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.     

Zebra mussel adhesion: structure of the byssal adhesive apparatus in the freshwater mussel, Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well.     

Byssus production rate of the White Sea blue mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Linnaeus, 1758) in the presence of metabolites of some hydrobionts

The byssus production of the blue mussel Mytilus edulis L. was studied in the laboratory in the presence of the metabolites of the following animals: a predator (a starfish Asterias rubens L.) and several species competing with the mussel in White Sea fouling communities (a bivalve Hiatella arctica L. and a solitary ascidian Styela rustica L.). The byssus threads and attachment plaques produced by each mussel per day were counted. The number of byssus threads and plaques was smallest in pure sea water and in the presence of metabolites produced by conspecific individuals.     

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．     

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.     

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.     

Byssogenesis in the juvenile pink heelsplitter mussel,Potamilus alatus (Bivalvia: Unionidae)

The North American pink heelsplitter (Potamilus alatus) differs from most freshwater mussels in China by the ability to secrete an ephemeral byssus during its juvenile stage. In the present study, light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to investigate this ephemeral byssal structure, and amino acid composition was analyzed and compared with that of other species. The results revealed that the byssus consists of a long byssal thread and a few adhesive plaques which are randomly set up along the thread and assembled by petioles. There is a thin but distinctive cuticle with a continuous homogeneous matrix surrounding the byssal thread. Structural variation occurred when the byssal thread was differentially stretched. Four‐stranded helical primary fasciculi, which form a stable rope‐like structure, become evident after removal of the cuticle. The primary fasciculi consist of bundles of hundreds of parallel secondary fasciculi, each measuring about 5 μm in diameter. All evidence indicates that the byssus of the pink heelsplitter has a significantly different macrostructure and microstructure than the permanent byssus of the marine mussel Mytilus. Byssogenesis ceases when juveniles exceed 30 mm in length, although it varies greatly even among juveniles of similar size. Byssus formation is influenced by substrate type. The unique characteristics of the byssus have important advantages for survival, transition, and aggregation during the early life history. This study not only provides first insight into the structure of the ephemeral byssus and its relationship to freshwater mussel development and growth, but also suggests possibilities for the synthesis of novel biopolymer materials particularly useful in freshwater ecosystems. J. Morphol. 276:1273–1282, 2015. © 2015 Wiley Periodicals, Inc.     

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。     

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.     

MECHANICAL PROPERTIES OF MUSSEL BYSSUS THREADS

The byssus threads of the common mussel, Mytilus edulis L.,have been tested mechanically and the results from the testsrelated to the ecology of the animal. The threads are mechanicallysimilar to other crystalline polymers such as polyethylene havinga modulus of about 10⁸N m^–2 and a long relaxation time.Resilience of 60% is similar to tendon; ultimate strain is aboutfive times that of tendon at 0.44. The thread is laid down witha prestrain of 10% and so guys the mussel in position. Calculationshows that a mussel with 50 byssus threads would be able toresist all but severe winter storms (Received 1 December 1978;     

Morphology of the Bivalve Mollusks Crenomytilus grayanus and Mytilus coruscus in Relation to Their Spatial Distribution in the Upper Subtidal Zone

The morphology of the shell and byssus threads was studied in two closely related mussel species Crenomytilus grayanus and Mytilus coruscus. The two species differ significantly from each other in the shell shape and in the degrees of development and deformation of byssus threads. These differences, in turn, determine (either directly or indirectly) the differences in strength of the byssal attachment and are discussed in terms of their functional morphology with respect to the spatial distribution of the mussels in marine coastal zones.     

Byssus attachment strength of two mytilids in mono-specific and mixed-species mussel beds

The mussel Xenostrobus securis is endemic to the brackish waters of New Zealand and Australia, but has successfully invaded the inner Galician Rías of NW Spain, where it coexists with the indigenous mussel Mytilus galloprovincialis. In this laboratory study, the plasticity of the byssus attachment strength of two mytilids was compared by manipulating substratum, salinity, and bed assembly. M. galloprovincialis showed stronger byssus detachment strength than X. securis, despite lower byssus coverage. Both species responded similarly to the substratum, with substantially lower byssus strength on methacrylate, which offered the lowest surface free energy. Byssus detachment values for M. galloprovincialis were lower at lower salinity. In mixed beds, a number of mussels moved upwards, eventually colonising the upper layers of the assemblage. This behaviour increased byssus strength but only for X. securis. X. securis is adapted to a wide spectrum of abiotic conditions, a trait that may promote its dissemination within estuarine environments.     

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.     

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.     

Green mussel Perna viridis L.: attachment behaviour and preparation of antifouling surfaces

The green mussel Perna viridis LINNE can be kept in simulated seawater for more than 6 months in good condition. The mussel forms many threads by secreting an adhesive protein from the foot, and attaches with more than 50 byssal threads, which makes most mussels clump together. In order to investigate the preparation of the antifouling surfaces toward green mussels, the attachment of mussels was tested using glass surfaces modified with silane coupling agents, together with non-treated material surfaces such as glass and silicone. The correlation between the attachment percentage and the mean number of the secreted byssus was highly significant, indicating that the mussel selects a favorable surface prior to the secretion of byssus. The relationships between the mussel attachment and the surface chemical parameters (surface free energy (sfe) and its dispersion and polar components) were examined based on a working hypothesis, which we have previously reported. The result of statistical regression test indicated that a certain correlation was found between the dispersion component and the mussel attachment, while the polar component did not correlate to the mussel attachment. The present surface chemical approach provided an additional clue for the preparation of ecologically clean antifouling materials that takes into account the combination of the wettability of both the marine adhesive proteins (MAP) and the modified surfaces.     

