Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders

Abstract

Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk spidroin-1 from Nephila spiders provide the basis for mechanical-elastic property studies of silk for biotechnological and biomedical potential applications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

The Biomedical Use of Silk: Past, Present, Future

“…Already, today, recombinant DNA technology, in combination with Escherichia coli expression systems, has revolutionized silk research and is opening up new commercial avenues ( Table 5 ). However, silk presents a number of specific challenges; for example, the silk molecules cannot be post‐translationally modified, but this is required to faithfully mimic the silk protein . The expression of native‐sized silk proteins is also not possible using standard E. coli expression systems because of the highly repetitive nature of the gene constructs, the very high glycine content of the protein, and the high molecular weight of the product (250–320 kDa).…”

Silk Spinning in Silkworms and Spiders

“…MaSp1 and MaSp2 isolated from N. clavipes dragline silk fibers contain phosphorylation sites in their respective repetitive regions [58,59], as does FibRep [38], the fibroin light chain and the P25 [60]. The impact of these phosphorylations on the silk formation process have not been thoroughly studied, but phosphorylation in general affect soluble proteins by altering conformational states.…”

Advances in understanding the properties of spider silk

“…Expression has been reported using the natural cDNAs as well as codon-optimized synthetic genes (Prince et al, 1995;Xia et al, 2010;Xu et al, 2007). Although endogenous spidroins expressed in MA glands have been shown to experience post-translational modifications (PTMs), including phosphorylation and hydroxylation, it is unclear whether these PTMs to spidroins are necessary for production of high-quality synthetic silk fibers (Dos Santos-Pinto et al, 2014). Using a bioreactor to optimize growth conditions, metabolically engineered bacterial strains have been generated to cope with intrinsically large numbers of Ala and Gly codons within the natural cDNAs, a strategy that has been effectively utilized to express some of the largest spidroin constructs (Xia et al, 2010).…”