厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析

贝壳是一种具有优异力学性能的生物硬组织,贝壳基质蛋白质对贝壳的形成具有重要意义。厚壳贻贝(Mytilus coruscus)贝壳中发现一种类似胶原蛋白质的新型贝壳基质蛋白质,命名为collagen-like protein 2（CLP-2）。然而,该蛋白质的结构与功能以及对贝壳形成的影响机制尚不清楚。为此,本研究对CLP 2开展了序列分析;进一步采取密码子优化结合原核重组表达策略,开展了CLP-2的重组表达;在此基础上分析了重组CLP-2对酸钙结晶的诱导、结晶速率抑制以及碳酸钙结合能力。对CLP-2的序列分析结果表明,该蛋白质序列中含有信号肽及两个Von Willebrand factor A（VWA）结构域。CLP-2在数据库中尚无高同源性蛋白质存在,表明这是一种较为新颖的贝壳基质蛋白。所获得的重组CLP-2对碳酸钙体外结晶表现出明显的诱导作用,扫描电镜以及傅里叶红外光谱结果表明,重组CLP-2可诱导碳酸钙晶体的形貌由立方体形转化为球形,并在高浓度下进一步转化为哑铃形;同时,重组CLP-2可促使碳酸钙晶体的晶型由方解石型向文石型转化;重组CLP-2在体外具有碳酸钙晶体结合作用;此外,重组CLP-2能显著抑制碳酸钙晶体的结晶速度（P<0.01）,并具有浓度依赖性。上述结果表明,厚壳贻贝贝壳CLP-2蛋白质在贝壳,特别是文石型肌棱柱层的生物矿化过程中具有重要作用。上述研究为深入了解贻贝贝壳的形成机制,以及胶原类蛋白质对生物矿化过程的影响奠定了基础。     

利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

Characterization and protection on acute liver injury of a polysaccharide MP-I from Mytilus coruscus

In this study, we analyzed a water-soluble polysaccharide MP-I isolated from Mytilus coruscus. MP-I was obtained by hot-water extraction, anion-exchange and gel-permeation chromatography. Complete hydrolysis, periodate oxidation, methylation analysis, as well as Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy were conducted to elucidate its structure. MP-I was subjected to investigate the protective effect on carbon tetrachloride (CCl(4)) induced liver damage in male Kunming mice. Based on the data obtained, MP-I was found to be an alpha-(1-->4)-D-glucan, branched with a single alpha-D-glucose at the C-6 position every eight residue, on average, along the main chain. Based on the calibration with Dextran, the glucan had a molecular weight of about 1.35 x 10(6) Da. Pharmacological studies revealed that MP-I could decrease serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and hepatic malondialdehyde aldehydes (MDA) levels, increase the hepatic total superoxide dismutase (T-SOD) activity, and improve hepatic damage in the CCl(4) induced liver injury in mice in a dose-dependent manner. The results suggest that the possible mechanism is due to its antioxidant activity of MP-I.     

厚壳贻贝足丝盘黏附蛋白mcofp-3的重组真核表达

厚壳贻贝(Mytilus coruscus)黏附蛋白分子mcofp-3(M.coruscusfoot protein-3)主要分布于贻贝足丝盘,贻贝在水环境下的黏附过程中起到关键作用,但因其难溶于水且在贻贝足丝盘中含量极低,故妨碍了对其进行深入研究。为建立厚壳贻贝足丝蛋白mcofp-3的真核表达体系,并获得足够的mcofp-3黏附蛋白进行后续研究,采用酵母表达体系对mcofp-3进行了重组表达。通过PCR方法克隆厚壳贻贝的mcofp-3基因,构建mcofp-3的酵母真核表达载体pVT102U/α/mcofp-3,鉴定结果表明,重组表达质粒pVT102U/α/mcofp-3由真核载体pVT102U/α和mcofp-3的成熟肽DNA片段组成,插入的mcofp-3成熟肽DNA片段与预期序列完全一致;采用LiAC转化法将重组表达质粒转化到S78酿酒酵母中,经过RT-PCR分析以及1.0%的琼脂糖凝胶电泳检测,结果表明,重组的mcofp-3得到了成功的转录;发酵菌液经阳离子交换柱及高效液相色谱分离,以及Tris-Tricine-SDS-PAGE检测,结果表明,重组的厚壳贻贝黏附蛋白分子mcofp-3得到了成功表达,表达...     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

厚壳贻贝Mytilus coruscus足丝盘及足丝的多巴分析

多巴(3,4-1-dihydroxyphenylalanine,DOPA)是贻贝足丝粘附蛋白中的一种特殊的氨基酸,由酪氨酸经羟化后生成,与贻贝足丝粘附蛋白的强粘附性能具有直接联系.目前,已鉴定的多种贻贝足丝蛋白序列中均发现有不同含量的DOPA存在.蛋白中DOPA的定量检测对于了解DOPA在蛋白粘附中的作用以及粘附蛋白的...     

