Minor Ampullate Silk Research Articles

Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein Based Nanovaccines via Subcutaneous Immunization

AbstractCoxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all‐trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide‐based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.

Spider minor ampullate silk protein nanoparticles: an effective protein delivery system capable of enhancing systemic immune responses.

Spider silk proteins (spidroins) are particularly attractive due to their excellent biocompatibility. Spider can produce up to seven different types of spidroins, each with unique properties and functions. Spider minor ampullate silk protein (MiSp) might be particularly interesting for biomedical applications, as the constituent silk is mechanically strong and does not super-contract in water, attributed to its amino acid composition. In this study, we evaluate the potential of recombinant nanoparticles derived from Araneus ventricosus MiSp as a protein delivery carrier. The MiSp-based nanoparticles were able to serve as an effective delivery system, achieving nearly 100% efficiency in loading the model protein lysozyme, and displayed a sustained release profile at physiological pH. These nanoparticles could significantly improve the delivery efficacy of the model proteins through different administration routes. Furthermore, nanoparticles loaded with model protein antigen lysozyme after subcutaneous or intramuscular administration could enhance antigen-specific immune responses in mouse models, through a mechanism involving antigen-depot effects at the injection site, long-term antigen persistence, and efficient uptake by dendritic cells as well as internalization by lymph nodes. These findings highlight the transnational potential of MiSp-based nanoparticle system for protein drug and vaccine delivery.

Helium Ion Microscopy and Sectioning of Spider Silk.

Focused ion beams have recently emerged as a powerful tool for ultrastructural imaging of biological samples. In this article, we show that helium ion microscopy (HIM), in combination with ion milling, can be used to visualize the inner structure of both major and minor ampullate silk fibers of the orb-web weaving spider Nephila madagascariensis. The internal nanofibrils were imaged in pristine silk fibers, with little or no damage to the sample structure observed. Furthermore, a method to cut/rupture the fibers using He+ ions combined with internal sample tension is presented. This showed that the stretching and rupturing of spider silk is a highly dynamic process with considerable material reorganization.

PH-dependent self-assembly mechanism of a single repetitive domain from a spider silk protein

Spider silk is self-assembled from full-length silk proteins, and some silk protein fragments can also form silk-like fibers in vitro. However, the mechanism underlying the silk fiber formation is not understood well. In this study, we investigated the fiber formation of a single repetitive domain (RP) from a minor ampullate silk protein (MiSp). Our findings revealed that pH and salt concentration affect not only the stability of MiSp-RP but also its self-assembly into fibers and aggregates. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the three-dimensional (3D) structure of MiSp RP in aqueous solution. On the basis of the structure and mutagenesis, we revealed that charge-dipole interactions are responsible for the pH- and salt-dependent properties of MiSp-RP. Our results indicate that fiber formation is regulated by a delicate balance between intermolecular and intramolecular interactions, rather than by the protein stability alone. These findings have implications for the design of silk proteins for mass production of spider silk.

Nanoscale imaging of major and minor ampullate silk from the orb-web spider Nephila Madagascariensis

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.

Composition of Minor Ampullate Silk Makes Its Properties Different from Those of Major Ampullate Silk.

Spider's minor ampullate silk, or MI-silk, exhibits distinct mechanical properties and water resistance compared to its major ampullate counterpart (MA-silk). The principal protein constituent of MI-silk is known as minor ampullate spidroin, or MiSp, and while its sequence has been deciphered and is thought to underlie the differences in properties with MA-silk, the composition of MI-silk and the relationship between its composition and properties remain elusive. In this study, we set out to investigate the mechanical properties, water resistance, and proteome of MA-silk and MI-silk from Araneus ventricosus and Trichonephila clavata. We also synthesized artificial fibers from major ampullate spidroin, MaSp1 and 2, and MiSp to compare their properties. Our proteomic analysis reveals that the MI-silk of both araneids is composed of MiSp, MaSp1, and spidroin constituting elements (SpiCEs). The absence of MaSp2 in the MI-silk proteome and the comparison of the water resistance of artificial fibers suggest that the presence of MaSp2 is the reason for the disparity in water resistance between MI-silk and MA-silk.

Spinning conditions affect structure and properties of Nephila spider silk

Raman spectroscopy is used to elucidate the effect of spinning conditions upon the structure and mechanical properties of silk spun by Nephila spiders from the major ampullate gland. Silk fibers produced under natural spinning conditions with spinning rates between 2 and 20 mm s−1 differed in microstructure and mechanical properties from fibers produced either more slowly or more rapidly. The data support the “uniform strain” hypothesis that the reinforcing units in spider silk fibers are subjected to the same strain as the fiber, to optimize the toughness. In contrast, in the case of synthetic high-performance polymer fibers, the both units and the fiber experience uniform stress, which maximizes stiffness. The comparison of Nephila major and minor ampullate silks opens an intriguing window into dragline silk evolution and the first evidence of significant differences between the two silks providing possibilities for further testing of hypotheses concerning the uniform strain versus uniform stress models.Impact statementIt is well established that the microstructure and mechanical properties of engineering materials are controlled by the conditions employed to both synthesize and process them. Herein, we demonstrate that the situation is similar for a natural material, namely spider silk. We show that for a spider that normally produces silk at a reeling speed of between 2 and 20 mm s−1, silk produced at speeds outside this natural processing window has a different microstructure that leads to inferior tensile properties. Moreover, we also show that the silk has a generic microstructure that is optimized to respond mechanically to deformation such that the crystals in the fibers are deformed under conditions of uniform strain. This is different from high-performance synthetic polymer fibers where the microstructure is optimized such that crystals within the fibers are subjected to uniform stress.Graphic abstract

Structural Basis of Oligomerization of N-Terminal Domain of Spider Aciniform Silk Protein.

Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from Nephila antipodiana (NA). Different from MaSpN, our biophysical characterization indicated that AcSpN assembles to form large oligomers, instead of a dimer, upon condition changes from neutral to acidic pH and/or from a high to low salt concentration. Our structural studies, by nuclear magnetic resonance spectroscopy and homology modelling, revealed that AcSpN and MaSpN monomers adopt similar overall structures, but have very different charge distributions contributing to the differential self-association features. The intermolecular interaction interfaces for AcSp oligomers were identified using hydrogen–deuterium exchange mass spectrometry and mutagenesis. On the basis of the monomeric structure and identified interfaces, the oligomeric structures of AcSpN were modelled. The structural information obtained will facilitate an understanding of silk fiber formation mechanisms for aciniform silk protein.

Strain-Dependent Structural Changes in Major and Minor Ampullate Spider Silk Revealed by Two-Photon Excitation Polarization.

Spider silk's mechanical properties make it an interesting material for many industrial applications. The structure and nanoscopic organization of its proteins are the basis of these qualities. In this study, the emission maxima of the autofluorescence from the protein core of major and minor ampullate silk fibers from the orb-web-weaving spider Nephila madagascariensis are determined and found to be 534 ± 11 and 547 ± 19 nm, respectively. Molecular conformational changes during applied strain are observed in both fiber types using two-photon excitation polarization measurements. Our findings showed that within the fibers the autofluorescent dipoles are separated into two distinct populations, one randomly orientated (amorphous regions) and one with aligned dipoles as found in crystalline structures. The crystalline-amorphous ratio was determined, and it was found that the crystalline dipoles made up around 30 and 20% of the autofluorescent dipoles in major and minor ampullate silk fibers, respectively. Using two-photon polarization measurements, it is possible to directly observe that the major and minor ampullate silk fibers structurally adapt to the applied stress, as well as discern different molecular conformational changes between major and minor ampullates. It was seen that the crystalline-amorphous ratio increased, with up to 9% for major fibers and 6% for minor fibers, as strain was applied, suggesting a conformational adaptation of the fiber, interpreted as noncrystalline 310-helices transforming into crystalline β-sheets.

Piezoresponse, Mechanical, and Electrical Characteristics of Synthetic Spider Silk Nanofibers.

This work presents electrospun nanofibers from synthetic spider silk protein, and their application as both a mechanical vibration and humidity sensor. Spider silk solution was synthesized from minor ampullate silk protein (MaSp) and then electrospun into nanofibers with a mean diameter of less than 100 nm. Then, mechanical vibrations were detected through piezoelectric characteristics analysis using a piezo force microscope and a dynamic mechanical analyzer with a voltage probe. The piezoelectric coefficient (d33) was determined to be 3.62 pC/N. During humidity sensing, both mechanical and electric resistance properties of spider silk nanofibers were evaluated at varying high-level humidity, beyond a relative humidity of 70%. The mechanical characterizations of the nanofibers show promising results, with Young’s modulus and maximum strain of up to 4.32 MPa and 40.90%, respectively. One more interesting feature is the electric resistivity of the spider silk nanofibers, which were observed to be decaying with humidity over time, showing a cyclic effect in both the absence and presence of humidity due to the cyclic shrinkage/expansion of the protein chains. The synthesized nanocomposite can be useful for further biomedical applications, such as nerve cell regrowth and drug delivery.

Punctuated evolution of viscid silk in spider orb webs supported by mechanical behavior of wet cribellate silk.

The origin of viscid capture silk in orb webs, from cribellate silk-spinning ancestors, is a key innovation correlated with significant diversification of web-building spiders. Ancestral cribellate silk consists of dry nanofibrils surrounding a stiff, axial fiber that adheres to prey through van der Waals interactions, capillary forces, and physical entanglement. In contrast, viscid silk uses chemically adhesive aqueous glue coated onto a highly compliant and extensible flagelliform core silk. The extensibility of the flagelliform fiber accounts for half of the total work of adhesion for viscid silk and is enabled by water in the aqueous coating. Recent cDNA libraries revealed the expression of flagelliform silk proteins in cribellate orb-weaving spiders. We hypothesized that the presence of flagelliform proteins in cribellate silk could have allowed for a gradual shift in mechanical performance of cribellate axial silk, whose effect was masked by the dry nature of its adhesive. We measured supercontraction and mechanical performance of cribellate axial silk, in wet and dry states, for two species of cribellate orb web-weaving spiders to see if water enabled flagelliform silk-like performance. We found that compliance and extensibility of wet cribellate silk increased compared to dry state as expected. However, when compared to other silk types, the response to water was more similar to other web silks, like major and minor ampullate silk, than to viscid silk. These findings support the punctuated evolution of viscid silk mechanical performance.