Byssus production ability and degree of byssal-gland development in the infaunal clam Ruditapes philippinarum

Byssus production of Ruditapes philippinarum clams becomes reduced with growth. This tendency is well recognized but has not been analysed in detail. Additionally, it remains uninvestigated whether the lack of competence to produce byssus threads in the adult stage is caused by atrophy of the byssal glands or not. The objective of this study was to evaluate the byssus production ability of clams through the juvenile to adult stages and to examine the importance of two endogenous factors (i.e. shell size, somatic condition) in determining the byssus production probability (proportion of clams with byssus production in a population). This study also histologically confirmed the presence of byssal glands in juvenile to adult clams. For these purposes, field surveys to investigate the relationship among byssus production, shell size and somatic condition of clams collected from four intertidal sites and a histological study for byssal glands of the clams was conducted. This study revealed that byssus production probability decreases with increasing shell size and declining somatic condition and that the lack of byssus production is not caused by the loss of the byssal glands.     

Coating proteins: structure and cross-linking in fp-1 from the green shell mussel Perna canaliculus

The protein family known as fp-1 provides mussel byssus with a protective outer coating and has drawn much attention for its water resistant bioadhesive properties in vitro. A new fp-l isolated from the green shell mussel Perna canaliculus (pcfp-1) reveals a composition dominated by only four amino acids: 3,4-dihydroxyphenyl-L-alanine (dopa), lysine, proline, and valine at approximately 20 mol % each. SDS-PAGE and MALDI-TOF mass spectrometry detected size variants at 48 and 52 kDa in preparations of purified Pcfp-1. The N-terminal sequence enabled construction of oligonucleotide primers for PCR and RACE-derived cDNAs from which the complete sequence of four variants was deduced. pcfp-1 deviates from all known homologues in other mussels in several notable respects: its mass is half, most of its sequence is represented by 75 tandem repeats of a tetrapeptide, i.e., PY*VK, in which Y* is dopa, prolines are not hydroxylated, and thiolate cysteines are clustered in homologous sequences at both the amino and carboxy termini. Amino acids in the repeat sequence show a striking resemblance to proline-rich cell wall proteins with tandemly repeated PPVYK pentapeptides [Hong, J. C., Nagao, R. T., and Key, J. L. (1987) J. Biol. Chem. 262, 8367-8376]. Cysteine plays a key role in cross-linking pcfp-1 by forming adducts with dopaquinone. Significant 5-S-cysteinyldopa and smaller amounts of 2-S-cysteinyldopa were detected in hydrolysates of the byssal threads of P. canaliculus. The cross-links could also be formed by oxidation of pcfp-1 in vitro using mushroom tyrosinase. Cysteinyldopa cross-links were present in trace amounts only in the byssus of other mussel species.     

Preferences of the Ponto-Caspian amphipod Dikerogammarus haemobaphes for living zebra mussels

A Ponto-Caspian amphipod Dikerogammarus haemobaphes has recently invaded European waters. In the recipient area, it encountered Dreissena polymorpha , a habitat-forming bivalve, co-occurring with the gammarids in their native range. We assumed that interspecific interactions between these two species, which could develop during their long-term co-evolution, may affect the gammarid behaviour in novel areas. We examined the gammarid ability to select a habitat containing living mussels and searched for cues used in that selection. We hypothesized that they may respond to such traits of a living mussel as byssal threads, activity (e.g. valve movements, filtration) and/or shell surface properties. We conducted the pairwise habitat-choice experiments in which we offered various objects to single gammarids in the following combinations: (1) living mussels versus empty shells (the general effect of living Dreissena ); (2) living mussels versus shells with added byssal threads and shells with byssus versus shells without it (the effect of byssus); (3) living mussels versus shells, both coated with nail varnish to neutralize the shell surface (the effect of mussel activity); (4) varnished versus clean living mussels (the effect of shell surface); (5) varnished versus clean stones (the effect of varnish). We checked the gammarid positions in the experimental tanks after 24 h. The gammarids preferred clean living mussels over clean shells, regardless of the presence of byssal threads under the latter. They responded to the shell surface, exhibiting preferences for clean mussels over varnished individuals. They were neither affected by the presence of byssus nor by mussel activity. The ability to detect and actively select zebra mussel habitats may be beneficial for D. haemobaphes and help it establish stable populations in newly invaded areas.     

Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc

The enzyme gland of the foot of the mussel Mytilus has been so far considered a gland producing and exporting a phenol oxidase catalysing the general tanning processes of byssus threads. In contrast, the present study shows that this gland produces mainly secretory granules which form the cortical layers of byssus threads. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, silver methenamine for sulphur-rich proteins demonstration) and enzyme digestion tests (pepsin, trypsin, alpha-chymotrypsin) indicate that secretory granules contain glycoproteins rich in sulphydryl groups and in aromatic amino acids. The cytochemical demonstration of phenol oxidase shows that enzyme activity is present in Golgi complex, whereas it is absent in secretory granules. For this reason, phenol oxidase does not seem to be exported and utilized for tanning of byssus threads, but it might rather be involved in the elaboration and tanning of the content of the secretory granules in the enzyme gland itself.