Minor Ampullate Spidroin Research Articles

Electrospun mini-MiSp spidroin/poly(L-lactide-co-ε-caprolactone) nanofibrous scaffolds for ARPE-19 cells

Age-related macular degeneration, a type of retinal degenerative disease, is the primary cause of severe visual impairment and irreversible blindness in people older than 50, due to age-related deterioration of the photoreceptors and the retinal pigment epithelium (RPE), which could be restored by cell therapy. However, the success of cell therapy depends on biocompatible scaffolds, which serve as carriers for the growth of RPE cells for repairing damaged or diseased retinas. In this study, the plasmid pET-SUMO-R2C for expressing a mini-spidroin, fusing the second repetitive region to the C-terminal domain of the minor ampullate spidroin gene from the spider Araneus ventricosus, was constructed and named R2C. The spidroin R2C was expressed, purified and wet spun into silk-like fibers, possessing a Young’s modulus of 3.51 ± 0.92 GPa and toughness of 24.67 ± 9.51 MPa. The spidroin R2C, mixed with poly(L-lactide-co-ε-caprolactone) (PLCL) at a ratio of 3:1, was electrospun into nanofibrous scaffolds, composed of nanometric fibers with a diameter of 751.70 ± 19.48 nm. After co-culturing with the R2C/PLCL scaffolds, the transcriptomic differences of the ARPE-19 cells were analyzed, showing that fourteen genes that are mainly related to inflammation, tumors and keratinization were upregulated more than three times, while seven genes that are mostly related to substance transport and homeostasis were downregulated more than three times. The R2C/PLCL scaffolds have the potential to be further studied to serve as a suitable carrier for RPE transplantation in the future.

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.

Characteristic Evaluation of Recombinant MiSp/Poly(lactic-co-glycolic) Acid (PLGA) Nanofiber Scaffolds as Potential Scaffolds for Bone Tissue Engineering.

Biomaterial-based nanofibrous scaffolds are the most effective alternative to bone transplantation therapy. Here, two recombinant minor ampullate spidroins (spider silk proteins), R1SR2 and NR1SR2C, were blended with Poly(lactic-co-glycolic) Acid (PLGA), respectively, to generate nanofiber scaffolds by electrospinning. The N-terminal (N), C-terminal (C), repeating (R1 and R2) and spacer (S) modules were all derived from the minor ampullate spidroins (MiSp). The physical properties and structures of the blended scaffolds were measured by scanning electron microscopy (SEM), water contact angle measurement, Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and Tensile mechanical testing. The results showed that blending of MiSp (R1SR2 and NR1SR2C) reduced the diameter of nanofibers, increased the porosity and glass transition temperatures of nanofibrous scaffolds, and effectively improved the hydrophilicity and ultimate strain of scaffolds. It is worth noting that the above changes were more significant in the presence of the N- and C-termini of MiSp. In cell culture assays, human bone mesenchymal stem cells (HBMSCs) grown on NR1SR2C/PLGA (20/80) scaffolds displayed markedly enhanced proliferative and adhesive abilities compared with counterparts grown on pure PLGA scaffolds. Jointly, these findings indicated recombinant MiSp/PLGA, particularly NR1SR2C/PLGA (20/80) blend nanofibrous scaffolds, is promising for bone tissue engineering.

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

Progress in the application of spider silk protein in medicine.

Spider silk protein has attracted much attention on account of its excellent mechanical properties, biodegradability, and biocompatibility. As the main protein component of spider silk, spidroin plays important role in spider spinning under natural circumstances and biomaterial application in medicine as well. Compare to the native spidroin which has a large molecular weight (>300 kDa) with highly repeat glycine and polyalanine regions, the recombinant spidroin was maintained the core amino motifs and much easier to collect. Here, we reviewed the application of recombinant spider silk protein eADF4(C16), major ampullate spidroin (MaSp), minor ampullate spidroin (MiSp), and the derivatives of recombinant spider silk protein in drug delivery system. Moreover, we also reviewed the application of spider silk protein in the field of alternative materials, repairing materials, wound dressing, surgical sutures along with advances in recombinant spider silk protein.

The common house spider, Parasteatoda tepidariorum, maintains silk gene expression on sub-optimal diet.

