for spidroin gene characterization. Only partial cDNA sequences for Minor ampullate silk have so far have been obtained, and recombinant expression and biomimetic silk preparation have not even been attempted. In order to enable recombinant production of MiSp and native-like minor ampullate silk spining, and also provide a new resource for research on spidroin evolution and gene regulation, I have characterized the A. ventricosus MiSp full-length gene. Based on the previous A. ventricosus fosmid gene library and STS/3D-PCR screening method, we obtained a positive clone containing the full-length A. ventricosus MiSp gene. The insert, about33kb, encompasses the full-length MiSp coding sequence as well as6647bp upstream of its start codon and14937bp downstream of its stop codon. The complete MiSp gene is composed of two exons and one unusually large intron, which is a novel finding for spidroin genes. The single intron in A. ventricosus MiSp DNA is5628bp in size and begins with the nucleotides "GT"(guanine, thymine) and ends with "AG"(adenine, guanine) and thus follows the GT-AG rule. The spliced full-length transcript of A. ventricosus MiSp coding gene is5440bp in size and encodes1766amino acid residues organized into conserved nonrepetitive N-and C-terminal domains and a central predominantly modular region (more than90%). A. ventricosus MiSp repetitive region (modular region), mainly consist of four motifs (Gly-X, Gly-Gly-X, Gly-Gly-Gly-X and poly-Ala) that are iterated in a non regular manner, and are interrupted by two nonrepetitive spacer regions (126residues), which share100%identity even at DNA level. Identification of the first full-length MiSp gene sequence reveals an unusually variable repetitive part, extremely conserved spacer regions, that the exon-intron organization differs between all so far characterized spidroin genes, and finally a new conserved element CACG for transcript regulation. Being the first full-length MiSp sequence (and the second full-length spidroin sequence), A. ventricosus MiSp full-length sequence fills a gap and provides a new gene blueprint for full-length MiSp recombinant production and biomimetic silk formation.
Part two:Structure and function of MiSp NT and CT, and silk assembly mechanism. Spider silk fibers are produced from soluble concentrated spidroins under ambient conditions in silk glands in a poorly understood complex process, in which physiological, physical and chemical conditions change progressively. Spidroins are highly repetitive in sequence but capped by non-repetitive NT (～130aa) and CT (～110aa) domains that have been suggested to regulate silk formation in similar manners (a hypothetic model from major ampullate spidroin, MaSp). In this dissertation, I show that the terminal domains from A. ventricosus MiSp respond in opposite ways to pH changes. For each spidroin, mainly the Rp region determines the mechanical properties, while NT and CT domains play very important roles in regulating the spidroin to silk transformation. Besides the three typical domains, A. ventricosus MiSp sequence contains two spacer domains of unknown function. From our recent work, the pH gradient in Nephila clavipes major ampullate gland has been determined and is found to be much broader than previously known, and the concentrations of some irons were also measured. Interestingly, carbonic anhydrase, which catalyses CO2+H2O←→HCO3-+H+, was unexpectedly found in the gland and it maintains the pH gradient. Herein, comprehensive studies of A. ventricosus MiSp NT, CT and spacer domains showed:1) MiSp NT dimerizes progressively when pH decreases form7.5to6.0, and the subsequent stabilization of NT dimers between pH6and5will result in the firm locking of spidroins into multimers in the distal part of the duct. NaCl in a physiological concentration shifts the dimerization process to lower pH. A. ventricosus MiSp NT is composed of5a-helices, Glu115makes an intramolecular salt bridge with Arg64, and cysteines located in helix1and4form an intramolecular disulfide bond. Glu84of Euprosthenops australis MaSp1NT, which is involved in the last step of dimer stabilization, lacks a counterpart in A, ventricosus MiSp NT. However, Asp109in MiSp NT is conserved, which possibly indicates a novel mechanism for MiSp NT dimerization;2) MiSp CT adopts a dimer conformation and, in