Novel Molecular and Mechanical Properties of Egg Case Silk from Wasp Spider, <i>Argiope bruennichi</i>

Zhao, Aichun; Zhao, Tianfu; Nakagaki, Koichi; Zhang, Yuansong; Sima, Yang-Hu; Miao, Yongwei; Shiomi, Kunihiro; Kajiura, Zenta; Nagata, Yoko; Takadera, Masayuki; Nakagaki, Masayuki

doi:10.1021/bi052414g

Cited by 61 publications

(57 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…N-terminal fragments (Ϸ200 bp) of TuSp1 were obtained, respectively, from a genomic DNA and cDNA library by PCR using degenerated primer sets (5Ј-ATG GTT TGG RTN ACN XYN ATQ GC-3Ј and 5Ј-RTC XTG XTG XTC XTG RAA NGG RAA-3Ј) designed on the basis of the conserved amino acid sequences of the N termini from known TuSp1s (1,28). One of the two fragments obtained from genomic DNA was the same as the one from the cDNA library.…”

Section: Methodsmentioning

confidence: 99%

Solution structure of eggcase silk protein and its implications for silk fiber formation

Lin

Huang

Zhang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with ␣-helical and random coil structures in silk glands into insoluble fibers with mainly ␤-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a Ϸ366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N-and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at >37°C and a protein concentration of >0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks.NMR ͉ spider silk ͉ structural transition ͉ TuSp1 S pider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. The orb-web spiders employ up to seven types of abdominal glands to produce silks for various purposes, ranging from prey capture to offspring protection in egg cases (1). Among seven different types of silks, tubuliform silk (eggcase silk) is unique because of its high serine and low glycine content (2, 3). Eggcase silk fibroins are synthesized only in female tubuliform (cylindrical) silk glands for the construction of protective egg cases, where eggs undergo development. A number of eggcase silk cDNA clones from orb-weaver superfamilies have recently been isolated (4-8). The length of eggcase silk cDNA varies from 10 to 13 kb in different species. The sequence of the inferred protein, tubuliform spidroin 1 (TuSp1) accounts for the amino acid (aa) composition of tubuliform silk very well, indicating that TuSp1 is the major component of eggcase silk (4-8). Both in situ hybridization and immunoblot analyses show that TuSp1 is expressed specifically in the tubuliform gland (4). Eggcase silk must be sufficiently robust to resist different threats, such as predator/parasitoid invasion or temperature fluctuations. Compared with the widely studied spider dragline silk, eggcase silk is slightly less tough but more resistant ...

show abstract

Section: Methodsmentioning

confidence: 99%

Solution structure of eggcase silk protein and its implications for silk fiber formation

Lin

Huang

Zhang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

show abstract

“…GPGX n repeats, which are found in MaSp2 and Flag silk, have been hypothesized to form ␤ turn structures, which provide extensibility to the threads (14). To date, the only reported full-length gene sequences from the spider silk family include masp1, masp2, and cysp1 (11,18). These gene sequences are known to encode large molecular mass proteins, with their corresponding predicted molecular masses representing 250, 311, and 277 kDa, respectively.…”

mentioning

confidence: 99%

Pyriform Spidroin 1, a Novel Member of the Silk Gene Family That Anchors Dragline Silk Fibers in Attachment Discs of the Black Widow Spider, Latrodectus hesperus

Blasingame

Tuton-Blasingame

Larkin

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed insolution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion.

show abstract

“…MS/MS 4 analyses of black widow peptides generated from solubilized egg case materials digested with trypsin have shown that the large diameter core fibers contain the fibroin TuSp1, as well as the egg case proteins ECP-1 and ECP-2 (egg case protein 1 or 2), which form a trimeric complex in cob weavers (14,16,17). Real time quantitative PCR and Northern blot analyses have demonstrated tubuliform-restricted patterns of expression for these gene products (14,(17)(18)(19). Although it has been widely accepted by the silk community that the aciniform glands produce egg case silk constituents and wrapping silk, no molecular evidence has been reported to demonstrate that aciniform-specific fibroins are directly assembled into these diverse materials (1).…”

mentioning

confidence: 99%

Aciniform Spidroin, a Constituent of Egg Case Sacs and Wrapping Silk Fibers from the Black Widow Spider Latrodectus hesperus

Vasanthavada

Falick

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct ϳ300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform glandrestricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.The ability to spin multiple task-specific silks is a defining feature of the diverse order Araneae (Ͼ37,000 described species). Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse mechanical properties (1). Spinning high performance fibers with different mechanical properties enable spiders to perform a wide range of functions, including prey capture, locomotion, and protection of developing offspring (2).Amino acid sequences of spider fibroins (spidroins) share a number of distinctive features. Repeats of four fundamental amino acid motifs characterize the majority of sequenced spider silks as follows: (i) alternating glycine and alanine ((GA) n ), (ii) polyalanine (A n ), (iii) GGX (X ϭ subset of residues), and (iv) GPGGX. Biochemical studies indicate that these motifs correspond to distinct structural modules, e.g. A n and (GA) n repeats form crystalline ␤-sheets, whereas ␤-spirals are generated from a series of concatenated ␤-turns from the repeat structure GPGGX (3). It has been proposed that these different structural modules contribute to the mechanics of the fibers. Combinations of these motifs form larger repetitive units termed ensemble repeats, which are organized in tandem copies throug...

show abstract

Novel Molecular and Mechanical Properties of Egg Case Silk from Wasp Spider, Argiope bruennichi

Cited by 61 publications

References 36 publications

Solution structure of eggcase silk protein and its implications for silk fiber formation

Solution structure of eggcase silk protein and its implications for silk fiber formation

Pyriform Spidroin 1, a Novel Member of the Silk Gene Family That Anchors Dragline Silk Fibers in Attachment Discs of the Black Widow Spider, Latrodectus hesperus

Aciniform Spidroin, a Constituent of Egg Case Sacs and Wrapping Silk Fibers from the Black Widow Spider Latrodectus hesperus

Contact Info

Product

Resources

About