贻贝酶解降压肽的降压活性及其安全性评价

以贻贝为原料制取降压肽,探讨了不同酶解条件对其ACE抑制活性的影响,以及不同活性修饰方法对其降血压活性的影响。以SHR饲喂试验分析其降血压活性,利用小鼠急性经口毒性试验对其安全性进行了评价。结果表明,以MA1修饰的2#降压肽降压效果较好,其在2~6 h之内均有显著降压效果,平均降低为16~28mmHg。在6 h处降压效果达到最大值为28 mmHg。后经毒理试验表明其安全无毒。此外利用高效液相色谱法对样品分子量进行了分析,发现2#降压肽的分子量在1700 Dal以下,十肽以下的短肽含量较高,约占50.4%,二三肽类物质含量为7.96%,对其降压活性影响显著。     

厚壳贻贝养殖群体与野生群体线粒体DNA的遗传分析(英文)

采川线粒体DNA COI基因序列对厚壳贻贝2个养殖群体与2个野生群体的遗传多样性进行了研究.4个群体共获得30个单倍型.结果显示:在养殖群体中单倍型的数量和遗传多样性要比野生群体的低,可能是由于只有少量的有效父母本对养殖群体的遗传变异有贡献所致.养贿群体与当地野生群体之间也未发生显著的遗传分化,可能足因为它们之间存在基因流.在今后厚壳贻贝养殖过程中,本研究可以用在对养殖群体进行遗传监测,从而保证养殖群体的遗传多样性水平.     

利用蛋白质组学手段鉴定新型贻贝足丝蛋白

贻贝通过足腺分泌特有的足丝并以此粘附于水下各种基质表面.贻贝足丝中富含各种粘附蛋白,其优异的水下粘附性能使其成为开发新型生物粘合剂的候选分子.厚壳贻贝足丝粘附能力强,本文采用尿素及盐酸胍抽提结合二维双向电泳技术（two-dimensional electrophoresis, 2-DE）,分别对厚壳贻贝足丝纤维和足丝盘的蛋白质进行分离及染色;采用串联质谱技术结合常规搜库和表达序列标签（EST) 数据库搜索,对分离获得的蛋白质点进行鉴定,从中获得了mfp-3、mfp-6、胶原蛋白以及3种未曾报道过的新型贻贝足丝蛋白成分.上述研究为深入了解厚壳贻贝足丝蛋白的分子多样性、探讨其粘附机理以及从中筛选具有应用前景的贻贝足丝蛋白奠定了基础.     

厚壳贻贝养殖群体与野生群体线粒体DNA的遗传分析(英文）

采用线粒体DNA COI基因序列对厚壳贻贝2个养殖群体与2个野生群体的遗传多样性进行了研究。4个群体共获得30个单倍型。结果显示：在养殖群体中单倍型的数量和遗传多样性要比野生群体的低,可能是由于只有少量的有效父母本对养殖群体的遗传变异有贡献所致。养殖群体与当地野生群体之间也未发生显著的遗传分化,可能是因为它们之间存在基因流。在今后厚壳贻贝养殖过程中,本研究可以用在对养殖群体进行遗传监测,从而保证养殖群体的遗传多样性水平。     

厚壳贻贝足丝黏附蛋白mfp-3的重组表达及黏附功能分析

厚壳贻贝（Mytilus coruscus）中富含各种黏附蛋白分子,其中贻贝足丝蛋白3（mussel foot protein-3, mfp-3）是贻贝用以与外界基质进行黏附的主要蛋白分子.贻贝足丝中天然的mfp-3的含量低,水溶性差,因此纯化困难.本文以厚壳贻贝足丝蛋白mfp-3的cDNA序列为目的基因,用PCR法扩增Mfp-3基因,并成功构建含有多聚组氨酸标签的重组mfp-3原核表达载体pET-21a/ Mfp-3.经IPTG（isopropylthio-β-D-galactoside）诱导表达出重组蛋白,利用亲和层析和反相高效液相色谱分离纯化,获得分子量为9.18 kD的重组蛋白.经酪氨酸酶催化、玻璃包被和石英晶体微天平（quartz crystal microbalance,QCM）分析.结果表明,重组厚壳贻贝mfp-3蛋白经酪氨酸酶催化后,L-3,4-二羟基苯丙氨酸(即多巴,L-3,4- dihydroxyphenylalanine, DOPA) 含量较高并且具有较好的黏附性能.上述研究为开发以mfp-3黏附蛋白为来源的生物粘合剂奠定了良好的基础.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

贝壳的有机质及生物矿化机制分析

以腹足纲贝壳香螺壳为研究对象,用弱酸去钙法进行蛋白提取,采用280纳米(A280)光吸收法测定蛋白含量,并通过聚丙烯酰胺(SDS-PAGE)凝胶电泳法对蛋白按照分子量大小的区别进行分离.实验结果表明香螺壳中蛋白含量和种类较少,其文石层比方解石层蛋白含量高的多,总量分别为0.89%和0.0533%;文石层分离出五种分子量的可溶性蛋白和四种分子量的不可溶性蛋白;而方解石层中分离出三种分子量的可溶性蛋白和三种分子量的不可溶性蛋白,且分子量不相同.正是这少量的蛋白质对贝壳的生物矿化过程和不同晶型的形成起着决定性作用.     