Cobweb weaving spiders and their relatives spin multiple task-specific fiber types. The unique material properties of each silk type result from differences in amino acid sequence and structure of their component proteins, primarily spidroins (spider fibrous proteins). Amino acid content and gene expression measurements of spider silks suggest some spiders change expression patterns of individual protein components in response to environmental cues. We quantified mRNA abundance of three spidroin encoding genes involved in prey capture in the common house spider, Parasteatoda tepidariorum (Theridiidae), fed different diets. After 10 days of acclimation to the lab on a diet of mealworms, spiders were split into three groups: (1) individuals were immediately dissected, (2) spiders were fed high-energy crickets, or (3) spiders were fed low-energy flies, for 1 month. All spiders gained mass during the acclimation period and cricket-fed spiders continued to gain mass, while fly-fed spiders either maintained or lost mass. Using quantitative PCR, we found no significant differences in the absolute or relative abundance of dragline gene transcripts, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), among groups. In contrast, prey-wrapping minor ampullate spidroin (MiSp) gene transcripts were significantly less abundant in fly-fed than lab-acclimated spiders. However, when measured relative to Actin, cricket-fed spiders showed the lowest expression of MiSp. Our results suggest that house spiders are able to maintain silk production, even in the face of a low-quality diet.

Novel Highly Soluble Chimeric Recombinant Spidroins with High Yield.

Spider silk has been a hotspot in the study of biomaterials for more than two decades due to its outstanding mechanical properties. Given that spiders cannot be farmed, and their low silk productivity, many attempts have been made to produce recombinant spidroins as an alternative. Herein, we present novel chimeric recombinant spidroins composed of 1 to 4 repetitive units of aciniform spidroin (AcSp) flanked by the nonrepetitive N- and C-terminal domains of the minor ampullate spidroin (MiSp), all from Araneus ventricosus. The spidroins were expressed in the form of inclusion body in E. coli with high yield. Remarkably, the aqueous solubility of the four spidroins ranged from 13.4% to over 50% (m/v). The four spidroins could self-assemble into silk-like fibers by hand-drawing. The secondary structures of these proteins, determined by circular dichroism spectrum (CD) and Fourier transform infrared spectrum (FTIR), indicated a prominent transformation from α-helix to β-sheet after fiber formation. The mechanical properties of the hand-drawn fibers showed a positive correlation with the spidroin molecular weight. In summary, this study describes promising biomaterials for further study and wide application.

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

BackgroundSpider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process.ResultsHere, we present a mild solubilization strategy—one-step heating method to solubilize spidroins from IBs, with combining spidroins’ high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70–90 °C for 20 min, pH 7.0–10, urea concentration 2–4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology.ConclusionsThe one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

Evaluation of the potential of chimeric spidroins/poly(L-lactic-co-ε-caprolactone) (PLCL) nanofibrous scaffolds for tissue engineering.

In this study, a novel type of chimeric spider silk proteins (spidroins) NTW1-4CT was blended with poly(L-lactic-co-ε-caprolactone) (PLCL) to obtain nanofibrous scaffolds via electrospinning. Spidroins are composed of a N-terminal module (NT) from major ampullate spidroins, a C-terminal module (CT) from minor ampullate spidroins and 1-4 repeat modules (W) from aciniform spidroins. Physical characteristics and structures of NTW1-4CT/PLCL (25/75, w/w) blend scaffolds were carried out by scanning electron microscope (SEM), water contact angles measurements, Fourier transform infrared (FTIR) spectroscopy and tensile mechanical tests. Results showed that blending with spidroins decreased diameters of nanofibers and increased porosity and wettability of scaffolds. Additionally, chimeric spidroins undergone a similar structural transition in electrospinning process as with the formation process of native and artificial spider silks from other spidroins. With amounts of W modules increasing, the tensile strength and elongation of blend scaffolds were also increased. Particularly, NTW4CT/PLCL (25/75) scaffolds revealed much higher breaking stress than pure PLCL scaffolds. In vitro experiments, human umbilical vein endothelial cells (HUVEC) cultured on NTW4CT/PLCL (25/75) scaffolds displayed significantly higher activity of proliferation and adhesion than on pure PLCL scaffolds. All results suggested that chimeric spidroins/PLCL, especially NTW4CT/PLCL (25/75) blend nanofibrous scaffolds had promising potential for vascular tissue engineering.