sharp contrast to MiSp NT, it is gradually destabilized when pH is lowered from7.5to5.0. At pH5.5and below, CT converts to a thioflavin T (ThT) positive state, which was not observed at higher pH, or for NT at any pH tested, and NaCl delays CT (3-sheet amyloid fibril formation. Characterization of the ThT positive aggregates by TEM and Congo red staining showed typical amyloid-like fibrils, which can trigger rapid polymerization of the spidroins. Low pH and increasing HCO3-concentration implies that pCO2is elevated along duct. The CO2analogue CS2interacts with specific, mainly nonpolar, residues distributed in helices2-4of A. ventricosus MiSp CT. Similar to MiSp NT, MiSp CT monomer is also composed of5a-helices, dimerizes mainly by hydrophobic interactions, and Arg43makes an intramolecular salt bridge with Glu87. Carbonic anhydrase activity, and NT and CT opposite stability changes emerge in the same region of the gland, suggesting a novel CO2dependent trigger mechanism of spider silk formation;3) In contrast to MiSp NT and CT domains, A. ventricosus MiSp spacer domain, folds into an a-helical conformation under different pH, and shows the same Tm of～50℃between pH7and5. The main quaternary structure of A. ventricosus MiSp spacer is a tetramer (small amounts of monomers are observed), which may be a way to connect spider silk protein molecules. NaCl can promote the spacer monomer, and prohibits tetramer formation. Interestingly, MiSp spacer self-assembles into short fibers visible to the naked eye, indicating that A. ventricosus MiSp spacer domain is able to form silks and may promote formation of solid silk fibers. According to the detailed determination of A. ventrisocus MiSp NT, CT and spacer domains, a novel spider silk formation mechanism has been suggested, which may lead to novel technology for biomimetic high-performance spider silk generation.
Part three:Directional ligation of full-length MiSp. Complete spidroin is one of the decisive factors for high-performance artificial spider silk preparation, however, so far recombinant spider silk proteins are based on partial genes or a few tandem repetitive motifs. As expected, biomimetic fibers made from such spidroins are inferior to native spider silk as regards mechanical properties. Different kinds of expression hosts have been tried in recent years, but no one has been found to support the expression of complete spidroin genes. A. ventricosus MiSp coding gene is extremely large, and Gly/Ala accounts for a huge proportion, making it very difficult to express complete spidroin in Escherichia coli. Here Rosetta2(DE3) was used firstly as host to try to express A. ventricosus MiSp full-length gene, but on SDS-PAGE no full-length protein was found, but lots of truncated MiSp proteins. In order to find an efficient method for preparing complete spider silk proteins, full-length A. ventricosus MiSp proteins were firstly directionally ligated, mediated by Rb intein. Full-length MiSp coding gene was divided into two fragments, and fused with Rbn and Rbc respectively which can be expressed in E. coli. The obtained fusion proteins are AvMiSpNTRbn and RbcAvMiSpCT. AvMiSpNTRbn and RbcAvMiSpCT were mixed at room temperature at equal proportions, whereby Rbn and Rbc should recognize each other in the presence of low concentration of DTT, and undergo a trans-splicing reaction (takes several hours), in which AvMiSpNT and AvMiSpCT are ligated in vitro into full-length MiSp via a peptide bond. The directional ligation of full-length A. ventricosus MiSp recombinant proteins mediated by intein is a novel in vitro technology, and make it possible to generate complete (high molecular weight). spider silk proteins, which is very useful for generating high-performance native-like spider silk preparation.
In this dissertation, the first-ever comprehensive and systematic characterization of A. ventricosus MiSp was carried, in order to remove bottlenecks in spider silk biomimetic preparation. The first full-length MiSp coding gene identification, a novel spider silk formation mechanism dependent on CO2, and a novel directional in vitro ligation strategy for generation of complete spider silk proteins are presented. These results lay solid foundations for novel biomimetic MiSp fiber generation, and also for production of other spidroin-based fibers.
蜘蛛生性偏爱独居(同类残食),纺丝量也远少于家蚕,因此无法像高密度饲养家蚕一样大规模获取蛛丝纤维,严重阻碍了蜘蛛丝在现代科技生活中的大规模应用。自二十世纪九十年代以来,随着生物技术日趋成熟,人们开始探索通过生物技术的方法制备人造蜘蛛丝,以满足现代科技发展对高品质纤维材料的需求。多年来国内外多家研究机构专注于重组蛛丝纤维的仿生制备,但到目前为止尚未有一家机构能够仿生类似天然蛛丝的人造蛛丝纤维,严重限制这种新型战略资源的产业化。
长期研究表明,国内外蛛丝蛋白纤维的人工仿生之所以进展缓慢,主要是因为现阶段面临三大瓶颈问题：1)蛛丝蛋白全长编码基因克隆滞后：蛛丝蛋白编码基因大、重复度高以及GC含量显著等,常规PCR和小型cDNA文库技术很难钓取全长编码基因,经过近25年的克隆研究国内外仅报道三种蛛丝蛋白全长基因序列(其中第二条为本论文克隆)；2)全长编码基因外源表达难以实现：蛛丝蛋白全长编码基因大,编码的丝素蛋白达到300kDa左右；基因序列重复度高,容易出现基因重组和缺失等；蛛丝蛋白基因GC含量高,容易形成特殊的mRNA二级结构,导致核糖体脱落和翻译提前终止等；蛛丝蛋白中Gly和Ala使用频率过高,表达过程中给宿主压力太大；3)成丝机理研究不足：蛛丝蛋白分泌腺尺寸太小,腺体内环境(生理及化学条件)尚没明确；蛛丝蛋白由N端和C端非重复调节区域以及中间重复区Rp组成,它们相互协调共同赋予液态蛛丝蛋白以及固态蛛丝纤维的高度可控特性和优良品质,现阶段相关NT和CT的结构和功能还没有解析完全,Rp与丝性能的关系也不明确；次壶腹腺丝蛋白(minor ampullate spidroin, MiSp)中还包含特有的spacer间隔区,结构和功能还有待研究；由于腺体内环境以及蛛丝蛋白成丝机理研究的严重不足,致使人工纤维的仿生方法比较单一。
为系统性突破上述三个瓶颈问题,为产业化高品质重组蛛丝纤维奠定基础,本论文首次以MiSp为研究对象,主要内容包括以下三个方面：
部分一：克隆鉴定MiSp全长编码基因。大腹园蛛(Araneus ventricosus)是我国广泛分布的蜘蛛种属之一,丝纤维综合品质优异,国内外多家研究机构均在进行A. ventricosus丝蛋白编码基因的鉴定分析。次壶腹腺丝作为一种新型的蛛丝蛋白纤维,研究相对滞后,除了少量短的cDNA序列报道外,表达和仿生均无涉及。为了促进MiSp蛋白成丝机理的解析、全长MiSp蛋白重组表达、开发及仿生应用、为蛛丝蛋白的进化和表达调控研究增添新成员,本章基于前期构建的A.ventricosus大型fosmid基因组文库和STS/3D-PCR技术筛选鉴定MiSp全长编码基因序列。经过多轮筛选,我们得到含有A. ventricosus MiSp全长编码基因的阳性克隆,外源插入片段约33kb左右(GenBank accession no. JX513956),覆盖MiSp全长编码基因及其上游6647bp和下游14937bp非编码序列。A. ventricosus MiSp全长编码基因长10.9kb,由两个外显子(exon)和一个在蛛丝蛋白基因中首次发现的基因内含子(intron)组成。A. ventricosus MiSp intron长5628bp,以GT起始AG终止,符合intron的GT-AG法则。A. ventricosus MiSp全长转录子为5440bp,编码的1766个氨基酸可划分为N端和C端非重复区以及中间占到90%以上的重复区,其中重复区含有四种重复模块(由Gly-X、Gly-Gly-X、Gly-Gly-Gly-X和poly-Ala组成)和两个特有的DNA水平相似度为100%的间隔区(spacer,126个氨基酸残基)。A. ventricosus MiSp全长编码基因组成结构揭示了蛛丝蛋白编码基因intron-exon结构多样性,上下游调控序列的比对分析为进一步研究蛛丝蛋白编码基因的转录调控增添了新的保守性调控元件CACG。A. ventricosus MiSp全长编码基因为国内外首条MiSp全长基因序列(世界上第二种全长蛛丝蛋白编码基因序列),填补了国内外就A. ventricosus丝蛋白全长编码基因克隆的空白,为国内外丝蛋白全长基因的研究增添新成员。
部分二：研究MiSp结构和功能、解析组装机制和成丝机理。常温下,高浓度蛛丝蛋白经过丝腺内部一系列物理和化学变化,在极短的时间内迅速转变成性能显著的固态蛛丝纤维,这一复杂的纤维化过程目前尚无系统性阐述。蛛丝蛋白主要由氨基酸水平高度重复的重复区域构成(Rp,占到整个蛋白90%以上),Rp两端分别为N端(NT,约110氨基酸残基)和C端非重复区(CT,约130个氨基酸残基)。Rp区域主要与蛛丝纤维机械性能和品质相关,NT和CT则主要参与高浓度蛛丝蛋白储液的维持及纤维化过程中对蛛丝蛋白的多种调节作用。前期研究推断拖丝蛋白(major ampullate spidroin, MaSp) NT和CT在蛛丝蛋白成丝过程中以相似的方式发挥调节作用,本论文研究则证实次壶腹腺丝蛋白MiSp NT和CT在蛛丝蛋白纤维化过程中以相反的作用方式分别扮演不同的角色。A.ventricosus MiSp除了拥有蛛丝蛋白典型的三个结构域NT、CT和Rp外,还含有两个spacer区域。我们前期对Nephila clavipes主壶腹腺体的pH值(pH7.6-5.7,这是到目前为止测得的最宽pH范围)和多种离子种类及浓度进行测试,并发现腺体导管中含有碳酸酐酶(催化CO2+H2O←→HCO3-+H+反应维持pH梯度)。参照腺体内环境变化趋势,我们对A. ventricosus MiSp NT、CT以及spacer的功能和结构进行了系统性研究：1)随着pH从7.5降至5.0,MiSp NT逐渐二聚化且二聚体稳定性逐渐增强,连接蛛丝蛋白单体形成牢固的蛛丝蛋白多聚体,NaCl可维持单体稳定性延缓NT二聚化；pH7.5至5.0,MiSp NT二级结构维持α-helix构象不变,高温可诱导NT由α-helix向random coil可逆转变。