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

BackgroundOrb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups.ResultsWe report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers.ConclusionsMiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Thermal characterization of natural and synthetic spider silks by both the 3ω and transient electrothermal methods

Thermal conductivity, thermal diffusivity, and volumetric heat capacity of three spider silks are measured in this paper as a benchmark for further studies. These silks include the major and minor ampullate silks of the Nephila clavipes spider, and a synthetic spider silk fiber made from recombinant dragline silk proteins purified from transgenic goats' milk. Two complementary measurement techniques are employed in the thermal characterization of these microscale single fibers for self-verification. One is the transient electrothermal technique (TET) and the other is the 3ω method. Experimental measurements indicate that thermal properties of the dragline silk are very close to those of the minor ampullate silk, whereas the ones for the synthetic silk are much lower due in part to its low crystallinity. The directly measured thermal conductivity, thermal diffusivity, and volumetric heat capacity of the major and minor ampullate silks are 1.2–1.26Wm−1K−1, 5.7–6×10−7m2s−1, and 2–2.17 MJm−3K−1, respectively. The thermal conductivity and thermal diffusivity of the as-spun synthetic silk are 0.24Wm−1K−1 and 1.6×10−7m2s−1 respectively. As part of this study, a detailed comparison of the TET and 3ω methods is provided showing the complementary nature of the techniques and illustrating the strengths and weaknesses of each.

Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.

Full-Length Minor Ampullate Spidroin Gene Sequence

Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level.

Minor Ampullate Silks from Nephila and Argiope Spiders: Tensile Properties and Microstructural Characterization

The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks

Synchrotron X-ray micro-diffraction experiments were carried out on Nephila clavipes (NC) and Argiope aurantia (AA) major (MA) and minor ampullate (MiA) fibers that make up dragline spider silk. The diffraction patterns show a semi-crystalline structure with β-poly(l-alanine) nanocrystallites embedded in a partially oriented amorphous matrix. A superlattice reflection 'S' diffraction ring is observed, which corresponds to a crystalline component larger in size and is poorly oriented, when compared to the β-poly(l-alanine) nanocrystallites that are commonly observed in dragline spider silks. Crystallite size, crystallinity and orientation about the fiber axis have been determined from the wide-angle X-ray diffraction (WAXD) patterns. In both NC and AA, the MiA silks are found to be more highly crystalline, when compared with the corresponding MA silks. Detailed analysis on the amorphous matrix shows considerable differences in the degree of order of the oriented amorphous component between the different silks studied and may play a crucial role in determining the mechanical properties of the silks.

Morphology, fibrous composition and tensile properties of drag-silk produced by two species of orb spider

Abstract Silk fibers produced by the orb spiders Argiope amoena and Nephila clavata were examined using scanning electron microscopy. The fibers were produced on a horizontal surface by unanesthetized spiders. The fibers have different morphologies, physical structures, and fibrous compositions broadly consisting of one to four filaments and numerous fibrilliform filaments with varying diameters. The fibers are composed of a wide range of different silk fibrils (e. g. major and minor ampullate or other gland silk). We examined a range of silks produced by orb spiders. The spiders produce different silks for purposes such as web mooring, web radial threads, scaffolding anchoring silk of egg cases. In addition fiber deposited when moving towards prey enmeshed in the web, fiber deposited when returning to the web center, and fiber used to hang vertically downwards from a branch are all different. The studies indicate that these two species of orb spider can spin fibers of diverse complex structures constructed from fibrils from different glands that vary in number, diameter, morphology, and conformation depending on application. We interpret the variation in the silk produced by relating it to the required tensile properties, a biological cost–benefit principle, and the functional requirements for different natural environments and applications.

Solid-state nuclear magnetic resonance (NMR) spectroscopy reveals distinctive protein dynamics in closely related spider silks

Structurally closely related spider silks, namely, major ampullate silk (MaS) reeled at 0.5 and 10 cm/s, minor ampullate silk (MiS), and cocoon silk have been investigated by solid-state nuclear magnetic resonance (NMR). These silks exhibit different mechanical properties that are not well explained with current structural data. NMR spectra confirm that the secondary structures of these silks are similar, but that differing relaxation dynamics exist at the alanine, glycine, and glutamine residue level. MaS reeled rapidly is made of more mobile proteins in both the β-sheet and amorphous regions, suggesting that reeling speed alters molecular rearrangement during spinning, with the chain packing being less organized in the fastpull fiber. Alanine and glycine relaxation dynamics increases as MaS < MiS < cocoon silk, showing that the whole fiber displays distinctive protein dynamics, chain packing, and intermolecular interactions. Besides molecular orientation, silk extensibility is related to a more mobile structural organization.

Diversity of Molecular Transformations Involved in the Formation of Spider Silks

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented β-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of α-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented β-sheets and α-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning.