Strukturelle und immunologische Charakterisierung der Spinnenseide von Nephila clavipes

Ziel der Arbeit war es, die Spinnenseide des Sicherungsfadens der Goldenen Radnetzspinne Nephila clavipes zu charakterisieren und eventuelle Zusammenhänge zwischen Fadenbildung, strukturellem Aufbau und biochemischer Konsistenz herauszuarbeiten. Dabei war vor allem die noch weitgehend unverstandene Rolle der beiden bekannten Spidroine MASp1 und MASp2 sowie ihrer repetitiven und konservierten C-terminalen Domänen von Interesse. Hierzu wurden Antiseren gegen rekombinante Polypeptide mit den entsprechenden Sequenzen aus beiden Bereichen der beiden Gene hergestellt, affinitätsgereinigt und in den biochemischen und histochemischen Untersuchungen eingesetzt. Mithilfe der Antikörper konnte erstmalig gezeigt werden, dass die hochkonservierten C-Termini auch in den hochmolekularen Proteinfraktionen der ausgesponnenen Fäden wieder zu finden sind und nicht etwa vor der Fadenbildung abgespalten werden. Experimente zeigten, dass Sie zur Bildung von Disulfidbrücken befähigt sind. Ihre Bedeutung sowie ihre phylogenetische Entwicklung wird im Zusammenhang mit Ergebnissen aus Sequenzvergleichen diskutiert. Aus den biochemischen Untersuchungen des Spinnguts ergab sich, dass dies überraschend heterogen ist und mehrere hochmolekulare, lösliche Proteine im Bereich von 220-270 kDa aufweist. Durch Anfärbung mit Lektinen wurde gezeigt, dass die Spidroine aller Wahrscheinlichkeit nach O-glycosyliert sind. Als weitere Ursache für die Heterogenität wurden N-terminale Abbauprozesse diskutiert, die sich aus den mit den Antiseren erzielten Ergebnissen und Versuchen N-terminaler Sequenzierung ergaben. Die Ergebnisse der strukturellen und der immunologischen Studien wurden in einem Modell zum Aufbau der Tragfäden von Nephila clavipes zusammengefasst. Neben einem Kern besitzt die Seide eine aus drei Schichten bestehende Hülle. Während die beiden äußeren Schichten labil sind, zeigt die dritte innere Schicht eine hohe Resistenz gegen Laugen, Säuren und chaotrope Agenzien und verleiht dem Faden Stabilität. Der Kern lässt sich aufgrund der Verteilung der Spidroine in zwei Zonen unterteilen. Während MASp1homogen verteilt in beiden Zonen vorliegt, fehlt MASp2 in der äußeren Zone und liegt in der inneren Zone konzentriert in kleinen Inseln vor. Dieses Verteilungsmuster wird im Zusammenhang mit der Rolle von MASp2 bei der Fadenbildung und dem Auftreten von Fibrillen diskutiert

Composition and Hierarchical Organisation of a Spider Silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material

Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family

Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibres. Here we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process

Biological responses to spider silk - antibiotic fusion protein

The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous implants were studied to access the in vivo biological response to 6mer + hepcidin, which were compared with controls of silk alone (6mer), polylactic–glycolic acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer + hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28603/2006, Chimera project (No. PTDC/EBB-EBI/109093/2008), Proteo-Light (No. PTDC/FIS/68517/2006), NIH (Grant No. P41 EB002520) Tissue Engineering Resource Center; and the NIH (Grant Nos EB003210 and DE017207).Tissue Engineering Resource Center - Bolsa No. P41 EB002520, bolsa Nos EB003210 and DE017207European - Projeto EXPERTISSUES (No. NMP3- CT-2004-500283

Glass transitions in native silk fibres studied by dynamic mechanical thermal analysis

Silks are a family of semi-crystalline structural materials, spun naturally by insects, spiders and even crustaceans. Compared to the characteristic β-sheet crystalline structure in silks, the non-crystalline structure and its composition deserves more attention as it is equally critical to the filaments' high toughness and strength. Here we further unravel the structure-property relationship in silks using Dynamic Mechanical Thermal Analysis (DMTA). This technique allows us to examine the most important structural relaxation event of the disordered structure the disordered structure, the glass transition (GT), in native silk fibres of the lepidopteran Bombyx mori and Antheraea pernyi and the spider Nephila edulis. The measured glass transition temperature Tg, loss tangent tan δ and dynamic storage modulus are quantitatively modelled based on Group Interaction Modelling (GIM). The "variability" issue in native silks can be conveniently explained by the different degrees of structural disorder as revealed by DMTA. The new insights will facilitate a more comprehensive understanding of the structure-property relations for a wide range of biopolymers

Mapping Nanostructural Variations in Silk by Secondary Electron Hyperspectral Imaging

Nanostructures underpin the excellent properties of silk. Although the bulk nanocomposition of silks has been well studied, direct evidence of the spatial variation of nanocrystalline (ordered) and amorphous (disordered) structures has remained elusive. Here we demonstrate that secondary electron hyperspectral imaging, can be exploited for direct imaging of hierarchical structures in carbon based materials which cannot be revealed by any other standard characterization methods. Applying this technique to silks from domesticated (Bombyx mori) and wild (Antheraea mylitta) silkworms, we report a variety of previously unseen features which highlight the local interplay between ordered and disordered structures. We conclude that our technique is able to differentiate composition on the nanoscale and enables in-depth studies into the relationship between morphology and performance of these complex biopolymer systems

Antibiotic Spider Silk: Site-Specific Functionalization of Recombinant Spider Silk Using “Click” Chemistry

The use of functionalised recombinant spider silk as a sustainable advanced biomaterial is currently an area of intense interest owing to spider silk’s intrinsic strength, toughness, biocompatibility and biodegradability. This paper demonstrates, for the first time, the site-specific chemical conjugation of different organic ligands that confer either antibiotic or fluorescent properties to spider silk. This has been achieved by the incorporation of the non-natural methionine analogue L-azidohomoalanine (L-Aha) using an E. coli methionine auxotroph and subsequent copper catalysed azide-alkyne cycloaddition (CuAAC) or ‘click chemistry’ functionalisation of 4RepCT3Aha. The 4RepCT3Aha protein can be modified either prior to, or post fibre formation increasing the versatility of this approach as demonstrated here by the formation of silk fibres bearing a defined ratio of two different fluorophores uniformly distributed along the fibres. Silk decorated with the fluoroquinone family broad spectrum antibiotic levofloxacin via a labile linker is shown to have significant antibiotic activity over a period of at least 5 days. The inherent low immunogenicity and pyrogenicity of spider silk should allow a diverse range of functionalised silks to be produced using these approaches that are tailored to applications including wound dressings and as tissue regeneration scaffolds

Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres