Conserved C-Termini of Spidroins Are Secreted by ... - ACS Publications

Are Secreted by the Major Ampullate Glands and Retained in the Silk Thread ... Morphology and Composition of the Spider Major Ampullate Gland and ...
0 downloads 0 Views 97KB Size
Download PDF
Biomacromolecules 2004, 5, 840-845

840

Conserved C-Termini of Spidroins Are Secreted by the Major Ampullate Glands and Retained in the Silk Thread Alexander Sponner, Eberhard Unger, Frank Grosse, and Klaus Weisshart*,† Institute of Molecular Biotechnology, Beutenbergstrasse 11, 07745 Jena, Germany Received September 26, 2003; Revised Manuscript Received December 22, 2003

The C-termini of Spidroins produced in the major and minor ampullate glands of spiders are highly conserved. Despite this conservation, no corresponding peptides have been identified in the spinning dopes or the silk filaments so far. To prove their presence or absence, polyclonal antibodies derived against fusion proteins containing the conserved C-terminal regions of both Spidroin 1 and 2 from the spider Nephila claVipes were generated. The antibodies reacted with high molecular weight polypeptides of the corresponding gland extracts and solubilized major ampullate filament and in addition to filament cross-sections. This demonstrates the existence of C-terminal specific peptides in the spinning dope and the mature Spidroins. Both the fusion proteins as well as the proteins contained within the gland lumen showed a reduction in their size under reducing conditions indicating the presence of disulfide bonds. Their high conservation and the biochemical data suggest crucial roles the C-termini play in the formation and/or structure of the corresponding silk filaments. Introduction Spider silks are composite materials of which the major constituents are fibrous proteins called Spidroins.1,2 The examination of spidroin cDNAs and spider silk genes sequenced so far has revealed that the Spidroins possess a highly hierarchical structure.3-11 Small common peptide motifs are iterated multiple times and are used in various combinations to form structural modules within the silk filaments.6,12,13 Rearrangements of the motifs have occurred during evolution and the repeat sizes do vary due to insertions or deletions of amino acid blocks within one repeat unit.3-6,13 This heterogeneity is based on the insertions or deletions of nucleotide triplet blocks in the 5′ parts of the genes coding for the repetitive elements.8,11,14 Some of the Spidroins possess nonrepetitive spacer regions that are interspersed between the repeat units.5,6 Nonrepetitive C-termini are common to the Spidroins synthesized in the major and minor ampullate as well as flagelliform glands of spiders belonging to the Araneoidea family, which comprises orb-weaving spiders.3-6,9,10 Their sequences within the ampullate and flagelliform Spidroins are highly conserved.10,11 A nonrepetitive N-terminal sequence has been identified for the Spidroins produced in the flagelliform and minor ampullate glands.5,6,15 They contain putative signal sequences that may have functions for the secretion of the Spidroins into the glandular lumen. These signal peptides are likely to be removed after secretion as suggested by the presence of appropriate consensus cleavage sites. Translational pauses occur during Spidroin synthesis giving rise to a ladder of smaller fragments that eventually * To whom correspondence should be addressed. † Present address: Carl Zeiss Jena GmbH, Carl-Zeiss-Promenade 10, 07745 Jena, Germany.

are converted into the full-length products,16-21 a process that resembles the fibroin synthesis in the silkworm Bombyx mori.21,22 The molecular weight of the glandular products have been reported to be in the range of 215-275 kDa23 as well as 320 kDa.17 The carboxy-termini produced in the major ampullate gland contain one or two cysteine residues that might serve to cross-link the spider silk fibroins by the formation of disulfide bridges.1 A reduction in size of fibroins extracted from the major ampullate glands from 275 to 180 kDa has indeed been observed under reducing conditions.23 The benchmark of Spidroins to date are the Spidroins 1 and 2 produced in the major ampullate gland of the orbweaving spider Nephila claVipes.2,24-32 Both contain an alteration of hydrophobic polyalanine and more hydrophilicic glycine-rich sequences that might adopt specific conformations.3,4,9,29,33-38 The periodicity in the hydropathy pattern within the repetitive parts might play a crucial role in the spinning process in analogy to the situation found in B. mori.50 B. mori fibroins possess highly hydrophilic ends as well as in the remaining parts smaller hydrophilic regions that are interspersed within larger hydrophobic blocks. Both, the ends and the alternating hydrophilic-hydrophobic parts contribute to the formation of micelles, which at higher protein concentrations aggregate into extended globules and finally gel like states. The gel like state is processed to an unsoluble thread or filament by applying shear and stress forces.50 If the N- and C-termini of the Spidroins play similar roles, it is currently unkown. N-terminal sequences are still lacking, and there is no proof presently if the C-termini are bona fide parts of the mature Spidroins.3-5,12 Their high conservation hints to an important function that might be regulatory rather than structural. However, an influence on the transition from the soluble spinning dope to the insoluble fiber during the spinning process cannot be ruled out.

