Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.
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.
Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine subrepeats, which account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in TuSp1. However, polyserine blocks and short polyalanine stretches were highly iterated within the primary sequence, and (13)C NMR spectroscopy demonstrated that the majority of alanine was found in a beta-sheet structure in post-spun egg case silk. The TuSp1 repeat unit does not display substantial sequence similarity to any previously described fibroin genes or proteins, suggesting that TuSp1 is a highly divergent member of the spider silk gene family.
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