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.     

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.     

厚壳贻贝低分子量足丝蛋白的初步鉴定(英文)

足丝蛋白是贻贝科(Mytilidae)所特有一种在水环境中也能表现出强黏附功能的蛋白,也是目前开发新型生物黏附剂的主要候选分子。厚壳贻贝(Mytilus coruscus)广泛分布于我国东部沿海,是我国具有重要经济价值的贻贝,其足丝粗硬,黏附力强,关于厚壳贻贝的足丝蛋白的研究目前尚未见报道。通过醋酸抽提结合反相高效液相色谱分离,从厚壳贻贝足丝盘中分离纯化到数种低分子量足丝蛋白,经质谱鉴定和氨基酸序列测定,其中三种足丝蛋白(分子量6 kD左右)属于贻贝足丝蛋白-3(mytilus foot protein-3,mfp-3)家族,且序列中富含DOPA,另有三种足丝蛋白为未知新型足丝蛋白。石英晶体微天平分析表明,厚壳贻贝低分子量足丝蛋白在金表面有较强的吸附能力,这与其黏附功能是直接相关的。以上工作为深入了解厚壳贻贝低分子量足丝蛋白的分子多样性以及黏附机制奠定了基础。     

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．     

一种新型贻贝足丝抗氧化蛋白的原核重组表达及功能

贻贝足丝及其足丝蛋白相关研究对于开发新型水下生物粘附剂具有重要的仿生学意义。足丝蛋白在其粘附过程中需要维持一定的还原态,而目前已报道的足丝抗氧化蛋白仅有MFP-6。此前在厚壳贻贝足丝中鉴定到一种新型的富含半胱氨酸和甘氨酸的足丝蛋白质,该蛋白质被命名为Cys/Gly-Rich-Protein（CGRP）,但是CGRP蛋白在足丝中的作用及机制尚不明确。为此,针对CGRP蛋白,在序列分析基础上,利用原核重组表达手段获得其重组蛋白质,采用2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picryl hydrazyl radical,DPPH）法检测CGRP重组蛋白经不同条件处理后的抗氧化活性。序列分析结果表明,CGRP蛋白含16.5%的半胱氨酸和10%的甘氨酸,其序列中含有两段半胱氨酸位置保守的重复序列,结构预测表明,其优势构象以无规卷曲为主。同源蛋白质搜索结果表明,CGRP蛋白在数据库中尚无高同源性蛋白质存在。通过密码子优化结合原核重组表达策略成功表达出CGRP重组蛋白,所获得的CGRP重组蛋白具有明显的抗氧化活性,且该活性在其半胱氨酸还原后显著增强(0.91±0.05 vs 0.71±0.11, P<0.01)而在半胱氨酸烷基化之后显著下降(0.08±0.03 vs 0.71±0.11, P<0.01),表明CGRP蛋白的抗氧化活性与其序列中半胱氨酸的自由巯基有关。本研究提示,CGRP蛋白是足丝中一种新的具有抗氧化功能的蛋白质,在足丝粘附过程中推测与MFP-6一起参与了富含多巴的足丝粘附蛋白的还原态维持,对贻贝足丝在固化和粘附过程中防止提前粘附具有重要意义。     

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...     

一种新型贻贝抗菌肽的分离纯化及鉴定

厚壳贻贝（Mytilus coruscus）广泛分布于我国东部海域,其体内富含各种抗菌肽分子,是研究软体动物免疫防御机制以及开发抗菌肽来源的新型生物抗生素的重要对象。采用多步反相高效液相色谱对厚壳贻贝血清进行分离纯化,获得一种分子量为6261.55 D的具有抗菌活性的多肽成分;经多肽N端测序和基因克隆,结果表明该抗菌肽由55个氨基酸残基构成,含6个半胱氨酸并形成三对二硫键。结构域分析表明该抗菌肽具有几丁质结合结构域（Chitin-biding domain）,因此将该抗菌肽命名为mytichitin-A。Mytichitin-A对革兰氏阳性菌具有较强的抑制作用,同时对真菌及革兰氏阴性菌也具有抑制作用。荧光定量PCR检测表明,mytichitin-A主要在厚壳贻贝的性腺组织中表达且在细菌诱导后12h其表达量达到峰值。研究为深入了解厚壳贻贝抗菌肽的分子多样性及免疫机制奠定了基础。       

