Dragline Silk: A Fiber Assembled with Low-Molecular-Weight Cysteine-Rich Proteins

Abstract

Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands

“…(b) Homologs of the five Cysteine Rich Proteins (CRPs) identified by Pham et al . 40 were identified by best BLASTP match with an e-value <1e-5 (see Supplementary File 1 ). Gland and species abbreviations, and sample sizes, as in Fig.…”

“…Other proteins known to form structural constituents of fibers include the Egg Case Proteins (ECP 1 & 2) 39 and Cysteine Rich Proteins (CRPs) 40 . We found ECPs were almost exclusively expressed in tubuliform glands, as expected, but CRPs, formerly only described from major ampullate silk 36 , 40 , were expressed in each of the individual gland types (Fig. 1b ) and are far more diverse than previously recognized.…”

“…We defined a transcript as spider-specific if there were no BLASTX alignments to a UniProt sequence outside the Araneae with an e-value <1e-5. A transcript was defined as encoding a known silk structural protein if it had a best BLASTX match ≤1e-5 to a UniProt defined spider silk protein, it had a best BlastP match to a Cysteine Rich Protein (CRP) 40 , or it was in the BlastClust cluster with such a transcript (Supplementary File 1 ).…”

Tick and Host Derived Compounds Detected in the Cement Complex Substance

“…The amino acid profile of dragline silk in Argiope trifasciata has a high Gly content (approximately 40%), which correlates with the Gly content (36.5%-43.9%) of the two major components of the spider dragline silk, MaSp1 and MaSp2 proteins [50]. A family of low-molecular weight Cysteine-rich proteins have been also characterized as involved in dragline silk formation [51]. However, Cys was an amino acid with a relative low representation (1.8%-2.0%) in tick cement (Table S3).…”

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers

“…However, recent biochemical evidence is emerging that supports additional proteins are spun into dragline silk. In fact, a new paradigm is surfacing that suggests dragline silk consists of additional spidroin family members as well as low molecular weight proteins that are spun with MaSp1 and MaSp2 [ 9 , 10 ]. With advances in technology, there is a pressing need to develop new biomaterials that have outstanding material properties and are environmentally friendly.…”