Distribution and Function of the Nacrein-Related Proteins Inferred from Structural Analysis

Nacrein is the first identified molluscan organic matrix (OM) component considered to be specifically involved in nacreous layer formation (Miyamoto et al. in Proc Natl Acad Sci USA 93:9657–9660, 1996); however, its localization in shell microstructures and phylogeny of molluscs and function still remain unclear. Therefore, to elucidate the functions of the nacrein-related proteins, we set up three experiments focused on (1) the primary structure of the nacrein-related proteins, (2) the tertiary structure of nacrein, and (3) in vitro crystallization of the proteins. In regard to the first experiment, our Western blot analysis and cDNA cloning clearly indicated for the first time the common occurrence of nacrein-related proteins both biochemically and genetically, independent of molluscan phylogeny and shell microstructures. Together with the data reported so far, we classified nacrein-related proteins into four types. Second, we determined the overall structure of nacrein via small-angle x-ray scattering via the program DAMMIN. This kind of research has never yet been attempted for the molluscan OM proteins. Our results inferred the structure of nacrein to be N-shaped based on the low-resolution solution dummy atom model structures that could be derived from the presence of the NG-repeat domain that was intercalated into two CA domains. Third, the result of the crystallization experiment revealed inhibitory activity of crystal formation for nacrein-related proteins when present in free state but the same molecule, when attached to the ISM, may regulate the form and size of aragonite crystal. These results demonstrate the fundamentally important function of nacrein-related proteins in molluscan shell formation. The nucleotide sequences reported in this article have been submitted to GenBank^TK/DDBJ with accession numbers AB252479 to AB252484.     

Characterization of a type I regulatory subunit of cAMP-dependent protein kinase from the bivalve mollusk Mytilus galloprovincialis

Two isoforms of the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), had been purified in our laboratory from two different tissues of the sea mussel Mytilus galloprovincialis. In this paper, we report the sequences of several peptides obtained from tryptic digestion of R(myt1). As a whole, these sequences showed high homology with regions of type I R subunits from invertebrate and also from mammalian sources, but homology with those of fungal and type II R subunits was much lower, which indicates that R(myt1) can be considered as a type I R isoform. This conclusion is also supported by the following biochemical properties: (1) R(myt1) was proved to have interchain disulfide bonds stabilizing its dimeric structure; (2) it failed to be phosphorylated by the catalytic (C) subunit purified from mussel; (3) it has a higher pI value than that of the R(myt2) isoform; and (4) it showed cross-reactivity with mammalian anti-RIbeta antibody.     

Purification of a novel nitric oxide inhibitory peptide derived from enzymatic hydrolysates of Mytilus coruscus

Shellfish contain significant levels of high quality protein and are therefore a potential source for biofunctional high-value peptides. To purify a novel anti-inflammatory peptide from Mytilus coruscus (M. coruscus), we applied enzymatic hydrolysis and tangential flow filtration (TFF) and investigated its nitric oxide inhibitory property. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Flavouzyme hydrolysates, which showed clearly superior nitric oxide inhibitory activity on lipopolysaccharide (LPS)-stimulated RAW264.7, were further purified using a TFF system and consecutive chromatographic methods. Finally, a novel anti-inflammatory peptide composed of 10 amino acid residues was obtained, and the sequence was identified as Gly-Val-Ser-Leu-Leu-Gln-Gln-Phe-Phe-Leu at N-terminal position. The peptide from M. coruscus effectively inhibited nitric oxide production on macrophage cells. This is the first report of an anti-inflammatory peptide derived from the hydrolysates of M. coruscus.     

Multi-domain protein families and domain pairs: comparison with known structures and a random model of domain recombination

There is a limited repertoire of domain families in nature that are duplicated and combined in different ways to form the set of proteins in a genome. Most proteins in both prokaryote and eukaryote genomes consist of two or more domains, and we show that the family size distribution of multi-domain protein families follows a power law like that of individual families. Most domain pairs occur in four to six different domain architectures: in isolation and in combinations with different partners. We showed previously that within the set of all pairwise domain combinations, most small and medium-sized families are observed in combination with one or two other families, while a few large families are very versatile and combine with many different partners. Though this may appear to be a stochastic pattern, in which large families have more combination partners by virtue of their size, we establish here that all the domain families with more than three members in genomes are duplicated more frequently than would be expected by chance considering their number of neighbouring domains. This duplication of domain pairs is statistically significant for between one and three quarters of all families with seven or more members. For the majority of pairwise domain combinations, there is no known three-dimensional structure of the two domains together, and we term these novel combinations. Novel domain combinations are interesting and important targets for structural elucidation, as the geometry and interaction between the domains will help understand the function and evolution of multi-domain proteins. Of particular interest are those combinations that occur in the largest number of multi-domain proteins, and several of these frequent novel combinations contain DNA-binding domains.Abbreviations:SCOP: Structural Classification of Proteins database, PDB: Protein DataBank, HMM: hidden Markov model