NMR高级结构显示A. ventricosus MiSp NT单体由5个α-helix构成,Arg64和Glu115形成分子内盐键,位于α-helix1和4上的两个Cys形成分子内二硫键。与Euprosthenops australis MaSp1NT不同,A. ventricosus MiSp NT中没有参与MaSp1NT第三步二聚化的保守Glu84,但Asp109高度保守,揭示了MiSp不一样的二聚化机制；2)我们首次发现随着pH值的降低,A. ventricosus MiSp CT始终维持二聚体构象但二聚体稳定性逐渐降低(与NT相反),与MiSp NT不同,pH7.5-6.5时CT的高温变性过程可逆,二级结构由α-helix向random coil转变,pH降至5.5时高温可诱导MiSp CT由α-helix向β-sheet不可逆转变。pH≤5.5时CT结构发生改变(解折叠)形成β-sheet淀粉状(amyloid)纳米纤维(通过透射电镜和Congo染色证实),作为重复区Rp快速纤维化的晶核,NaCl对纳米纤维的形成具有抑制作用,NT不具备形成β-sheet amyloid纳米纤维的能力(pH7.5-5.0). NMR高级结构证实A. ventricosus MiSp CT单体与MiSp NT相似,由5个α-helix构成,α-helix2和4通过Arg43和Glu87形成盐键,单体之间主要依靠疏水作用二聚化,其中α-helix5申向对应单体内部形成发夹结构。导管末端pH值低以及HC03-浓度上升,因此导管末端的pC02较高,我们在分子水平证实CS2(CO2类似物,CO2难以操控以及改变pH值)会与CT内部区域保守的疏水氨基酸结合改变蛋白高级结构,促进去折叠纤维化,NT则不受CS2的影响。碳酸酐酶、NT稳定性增强以及CT稳定降低的起始部位均发生在腺体的相同位置,证实CO2和质子依懒型的蛛丝蛋白成丝新机制；3)不同于NT和CT, A. ventricosus MiSp spacer结构域不受pH值影响,维持α-helix构象不变,Tm值均为50℃左右,高温可诱导spacer蛋白由α-helix向random coil转变；A. ventricosus MiSp spacer主要为蛋白四聚体(存在少量单体构象),可拉近蛛丝蛋白分子形成复杂的蛛丝蛋白网链,增加丝纤维力学性能；NaCl可稳定spacer单体构象,但不能完全抑制蛋白四聚体的生成；A. ventricosus MiSp spacer可快速自组装并纤维化,形成肉眼可见的短小丝纤维。通过对A. ventricosus MiSp NT、CT和spacer结构域系统性的结构和功能研究,建立了蛛丝蛋白全新的成丝机理模型,为高品质蛛丝蛋白纤维的仿生提供了新的制备工艺。
部分三：实现全长蛛丝蛋白的外源定向拼接。完整的蛛丝蛋白是制备高品质蛛丝纤维的保障,目前蛛丝蛋白的外源表达均是部分基因产物或是重复模块的嵌合体,与天然蛛丝蛋白存在较大差异,仿生的单丝纤维性能远不及天然蛛丝。在过去十几年科学家们多次尝试不同的表达系统制备蛛丝蛋白,但效果均不理想。为了寻求适合全长蛛丝蛋白的制备方法,本论文首次以A. ventricosus MiSp全长编码基因为研究对象,分别尝试不同的外源表达方法制备全长蛛丝蛋白。A.ventricosus MiSp全长编码基因大、Gla/Ala使用频率高等,很难在大肠杆菌中全长表达。本论文首先1)以Rosetta2(DE3)为宿主表达A. ventricosus MiSp全长编码基因,SDS-PAGE显示MiSp全长编码基因难以表达或者表达量及其微小,而且还伴随大量翻译不完全的MiSp蛋白分子。为了寻求简单高效的全长蛛丝蛋白的制备方法,本论文2)借助Rb intein的反式剪接技术,在国内外首次尝试体外定向拼接全长A. ventricosus MiSp重组蛋白。我们将MiSp全长编码基因分成两部分,并分别与Rbn和Rbc融合表达,表达产物分别为AvMiSpNTRbn和RbcAvMiSpCT。AvMiSpNTRbn和RbcAvMiSpCT按等比例混合后,Rbn和Rbc在体外低浓度DTT诱导下相互识别并定向剪接,AvMiSpNT和AvMiSpCT随着剪接反应通过肽键连接成为完整的MiSp蛋白。Intein介导的A. ventricosus MiSp全长蛋白拼接是一种全新的合成方法,可以有效的实现全长蛛丝蛋白(高分子量)的外源制备,为后续高品质重组蛛丝纤维的仿生和应用提供技术保障。
本论文针对当前蛛丝蛋白重组制备和仿生领域的三大瓶颈问题开展系统性研究,在国内外首次克隆MiSp全长编码基因、建立全新的蛛丝蛋白CO2和质子依赖型成丝机制以及全长蛛丝蛋白外源制备新策略,为仿生新型高品质MiSp蛋白纤维奠定坚实的基础,也为其它蛛丝蛋白纤维的制备提供强有力的技术支持。
