Correlation between fibroin amino acid sequence and physical silk properties

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.     

Construction of silk fiber core in lepidoptera

The formation and properties of lepidopteran silk fibers depend on amino acid repeats in the principal protein, heavy chain fibroin (H-fibroin). In H-fibroins of the "bombycoid" type, concatenations of alanine or of the GAGAGS crystalline motifs (1st tier repeats) and adjacent sequences breaking periodicity make 2nd tier repeats. Two to six such repeats comprise a 3rd tier assembly, and 12 assemblies, linked by an amorphous sequence, constitute the repetitive H-fibroin region. Heterogeneity in the repeat length and intercalation of amorphous regions prevent excessive crystallization. In the "pyraloid" H-fibroins, iterations of simple motifs are absent and assemblies of several complex motifs constitute highly regular repeats that are organized in about 12 highest order reiterations without specific spacers. Repeat homogeneity appears crucial for the alignment and interaction of the disjunct motifs that must be registered precisely to form crystallites; repeat heterogeneity is associated with decreased fiber strength. Both H-fibroin types are typically hydrophobic, and their secretion requires disulfide linkage to light chain fibroin and participation of another protein, P25. These auxiliary proteins are absent in saturniid moths with amphiphilic H-fibroin repeats. The selection at nucleic acid and protein levels and the availability of nutrients play roles in H-fibroin evolution.     

Protein composition of silk filaments spun under water by caddisfly larvae

Silk fiber produced by the larvae of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) is composed of two filaments embedded in a layer of glue proteins. In an aerial environment Lepidoptera spin silk filaments assembled from heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and the glycoprotein P25. The silk filament of caddisflies, which is produced and persists in water, contained homologues of H-fibroin (>500 kDa) and L-fibroin (25 kDa) but not of P25. The amphiphilic nature of H-fibroin and its high content of charged amino acids probably facilitate the secretion and storage of a covalently linked L-fibroin/H-fibroin dimer in the absence of P25. Several types of short amino acid motifs were arranged in orderly fashion in the regularly reiterated repeats that made up more than 95% of the length of H-fibroin. The H-fibroins of Hydropsyche angustipennis and Limnephilus decipiens from different caddisfly suborders contained GPXGX, SXSXSXSX, and GGX motifs such as the lepidopteran and spider silks but differed from them by a lack of poly(A) and poly(GA) motifs. H-fibroins of both caddisfly species harbored a conserved repeat of 31 residues but were distinguished by a few species-specific motifs and their organization in higher order repeats. Structural differences may be related to the silk function as a catching net in H. angustipennis and a stitching fiber in L. decipiens.     

The Design of Silk Fiber Composition in Moths Has Been Conserved for More Than 150 Million Years

The silk of caterpillars is secreted in the labial glands, stored as a gel in their lumen, and converted into a solid filament during spinning. Heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and P25 protein constitute the filament core in a few species that have been analyzed. Identification of these proteins in Yponomeuta evonymella, a moth from a family which diverged from the rest of Lepidoptera about 150 million years ago, reveals that the mode of filament construction is highly conserved. It is proposed that association of the three proteins is suited for long storage of hydrated silk dope and its rapid conversion to filament. Interactions underlying these processes depend on conserved spacing of critical amino acid residues that are dispersed through the L-fibroin and P25 and assembled in the short ends of the H-fibroin molecule. Strength, elasticity, and other physical properties of the filament are determined by simple amino acid motifs arranged in repetitive modules that build up most of the H-fibroin. H-Fibroin synergy with L-fibroin and P25 does not interfere with motif diversification by which the filament acquires new properties. Several types of motifs in complex repeats occur in the silks used for larval cobwebs and pupal cocoons. Restriction of silk use to cocoon construction in some lepidopteran families has been accompanied by simplification of H-fibroin repeats. An extreme deviation of the silk structure occurs in the Saturniidae silkmoths, which possess modified H-fibroin and lack L-fibroin and P25. [Reviewing Editor: Dr. David Pollok]     

Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly <Emphasis Type="Italic">Stenopsyche marmorata</Emphasis> (Trichoptera; <Emphasis Type="Italic">Stenopsychidae</Emphasis>)

The silks of both Lepidoptera and its sister order Trichoptera contain a homologue of heavy chain (H-fibroin), which is assumed to determine the physical properties of the fiber, such as elasticity and toughness. The long repetitive region of the H-fibroin caddisfly Stenopsyche marmorata shows a conspicuous hierarchical structure that is composed of huge units, which are mainly constructed from four large blocks (SA, SB, SC and SD) arranged in an orderly fashion. Each block contains short, distinct motifs such as SXSXSX(SX), GPXG(X)_1–3 or triplet GGX, which also occur in lepidopteran and spider filaments. The SA, SB and SC blocks have nearly fixed amino acid numbers, while the length of the SD block varies, usually due to a variable number of GPXGXXX repeats. The multiple sandwich structure that occurs in the SB block is assumed to be unique to the caddisfly and may be related to the use of silk in an aqueous environment. The overall average of hydrophilicity in the repetitive H-fibroin region of S. marmorata is −0.609, whereas hydrophobicity prevails in most lepidopteran H-fibroins. Gly (29.51%), Pro (11.28%) and Ser (10.90%) are the three predominant amino acids of H-fibroin, and the high content of essential amino acids reflects the energy-rich food resources of the caddisfly. The H-fibroin of S. marmorata is about 400–500 kDa and expressed in both the middle and posterior silk glands, which is different from the expression pattern in Lepidoptera species.     

