Molecular mechanisms of the high performance of spider silks revealed through multi-omics analysis

Abstract

Spider silk is considered a promising next-generation biomaterial due to its exceptional toughness, coupled with its renewability and biodegradability. Contrary to the conventional view that spider silk is mainly composed of two types of silk proteins (spidroins), MaSp1 and MaSp2, multi-omics strategies are increasingly revealing that the inclusion of complex components confers the higher mechanical properties to the material. In this review, we focus on several recent findings that report essential components and mechanisms that are necessary to reproduce the properties of natural spider silk. First, we discuss the discovery of MaSp3, a newly identified spidroin that is a major component in the composition of spider silk, in addition to the previously understood MaSp1 and MaSp2. Moreover, the role of the Spider-silk Constituting Element (SpiCE), which is present in trace amounts but has been found to significantly increase the tensile strength of artificial spider silk, is explored. We also delve into the process of spidroin fibril formation through liquid-liquid phase separation (LLPS) that forms the hierarchical structure of spider silk. In addition, we review the correlation between amino acid sequences and mechanical properties such as toughness and supercontraction, as revealed by an analysis of 1,000 spiders. In conclusion, these recent findings contribute to the comprehensive understanding of the mechanisms that give spider silk its high mechanical properties and help to improve artificial spider silk production.

Keywords: Spider Silkome Database; liquid–liquid phase separation; spider silk; spidroin; structural proteins.

Figure 1

(A) Schematic illustration of the spinning process and the hierarchical structure of spider silk. The major ampullate gland contains liquid-phase proteins that constitute the silk, including spidroins, cysteine-rich proteins (CRP) and spider silk constituting elements (SpiCE). The final solid-state silk released is composed of a surface coating skin and the core bundle of nanofibrils. (B) Spidroin is composed of repeating domains consisting of crystalline and amorphous regions and N/C-terminal domains that play roles in the fibrillization process. (C) The repeat sequences and exonic regions of MaSp1, MiSp, and AgSp in Araneus ventricosus. The composition, length and number of repeated amino acids vary depending on the type of spidroin.

Figure 2

Multi-omics analysis combining high-resolution genomic analysis including full-length spider silk gene sequences, transcriptomic analysis of silk glands, and proteomic analysis of dragline silks identified the presence of a novel Spidroin (MaSp3) and a small molecular weight protein, SpiCE. Reprinted from Ref. 24 Copyright (2021) National Academy of Sciences.

Figure 3

(A) Artificial spider silk film made with SpiCE and MaSp2, where the addition of 1% SpiCE (w/w) more than doubles the tensile strength (right graph). (B) Stress-strain curve of artificial spider silk produced by combining SpiCE and MaSp2, where the addition of SpiCE (right) causes the characteristic yield point of the spider silk. Modified from Ref. 24 Copyright (2021) National Academy of Sciences.

Figure 4

(A) The schematic figure of the conformational changes of spidroins as they pass through the Major Ampullate Gland, influenced by the pH gradient (from neutral to acidic) and ion exchange, causing the CTD and NTD to dimerize. (B) Spider silk proteins undergo liquid-liquid phase separation under phosphate conditions at neutral pH, and further fibrillation occurs when the pH shifts to acidic conditions. Shear stress in this fibrillated state leads to the formation of a hierarchical fiber structure. Scale bar is 10 μm, Reproduced and modified from Ref. 42 under Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Figure 5

Overview of the physical properties of 446 spider silk samples. (A) Pearson correlation heatmap of the physical properties of dragline silk fibers measured in Arakawa et al. [46]. Toughness is not only correlated with tensile strength and strain at break but also correlated with Young’s modulus. Supercontraction is correlated with strain at break. (B) Scatter plot of toughness versus strain at break (with spot size proportional to tensile strength). The collected samples represent an almost continuous spectrum of toughness from <0.01 to >0.40 GJ/m3. Spots are colored according to broad phylogenetic grouping: Araneoidea (red), RTA clade (light blue). (C) Screenshots of the Spider Silkome Database (https://spider-silkome.org). Reprinted from Ref. 46 under Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Figure 6

Scatterplot of physical properties (toughness or supercontraction) as a function of the average motif abundance per repeat of certain amino acid motifs. AGQG motif in MaSp1 is positively correlated with supercontraction, AAAAAAAA motif of MaSp2 is negatively correlated, and YGQGG motif in MaSp1 is positively correlated with toughness. Reprinted and modified from Ref. 46 under Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).