Orb-weaving spider utilize up to six different protein-based silks, each secreted from a specialized gland, and each evolved to fulfill a certain task. The minor ampullate silk, used for auxiliary spiral and prey wrapping, displays intriguing mechanical properties and excellent biocompatibility. Particularly, minor ampullate silk does not supercontract when hydrated, which thereby avoid an obvious drawback if implantation is aimed for. Minor ampullate silk is therefore considered to be a strategic resource that can be developed for applications in high-technology fields including military, aerospace and biomedical areas.
Due to the cannibalism of most spiders and small amounts of silks, large-scale production of spider silk is not possible by housing spiders at high densities, as for silkworms, which limits their utilization. Since1990scientists have tried to move the application of spider silks forward by biotechnological means, in order to exploit the outstanding fibers in modern technology. However, biomimetic spider silk with similar properties to native silk has not yet been developed, delaying the industrialization of this novel strategic resource.
Experience from the previous work shows mainly three bottlenecks in producing native-like spider silks artificially:1) Full-length gene cloning of spider silk proteins (spidroins) is limited. Genes coding for spidroins are extraordinarily large and repetitive with high GC content, and complete gene characterization by PCR and cDNA library is not practically feasible. From1990to2014, only three spidroin full-length genes have been sequenced (the second one is from this dissertation);2) Recombinant production of complete spidroins is not yet achieved. Spidroins are up to300kDa in size and pronouncedly modular (repetitive) with high GC content in the coding genes, which generally cause problems such as genetic instability and unwanted mRNA secondary structures. Also, the high frequency of Gly and Ala makes the spidroins difficult to express in heterologous hosts;3) Silk formation mechanism is still not unraveled. The extremely small inner diameter (micron-sized) complicates the measurements of the physiological and chemical microenvironments of silk secreting glands. Furthermore, spidroins are highly repetitive in sequence but capped by non-repetitive N-and C-terminal domains (NT and CT), and it is to date not known in detail what roles the terminal domains play in the silk formation process. Additionally, the structure and function of the unique spacer domains in minor ampullate spidroin (MiSp) are entirely unknown. Compared to the microenvironments. of silk glands and native silk formation mechanisms, the spinning technologies used so far are primitive.