Molecular characterization and evolutionary study of spider tubuliform (eggcase) silk protein

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the only silk produced just during a short period of time, the reproductive season, in the spider's life. To understand the molecular characteristics of the proteins composing this silk, we constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Sequencing of tubuliform silk cDNAs reveals the repetitive architecture of its coding sequence and novel amino acid motifs. The inferred protein, tubuliform spidroin 1 (TuSp1), contains highly homogenized repeats in all three spiders. Amino acid composition comparison of the predicted tubuliform silk protein sequence to tubuliform silk indicates that TuSp1 is the major component of tubuliform silk. Repeat unit alignment of TuSp1 among three spider species shows high sequence conservation among tubuliform silk protein orthologue groups. Sequence comparison among TuSp1 repetitive units within species suggests intragenic concerted evolution, presumably through gene conversion and unequal crossover events. Comparative analysis demonstrates that TuSp1 represents a new orthologue in the spider silk gene family.     

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.     

The synthesis of RNA and silk protein in Galleria mellonella silk glands

The silk protein synthesis in silk glands of Galleria mellonella is preceded by the increase of total RNA content. The levels of the main RNA classes: 28S and 18S rRNA, tRNA as well as poly(A) + RNA change proportionally to the total RNA pool of glands. The fibroin-like silk protein of molecular weight of about 240 000 is characterized by the high content of four amino acids: glycine, alanine, serine and leucine, which account for more than 70% of amino acid residues. This fibroin-like protein is present in the posterior, middle and anterior parts of silk gland of the last instar larvae.     

Sequence analysis corresponding to the PPE and PE proteins in<Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and other genomes

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC 1551 strains of theMycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and β-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40–100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of theMethanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function inArchaea andBacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 β-strands. We suggest that proteins with AB repeats inMycobacterium tuberculosis and other genomes may be associated as surface antigens. TheM. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in theM. leprae genome compared to theM. tuberculosis genome.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.     

Silk tape nanostructure and silk gland anatomy of trichoptera

Caddisflys (order Trichoptera) construct elaborate protective shelters and food harvesting nets with underwater adhesive silk. The silk fiber resembles a nanostructured tape composed of thousands of nanofibrils (～ 120 nm) oriented with the major axis of the fiber, which in turn are composed of spherical subunits. Weaker lateral interactions between nanofibrils allow the fiber to conform to surface topography and increase contact area. Highly phosphorylated (pSX)(4) motifs in H-fibroin blocks of positively charged basic residues are conserved across all three suborders of Trichoptera. Electrostatic interactions between the oppositely charged motifs could drive liquid-liquid phase separation of silk fiber precursors into a complex coacervates mesophase. Accessibility of phosphoserine to an anti-phosphoserine antibody is lower in the lumen of the silk gland storage region compared to the nascent fiber formed in the anterior conducting channel. The phosphorylated motifs may serve as a marker for the structural reorganization of the silk precursor mesophase into strongly refringent fibers. The structural change occurring at the transition into the conducting channel makes this region of special interest. Fiber formation from polyampholytic silk proteins in Trichoptera may suggest a new approach to create synthetic silk analogs from water-soluble precursors.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.     

Evidence for unequal crossing over in the evolution of the neurofilament polypeptide H.

Two allelic forms of the rabbit neurofilament protein H, designated H1 and H2, differ by approximately 6% in their electrophoretic mobilities. We have used techniques of peptide and nucleic acid analysis to determine that this difference is located in the central portion of the COOH-terminal tail, a region of the H protein that can project from the filament, and may form cross-bridges. This region comprises tandem amino acid motifs containing the sequence KSP, in which the serine residues are sites of phosphorylation. The sequence of this repetitive region of H2 is 132 nucleotides (44 amino acids) shorter than H1 and differs significantly in the arrangement of the repeated motifs. The difference suggests that both forms have evolved from a common ancestor over the past several million years by the process of unequal crossing over and that this process figured importantly in generating the repetitive region. In addition, the results indicate that this region is functionally redundant and that the length and structure of the tail of rabbit H resemble H from other mammalian species more closely than was suggested by a previously reported cDNA, on account of a single nucleotide difference.     

The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture

Sericin is the silk protein that covers fibroin fibers and functions as a `glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 g sericin hydrolysate and 350 g SerD (the peptide containing the two repetitive units) ml^–1, respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation.     

Dynamic Rearrangement Within the Antheraea pernyi Silk Fibroin Gene Is Associated with Four Types of Repetitive Units

We characterized a full-length gene encoding wild silkmoth Antheraea pernyi fibroin (Ap-fibroin) to clarify the conformation of repetitive sequences. The gene consisted of a first exon encoding 14 amino acid residues, a short intron (120 bp), and a long second exon encoding 2,625 amino acid residues. Three amino acids, alanine, glycine, and serine, amounted to 81% of the Ap-fibroin sequence. The Ap-fibroin, except for 155 residues of the amino terminus, was composed of 80 tandemly arranged polyalanine-containing units (motifs). A motif was a doublet of a polyalanine block (PAB) and a nonpolyalanine block (NPAB). Seventy-eight of the 80 motifs were classified into four types based on differences in the NPAB sequences. Although respective motifs were significantly conserved, many rearrangements were observed within the second exon, i.e., the triplication of a 558-bp-long sequence and other duplication events of shorter sequences. Chi-like sequences, GCTGGAG, might contribute to the rearrangement within the gene as described in human minisatellite loci, because they were found at specific sites of NPAB-encoding sequences in three of four types of motifs. The present results support the idea that the Ap-fibroin gene is unstable like minisatellite sequences and that the evolution of this gene is strongly associated with its instability. Received: 18 February 2000 / Accepted: 30 June 2000