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.     

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.     

海洋贻贝粘附蛋白类的结构与功能

海洋贻贝粘附蛋白具有高强度、高韧性和防水性,以及极强的黏附基体的功能,这与其特殊的分子结构、多巴(DOPA)介导的链间交联和与底材之间的相互作用方式有关,并且,它还具有很好的生物相容性和可降解性,是一类极具优势和潜力的生物胶黏剂.本文主要就粘附蛋白分子的结构和功能、粘附蛋白的粘附机理以及有关粘附蛋白生物粘剂等问题对其进行综述     

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...     

贻贝足腺细胞的超强度粘液

贻贝足腺细胞能分泌出超强度粘液 (ｓｕ ｐｅｒｇｌｕｅ) ,该粘液遇海水后即变成贝壳素性质的足丝 ,用以固着在附着物上。因其粘合力及防水性能极强 ,是现有任何粘合剂均无法比拟的 ,故而引起了学者们的极大兴趣[1,2 ] 。贻贝的超强度粘液具有非常优秀的抗水性能 ,且凝固速度极快 ,因此也可被用来制作水下密封胶、防腐剂和粘合剂 ,如在国防和海洋工程领域可直接用作船舶、潜艇和海水养殖设备的防水防附剂和粘结剂 ,可以替代螺丝和铆焊等常规固定手段。此外 ,贻贝足腺细胞所分泌的粘液蛋白对细胞没有毒害作用 ,也不引起人体产生免疫反应 ,因…     

盐度对企鹅珍珠贝足丝分泌的影响

企鹅珍珠贝（Pteria penguin）是生产附壳珍珠的大型海水经济贝类,其依靠强壮的足丝将自身固定在硬质基底上,抵抗水流的冲击和抵御被捕食等。足丝分泌和足丝的形状很容易受到环境的影响,本实验采用盐度30为低盐度组、盐度35为中盐度组和盐度40为高盐度组,研究这3种盐度对企鹅珍珠贝足丝分泌、足丝直径和足丝拉力的影响,通过单因素方差分析法（LSD法）分析这三个足丝相关指标在3种盐度组间是否存在显著性差异。结果显示,3种盐度下企鹅珍珠贝足丝附着率无显著差异,但在整个实验周期72 h内,中盐度组的足丝分泌总数为（48.7 ± 15.1）根,显著高于低盐度组的（24.7 ± 5.0）根和高盐度组的（13.3 ± 1.5）根。在实验的前6 h内,中盐度组的足丝首次附着率显著高于低盐度组和高盐度组（P < 0.05）,但在后续的12 h、18 h、30 h、42 h、54 h和66 h这6个时间点,3个盐度组的足丝首次附着率均无显著性差异。足丝直径未受盐度变化的影响,但盐度对足丝拉力具有显著影响,中盐度组的足丝拉力显著高于低盐度组和高盐度组（P < 0.05）。上述结果表明,企鹅珍珠贝为适应一定范围内盐度的改变,会在短时间内通过抑制足丝分泌来减少能量消耗,随着对环境的适应足丝分泌会恢复。盐度影响足丝分泌且对足丝拉力影响显著,但对足丝直径无明显影响。本研究可以为企鹅珍珠贝养殖及珍珠插核培育提供理论基础。     

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.     

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.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Polyphosphoprotein from the adhesive pads of Mytilus edulis

Achieving a satisfactory biochemical explanation for the opportunistic underwater adhesion of marine invertebrates such as mussels and barnacles requires a detailed characterization of proteins extracted from holdfast structures produced by these organisms. Mefp-5 is an adhesive protein derived from the foot of the common mussel, Mytilus edulis, and deposited into the byssal attachment pads. Purification and primary structure of mefp-5 was determined by peptide mapping and cDNA sequencing. The protein is 74 residues long and has a mass of about 9500 Da. Mefp-5 composition shows a strong amino acid bias: aromatic amino acids, lysine, and glycine represent 65 mol % of the composition. More than a third of all the residues in the protein are posttranslationally modified by hydroxylation or phosphorylation. The conversion of tyrosine to 3, 4-dihydroxyphenyl-L-alanine (DOPA) and serine to O-phosphoserine accounts for the hydroxylation and phosphorylation, respectively. Neither modification is complete since variations in the extent of phosphorylation and hydroxylation can be detected by mass spectrometry. More than 75% of the DOPA is adjacent to basic residues, e.g., Lys-DOPA and DOPA-Lys. Phosphoserine occurs in sequences strikingly reminiscent of acidic mineral-binding motifs that appear in statherin, osteopontin, and others. This may be an adaptation for adhesion to the most common substrata for mussels, i.e., calcareous materials.     

Self-assembly and adhesion of DOPA-modified methacrylic triblock hydrogels

Marine mussels anchor to a variety of surfaces by secreting liquid proteins that harden and form water-resistant bonds to a variety of surfaces. Studies have revealed that these mussel adhesive proteins contain an unusual amino acid, 3,4-dihydroxy-L-phenylalanine (DOPA), which is believed to be responsible for the cohesive and adhesive properties of these proteins. To separate the cohesive and adhesive roles of DOPA, we incorporated DOPA into the midblock of poly(methyl methacrylate)-poly(methacrylic acid)-poly(methyl methacrylate) (PMMA-PMAA-PMMA) triblock copolymers. Self-assembled hydrogels were obtained by exposing triblock copolymer solutions in dimethyl sulfoxide to water vapor. As water diffused into the solution, the hydrophobic end blocks formed aggregates that were bridged by the water-soluble midblocks. Strong hydrogels were formed with polymer weight fractions between 0.01 and 0.4 and with shear moduli between 1 and 5 kPa. The adhesive properties of the hydrogels on TiO2 surfaces were investigated by indentation with a flat-ended cylindrical punch. At pH values of 6 and 7.4, the fully protonated DOPA groups were highly adhesive to the TiO2 surfaces, giving values of approximately equal to 2 J/m2 for the interfacial fracture energy, which we believe corresponds to the cohesive fracture energy of the hydrogel. At these pH values, the DOPA groups are hydrophobic and have a tendency to aggregate, so contact times of 10 or 20 min are required for these high values of the interfacial strength to be observed. At a pH of 10, the DOPA groups were hydrophilic and highly swellable, but less adhesive gels were formed. Oxidation of DOPA groups, a process that is greatly accelerated at a pH of 10, decreased the adhesive performance of the hydrogels even further.     

3,4-dihydroxy-L-phenylalanine as a cell adhesion molecule in serum-free cell culture

In this article, we examined the feasibility of using 3,4‐dihydroxy‐L ‐phenylalanine (DOPA) as a cell adhesion molecule in serum‐free cultures of anchorage‐dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X‐ray photoelectron spectroscopy and energy‐dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA‐coated, fibronectin‐coated, or no material‐coated culture plates in serum‐free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell‐adhesion molecule for serum‐free culture. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1055–1060, 2012     

Surface-enhanced Raman spectroscopy of DOPA-containing peptides related to adhesive protein of marine mussel, Mytilus edulis

The common blue marine mussel adheres to underwater surfaces using an adhesive protein (Mefp-1) extruded from its foot. This highly hydroxylated protein contains a number of unusual amino acids, including 3,4-dihydroxyphenylalanine (DOPA), which is thought to contribute to the crosslinking of the extruded threads and adhesion to the substratum. Mefp-1 adheres to a wide variety of surfaces and is ultimately biodegradable. In this study we use surface-enhanced Raman spectroscopy (SERS) to characterize the adsorption of DOPA-containing peptides on colloidal gold. The peptides are simplified fragments of the Mefp-1 consensus decapeptide repeat, Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys. Our results show that the peptides TDeltaKA, PTDeltaKA, and PPTDeltaKA (where Delta represents DOPA) coordinate to the gold surface through the catechol oxygens of the DOPA residue and through primary amine groups. The diproline sequence introduces conformational constraints that influence the conformations of the adsorbed peptides. These findings lay the groundwork for developing synthetic adhesives for underwater and medical applications.