Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization

Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into “attachment discs” that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (∼100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin type, while motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive “linker”, which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers.     

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori

Sericins are soluble silk components encoded in Bombyx mori by three genes, of which Ser1 and Ser3 have been characterized. The Ser1 and Ser3 proteins were shown to appear later in the last larval instar as the major sericins of cocoon silk. These proteins are, however, virtually absent in the highly adhesive silk spun prior to cocoon spinning, when the larvae construct a loose scaffold for cocoon attachment. We show here that the silk-gland lumen of the feeding last instar larvae contains two abundant adhesive proteins of 230 kDa and 120 kDa that were identified as products of the Ser2 gene. We also describe the sequence, exon–intron structure, alternative splicing and deduced translation products of this gene in the Daizo p50 strain of B. mori. Two mRNAs of 5.7 and 3.1 kb are generated by alternative splicing of the largest exon. The predicted mature proteins contain 1740 and 882 amino acid residues. The repetitive amino acid sequence encoded by exons 9a and 9b is apparently responsible for the adhesiveness of Ser2 products. It has a similar periodic arrangement of motifs containing lysine and proline as a highly adhesive protein of the mussel Mytilus edulis.     

Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and β-cyclodextrin

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.     

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.     

Spider silk fibroin enhances insulin secretion and reduces blood glucose levels in diabetic mice

Fibroin silk proteins make up the cocoons of silkworms and spider webs and are rich in glycine and alanine residues. Recent studies have shown that silk fibroin hydrolysate from silkworms improves blood glucose and lipid metabolism. In the present study, we investigated the anti-diabetic effects of spider silk fibroin protein in type 2 diabetic mice. Recombinant AvMaSp-R, which consists of the 240 amino acid repetitive domain of major ampullate silk protein (AvMaSp) from the spider Araneus ventricosus, was produced in baculovirus-infected insect cells. We tested the effects of oral AvMaSp-R administration on serum insulin and blood glucose levels in diabetic mice and found that AvMaSp-R increases serum insulin levels and reduces blood glucose levels in diabetic mice. Consequently, our results are the first to provide evidence that silk fibroin protein from spiders enhances insulin secretion, which leads to reduced blood glucose levels in type 2 diabetic mice.     

Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties

Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.     

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [¹⁴C]xylose transferred from UDP-d-[¹⁴C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V_max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K_m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [¹⁴C]xylose transfer to silk was alkali labile, and [¹⁴C]xylitol was formed when [¹⁴C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [¹⁴C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [¹⁴C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [¹⁴C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V_max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V_max value close to 20% of that of intact fibroin.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.     

Intramolecular homologous recombination event occurred in the spider egg case silk gene CySp2 of wasp spider Argiope bruennichi

To gain further understanding of egg case silk proteins gene family, Zhao et al. (2006) isolated two full-length cDNAs for egg case silk proteins, cylindrical silk protein 1 (CySp1) and cylindrical silk protein 2 (CySp2), from the wasp spider, Argiope bruennichi. CySp2 was reported to contain no apparent Signal peptide sequences, and the CySp1-CySp2 complex, which would possess a signal peptide, would be transported across the endoplasmic reticulum and secreted to the Golgi. According to a report by Hayashi, genomic DNA sequencing is one approach that can be successfully utilized to retrieve 5′ ends of silk genes; using this method, we retrieved the 5’ end of CySp1. We found that CySp2 contained a typical signal peptide similar to that found in CySp1; thus, due to technical limitations, an artificial error had occurred in the CySp2 sequence reported by Zhao et al.     

Microbial production of spider silk proteins

The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.     

The amino acid sequence of cytochrome c from niger-seed, Guizotia abyssinica

The amino acid sequence of cytochrome c from niger-seed has been determined by sequence analysis of chymotryptic and tryptic peptides using the dansyl-phenylisothiocyanate method and by qualitative analysis of peptide composition by the dansyl method. Although the spectral ratios indicated the protein was not completely pure, no indication of impurity was found during the sequence analysis and no peptides in addition to those given here were obtained. In certain cases the alignment of peptides was by homology with other cytochromes c. Four residues in the proposed sequence, alanine-1, cysteine-25, histidine-26 and lysine-61 were identified only from peptide compositions. The amino-terminus of the protein is acetylated. The sequence contains two residues of ϵ-N-trimethyllysine.     

Rise and fall of the delta globin gene

The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established. The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension. This peptide has the general characteristics of a signal sequence. The promoter region of phoE has no homlogy with the consensus sequence of E. coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed. The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782. A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed. Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212.When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found. Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris)

The amino acid sequence of the plastocyanin from French bean (Phaseolus vulgaris) was determined. The protein consists of a single polypeptide chain of 99 residues, and the sequence was determined by characterization of CNBr, tryptic, chymotryptic and thermolysin peptides. When the sequence is compared with that from the plastocyanin of the unicellular green alga Chlorella fusca, the French-bean protein shows the deletion of the N-terminal residue, a two residue insertion and 53 identical residues. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50037 (16pp., 1 microfiche) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.