Untangling spider silk evolution with spidroin terminal domains.

Jessica E Garb,Nadia A Ayoub,Cheryl Y Hayashi

doi:10.1186/1471-2148-10-243

Abstract

BackgroundSpidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains.ResultsWe report 11 additional spidroin N-termini found by sequencing ~1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs.ConclusionsA non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk assembly. Phylogenetic analyses of the conserved N- and C-terminal domains illustrate dramatic radiation of the spidroin gene family, involving extensive duplications, shifts in expression patterns and extreme diversification of repetitive structural sequences that endow spider silks with an unparalleled range of mechanical properties.

Highlights

Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers
Total RNA was extracted from each tissue type by homogenization in TRIzol (Invitrogen, Carlsbad, CA) and further purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). mRNA was isolated with oligo-(dT)-tagged magnetic beads (Invitrogen). cDNA was synthesized using Invitrogen’s SuperScript Choice protocol, starting with the anchored oligonucleotide18V. cDNAs were fractionated by size using ChromaSpin 1000 columns (Clontech, Mountain View, CA), blunt-end ligated into pZErO-2 vector (Invitrogen), and electroporated into TOP10 Escherichia coli (Invitrogen)
Fourteen additional published N-terminal spidroin sequences, and one unpublished N-terminal sequence reported in GenBank as a “major ampullate dragline silk protein” (AY945306) but which we attribute to Flag because it flanks repetitive sequence characteristic of Flag spidroins, were included in subsequent analyses (Table 1)

Summary

Introduction

Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. There are numerous types of spider silks and each has its own suite of mechanical properties, including exceptional tensile strengths, extensibilities, and toughness [1,2] This mechanical diversity is associated with the distinct functional demands of the different silk types and largely stems from variation in the molecular composition of the silk proteins [3,4]. Flanking a spidroin’s long core region of iterated repeats are short, non-repetitive amino (N) and carboxy (C) terminal domains (Figure 1). Sequence conservation of these terminal domains across spidroins, and their presence in silk fibers [16,17,18], imply they serve some critical role. Predicted signal peptides in the N-terminal domain are thought to regulate spidroin secretion from silk gland cells [19,20,21]; whereas experimental data suggest the N- and Cterminal domains contribute to fiber assembly [22,23,24,25,26,27,28]

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