In order to remove the three bottlenecks above for generating high-performance biomimetic spider silks, in this dissertation Araneus ventricosus MiSp was studied firstly in order to lay the foundations for preparation of artificial spider silks with superior properties:
Part one:full-length MiSp gene characterization. A. ventricosus, one of the spider species widely distributed in China, the silks of which exhibit striking performance, has been used for spidroin gene characterization. Only partial cDNA sequences for Minor ampullate silk have so far have been obtained, and recombinant expression and biomimetic silk preparation have not even been attempted. In order to enable recombinant production of MiSp and native-like minor ampullate silk spining, and also provide a new resource for research on spidroin evolution and gene regulation, I have characterized the A. ventricosus MiSp full-length gene. Based on the previous A. ventricosus fosmid gene library and STS/3D-PCR screening method, we obtained a positive clone containing the full-length A. ventricosus MiSp gene. The insert, about33kb, encompasses the full-length MiSp coding sequence as well as6647bp upstream of its start codon and14937bp downstream of its stop codon. The complete MiSp gene is composed of two exons and one unusually large intron, which is a novel finding for spidroin genes. The single intron in A. ventricosus MiSp DNA is5628bp in size and begins with the nucleotides "GT"(guanine, thymine) and ends with "AG"(adenine, guanine) and thus follows the GT-AG rule. The spliced full-length transcript of A. ventricosus MiSp coding gene is5440bp in size and encodes1766amino acid residues organized into conserved nonrepetitive N-and C-terminal domains and a central predominantly modular region (more than90%). A. ventricosus MiSp repetitive region (modular region), mainly consist of four motifs (Gly-X, Gly-Gly-X, Gly-Gly-Gly-X and poly-Ala) that are iterated in a non regular manner, and are interrupted by two nonrepetitive spacer regions (126residues), which share100%identity even at DNA level. Identification of the first full-length MiSp gene sequence reveals an unusually variable repetitive part, extremely conserved spacer regions, that the exon-intron organization differs between all so far characterized spidroin genes, and finally a new conserved element CACG for transcript regulation. Being the first full-length MiSp sequence (and the second full-length spidroin sequence), A. ventricosus MiSp full-length sequence fills a gap and provides a new gene blueprint for full-length MiSp recombinant production and biomimetic silk formation.
Part two:Structure and function of MiSp NT and CT, and silk assembly mechanism. Spider silk fibers are produced from soluble concentrated spidroins under ambient conditions in silk glands in a poorly understood complex process, in which physiological, physical and chemical conditions change progressively. Spidroins are highly repetitive in sequence but capped by non-repetitive NT (～130aa) and CT (～110aa) domains that have been suggested to regulate silk formation in similar manners (a hypothetic model from major ampullate spidroin, MaSp). In this dissertation, I show that the terminal domains from A. ventricosus MiSp respond in opposite ways to pH changes. For each spidroin, mainly the Rp region determines the mechanical properties, while NT and CT domains play very important roles in regulating the spidroin to silk transformation. Besides the three typical domains, A. ventricosus MiSp sequence contains two spacer domains of unknown function. From our recent work, the pH gradient in Nephila clavipes major ampullate gland has been determined and is found to be much broader than previously known, and the concentrations of some irons were also measured. Interestingly, carbonic anhydrase, which catalyses CO2+H2O←→HCO3-+H+, was unexpectedly found in the gland and it maintains the pH gradient. Herein, comprehensive studies of A. ventricosus MiSp NT, CT and spacer domains showed:1) MiSp NT dimerizes progressively when pH decreases form7.5to6.0, and the subsequent stabilization of NT dimers between pH6and5will result in the firm locking of spidroins into multimers in the distal part of the duct. NaCl in a physiological concentration shifts the dimerization process to lower pH. A. ventricosus MiSp NT is composed of5a-helices, Glu115makes an intramolecular salt bridge with Arg64, and cysteines located in helix1and4form an intramolecular disulfide bond. Glu84of Euprosthenops australis MaSp1NT, which is involved in the last step of dimer stabilization, lacks