Production and Properties of Triple Chimeric Spidroins.

All spider silk proteins (spidroins) are composed of N- and C-terminal domains (NT and CT) that act as regulators of silk solubility and assembly and a central repetitive region, which confers mechanical properties to the fiber. Among the seven types of spider silks, aciniform silk has the highest toughness. Herein, we fused NT and CT domains from major and minor ampullate spidroins (MaSps and MiSps), respectively, to 1-4 repeat domains (W) from another type of spidroin, aciniform spidroin 1(AcSp1). Although the three domains originate from distantly related spidroin types, they keep their respective characteristics in the chimeric spidroins. Furthermore, all chimeric spidroins could form silk-like fibers by manual-drawing. In contrast to fibers made in the same manner from W domains only, NTW1-4CT fibers show superior mechanical properties. Our results suggest that chimeric spidroins with NT, CT, and repeat domains can be designed to form fibers with various mechanical properties.

Silk gene expression of theridiid spiders: implications for male-specific silk use

Spiders (order Araneae) rely on their silks for essential tasks, such as dispersal, prey capture, and reproduction. Spider silks are largely composed of spidroins, members of a protein family that are synthesized in silk glands. As needed, silk stored in silk glands is extruded through spigots on the spinnerets. Nearly all studies of spider silks have been conducted on females; thus, little is known about male silk biology. To shed light on silk use by males, we compared silk gene expression profiles of mature males to those of females from three cob-web weaving species (Theridiidae). We de novo assembled species-specific male transcriptomes from Latrodectus hesperus, Latrodectus geometricus, and Steatoda grossa followed by differential gene expression analyses. Consistent with their complement of silk spigots, male theridiid spiders express appreciable amounts of aciniform, major ampullate, minor ampullate, and pyriform spidroin genes but not tubuliform spidroin genes. The relative expression levels of particular spidroin genes varied between sexes and species. Because mature males desert their prey-capture webs and become cursorial in their search for mates, we anticipated that major ampullate (dragline) spidroin genes would be the silk genes most highly expressed by males. Indeed, major ampullate spidroin genes had the highest expression in S. grossa males. However, minor ampullate spidroin genes were the most highly expressed spidroin genes in L. geometricus and L. hesperus males. Our expression profiling results suggest species-specific adaptive divergence of silk use by male theridiids.

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.

Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types.

Structure and Function of C-Terminal Domain of Aciniform Spidroin

C-terminal domains (CTDs) of various spidroins are relatively conserved in the amino acid sequence and have been suggested to perform similar functions. Here, we solved the structure of the CTD of an aciniform spidroin using NMR spectroscopy with a two-point mutant and studied its functional role with several constructs. The CTDs of aciniform, major, and minor ampullate spidroins adopt the same domain-swapping dimeric folding although their sequence identities are 24-40%. Unlike CTDs of major and minor ampullate spidroins, the aciniform CTD had no obvious effects on preventing spidroins from aggregation in storage but slightly enhanced protein assembly under shear force. The differential functions may result from significant differences in detailed structures and properties of repetitive regions of various types of spidroins. Nevertheless, all CTDs may have a common functional role: dimerization of the CTD doubles the size of silk proteins, reduces the number of chain ends, and thus leads to fewer chain end defects in the fibers formed.

Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTDMi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTDMi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTDMi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.

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.

Molecular dynamics simulations of the minor ampullate spidroin modular amino acid sequence from Parawixia bistriatra: insights into silk tertiary structure and fibre formation

Spider fibres are primarily composed of proteins that are secreted by specialised glands found in different groups of arthropods. Because of their unique mechanical characteristics, it is of great interest to understand how the influence of repetitive modules within the fibres affects the final protein structure. Because each fibre is composed of a diverse set of repeated modular sequences, the differences between fibres allow for their structural comparison and, thereby, their functional comparison. Herein, we present molecular dynamics simulations of partial sequences from minor ampullate Spidroin (MiSp) silk of the Brazilian species Parawixia bistriata. Our data show that the formation of β-sheet structures is directly related to the N-termini alignment of the modules. The N-terminal alignment gives rise to a high number of hydrogen bonds whose formation is driven by repeated alanine (Ala) sequences, which, in turn, lead to increased fibre strength. This increased fibre strength contributes significantly to the final tertiary structure of the silk.