重组贻贝足蛋白的研究进展与应用*

贻贝足蛋白是一类通过贻贝足腺分泌的蛋白质复合物,与基质表面发生反应而产生极强的黏附作用。其在海洋环境中具有强黏附能力、可降解性和优秀的生物相容性等优点,因此常被用做生物医药黏合剂。但提取天然蛋白质受原料来源限制,且工艺烦琐导致价格高昂,阻碍了贻贝足蛋白的进一步应用发展。微生物合成的最新进展为贻贝足蛋白的产出提供了一种新思路,并且具有扩大规模生产的意义。主要综述了贻贝足蛋白的基因工程生产方法,总结了重组蛋白在黏附抗污涂层、组织工程材料等领域的应用现状。同时对其研究方向进行了展望,指出重组贻贝足蛋白的进一步发展的关键技术是解析蛋白质的构效和层级结构,在此基础上提高其异源表达水平,以获得更多生物功效性的衍生产品。     

重组贻贝足蛋白的研究进展与应用*

贻贝足蛋白是一类通过贻贝足腺分泌的蛋白质复合物,与基质表面发生反应而产生极强的黏附作用。其在海洋环境中具有强黏附能力、可降解性和优秀的生物相容性等优点,因此常被用做生物医药黏合剂。但提取天然蛋白质受原料来源限制,且工艺烦琐导致价格高昂,阻碍了贻贝足蛋白的进一步应用发展。微生物合成的最新进展为贻贝足蛋白的产出提供了一种新思路,并且具有扩大规模生产的意义。主要综述了贻贝足蛋白的基因工程生产方法,总结了重组蛋白在黏附抗污涂层、组织工程材料等领域的应用现状。同时对其研究方向进行了展望,指出重组贻贝足蛋白的进一步发展的关键技术是解析蛋白质的构效和层级结构,在此基础上提高其异源表达水平,以获得更多生物功效性的衍生产品。     

Novel Proteins Identified in the Insoluble Byssal Matrix of the Freshwater Zebra Mussel

The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.     

Location and analysis of byssal structural proteins of Mytilus edulis

The acellular attachment organ (byssus) of the marine mussel Mytilus edulis L. is composed of threads that emanate from the body of the mussel to adhesive discs that anchor the threads to rocks, sand and other mussels. Three proteins have been purified by immunohistological methods and located to specific regions of the byssus. A collagenous protein with subunit molecular weights of 53,000, 55,000 and 65,000 is found in the matrix of the elastic thread region. Its 73,000-MW precursor was extracted from foot glands in the area proximal to the animal body and was identified by immune cross-reactivity. A cystine-rich, acidic protein was found in all regions of the byssus associated with a third protein, the polyphenolic protein. The L-dopa-containing polyphenolic protein appears in the cortex of the entire thread and adhesive plaque and at the substrate-plaque interface. Antiserum to this protein stains spherical vesicles in the phenol gland of the foot. Using immuno-electrophoretic methods, the polyphenolic protein and the cystine-rich protein were shown to form high molecular weight aggregates with aging of the byssus.     

Investigation of the loss of byssus in Mytilus galloprovincialis from mussel farms in the Adriatic Sea

A fungal infection has been found in the mussel Mytilus galloprovincialis from Adriatic Sea mussel farms. The infection ultimately results in the loss of the byssus, with serious consequences for mussel farming yield. The pathogen provokes the progressive destruction of the foot muscles, also damaging related structures such as the intra-organism part of the byssus apparatus, resulting in loss of the thread component. The affected health status of the animal is also sustained by modifications in the digestive gland structure, ranging from hyperactivity to extreme cell death in the tubula. At present, the identity of the harmful fungus is unknown.     

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.     

Application of proteomics for fast identification of species-specific peptides from marine species

In this work, a novel approach based on proteomics is applied for the analysis of the three European marine mussel species: Mytilus edulis (ME), Mytilus galloprovincialis (MG) and Mytilus trossulus (MT), which are of interest in biotechnology and food industry. The proteomes of these species are poorly described in databases, are difficult to diagnose, and have a controversial taxonomy, To characterise species-specific peptides, we compared 51 matrix-assisted laser desorption/ioization-time of flight peptide mass maps generated from 6 random selected prominent spots derived from the two-dimensional electrophoresis analysis of foot protein extracts from several individuals. Minor species-specific differences in the peptide maps were detected in only one of the spots, corresponding to tropomyosin. Two peptides were unique to ME and MG individuals, whereas another peptide was present only in MT individuals. The sequence of these peptides was characterised by, nanoelectrospray ionization-ion trap (nanoESI-IT) tandem mass spectrometry (MS/MS) analysis followed by database searching and de novo sequence interpretation. We detected a single T to D amino acid substitution in MT tropomyosin. Unambiguous and highly-specific species identification was then demonstrated by analysing peptide extracts from tropomyosin spots by micro high-performande liquid chromatography (microHPL) ESI-IT mass spectrometry using the selected ion monitoring configuration, focused on these peptides, in continuous MS/MS operation. Our results suggest that proteomics may be successfully applied for the identification of species whose proteome is not present in databases.     

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7-7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.     

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7–7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.