a counterpart in A, ventricosus MiSp NT. However, Asp109in MiSp NT is conserved, which possibly indicates a novel mechanism for MiSp NT dimerization;2) MiSp CT adopts a dimer conformation and, in sharp contrast to MiSp NT, it is gradually destabilized when pH is lowered from7.5to5.0. At pH5.5and below, CT converts to a thioflavin T (ThT) positive state, which was not observed at higher pH, or for NT at any pH tested, and NaCl delays CT (3-sheet amyloid fibril formation. Characterization of the ThT positive aggregates by TEM and Congo red staining showed typical amyloid-like fibrils, which can trigger rapid polymerization of the spidroins. Low pH and increasing HCO3-concentration implies that pCO2is elevated along duct. The CO2analogue CS2interacts with specific, mainly nonpolar, residues distributed in helices2-4of A. ventricosus MiSp CT. Similar to MiSp NT, MiSp CT monomer is also composed of5a-helices, dimerizes mainly by hydrophobic interactions, and Arg43makes an intramolecular salt bridge with Glu87. Carbonic anhydrase activity, and NT and CT opposite stability changes emerge in the same region of the gland, suggesting a novel CO2dependent trigger mechanism of spider silk formation;3) In contrast to MiSp NT and CT domains, A. ventricosus MiSp spacer domain, folds into an a-helical conformation under different pH, and shows the same Tm of～50℃between pH7and5. The main quaternary structure of A. ventricosus MiSp spacer is a tetramer (small amounts of monomers are observed), which may be a way to connect spider silk protein molecules. NaCl can promote the spacer monomer, and prohibits tetramer formation. Interestingly, MiSp spacer self-assembles into short fibers visible to the naked eye, indicating that A. ventricosus MiSp spacer domain is able to form silks and may promote formation of solid silk fibers. According to the detailed determination of A. ventrisocus MiSp NT, CT and spacer domains, a novel spider silk formation mechanism has been suggested, which may lead to novel technology for biomimetic high-performance spider silk generation.
Part three:Directional ligation of full-length MiSp. Complete spidroin is one of the decisive factors for high-performance artificial spider silk preparation, however, so far recombinant spider silk proteins are based on partial genes or a few tandem repetitive motifs. As expected, biomimetic fibers made from such spidroins are inferior to native spider silk as regards mechanical properties. Different kinds of expression hosts have been tried in recent years, but no one has been found to support the expression of complete spidroin genes. A. ventricosus MiSp coding gene is extremely large, and Gly/Ala accounts for a huge proportion, making it very difficult to express complete spidroin in Escherichia coli. Here Rosetta2(DE3) was used firstly as host to try to express A. ventricosus MiSp full-length gene, but on SDS-PAGE no full-length protein was found, but lots of truncated MiSp proteins. In order to find an efficient method for preparing complete spider silk proteins, full-length A. ventricosus MiSp proteins were firstly directionally ligated, mediated by Rb intein. Full-length MiSp coding gene was divided into two fragments, and fused with Rbn and Rbc respectively which can be expressed in E. coli. The obtained fusion proteins are AvMiSpNTRbn and RbcAvMiSpCT. AvMiSpNTRbn and RbcAvMiSpCT were mixed at room temperature at equal proportions, whereby Rbn and Rbc should recognize each other in the presence of low concentration of DTT, and undergo a trans-splicing reaction (takes several hours), in which AvMiSpNT and AvMiSpCT are ligated in vitro into full-length MiSp via a peptide bond. The directional ligation of full-length A. ventricosus MiSp recombinant proteins mediated by intein is a novel in vitro technology, and make it possible to generate complete (high molecular weight). spider silk proteins, which is very useful for generating high-performance native-like spider silk preparation.
In this dissertation, the first-ever comprehensive and systematic characterization of A. ventricosus MiSp was carried, in order to remove bottlenecks in spider silk biomimetic preparation. The first full-length MiSp coding gene identification, a novel spider silk formation mechanism dependent on CO2, and a novel directional in vitro ligation strategy for generation of complete spider silk proteins are presented. These results lay solid foundations for novel biomimetic MiSp fiber generation, and also for production of other spidroin-based fibers.
引文
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