10.1021/bm034378b CCC: $27.50 © 2004 American Chemical Society Published on Web 01/30/2004

Conserved C-Termini of Spidroins

In a first step to elucidate potential roles of the C-termini, we investigated in this study their presence in the spinning dope and the filament. Specific anti-sera reacted to high molecular weight products in gland extracts and the solubilized major ampullate filament demonstrating that the C-termini are an integral part of the mature Spidroins. Fusion peptides expressing the corresponding C-termini of both Spidroin 1 and 2 readily underwent disulfide bridge formation as was also seen with the high molecular weight proteins present in glandular extracts. Our biochemical data suggest that the conserved C-termini of Spidroins contribute to the formation and properties of the spider’s major ampullate filament. Material and Methods Spiders. The golden orb-weaving spider Nephila claVipes was obtained from Mascarino Tarantulas (Los Angeles, CA). The animals were kept at 80% humidity in little cages to discourage web building. For the experiments adult female animals at the 6th to 9th instar state were used. Cloning, Expression, and Purification of C-Terminal Spidroin Sequences. A cDNA clone for Spidroin 1 and one for Spidroin 2 were used to amplify the respective C-terminal sequences. The employed primers provided a distal EcoRI and a proximal NdeI site to an ATG in frame codon on the 5′-end and a BamHI site after the STOP codon at the 3′end. Sequences of the primer pairs were 5′-TTT GAA TTC CAT ATG GCT GCC TCT GCA GCT GCA TCC-3′/5′TTT GGA TCC TTA ACC TAG GGC TTG ATA AAC TG3′ for Spidroin 1 and 5′-TTT GAA TTC CAT ATG GGG CCA GGT TCT CAA GCT TCC GCT GC-3′/5′-TTT GGA TCC TTA AAA TGC ACT CAA AAC AGA TTG G-3′ for Spidroin 2, respectively. In the case of Spidroin 1, this leads to the amplification of nucleotides 1933-2247, which correspond to amino acids 645-748 according to the published sequences.10,14 For Spidroin 2, nucleotides 1561-1884 corresponding to amino acids 521-62739 were amplified. The sequences were cloned via EcoRI and BamHI into pBlueskript KS II+ (Stratagene) to yield plasmids pBS/S1C and pBS/ S2C, respectively. Clones were confirmed by sequencing. Sequences were further sub-cloned in the bacterial pET15a expression vector40 via the NdeI and BamHI sites to obtain pET/S1C and pET/S2C. pET15a provides an N-terminal histidine tag. Expression was done in the E. coli BL21(DE3) strain and the peptides purified on Talon resin (ClonTech) according to the supplier’s manual. Generation and Characterization of Polyclonal Serum against C-Terminal Spidroin Sequences. Polyclonal sera were generated in rabbits according to standard procedures41 by Biogenes (Berlin, Germany). For western blots, 2 µg of fusion proteins were separated by 12.5% SDS-PAGE, transferred to Nitrocellulose sheets, and incubated with the respective antibodies.42,43 For detection, the enhanced chemiluminescent (ECL) system purchased from Amersham Plc was used. To remove reactivities against bacterial proteins, the sera were incubated with a nitrocellulose (NC)-filter strip to which the antigen had been blotted. Bound antibodies were eluted with an acidic buffer and neutralized.41 Cross-

Biomacromolecules, Vol. 5, No. 3, 2004 841

reactive antibodies were adsorbed to an NC strip to which the cross-reactive protein had been immobilized. Characterization of the Protein Content of Glands and Threads. For dot blots, 1 µL of a solution of silk threads (ca. 10 mg/mL) in saturated LiSCN or 1 µL of a gland extract in 8 M urea (4 glands in 100 µL) were spotted onto NC filters. Extracts were prepared in the presence of a mixture of protease inhibitors (50 µg/mL antipain-dihydrochloride, 5 µg/mL aprotinin, 5 µg/mL bestatin, 10 µg/mL chymostatin, 50 µg/mL E-64, 100 µg/mL EDTA-Na2, 5 µg/mL leupeptin, 200 µg/mL Pefabloc SC, 5 µg/mL pepstatin, 30 µg/mL phosphoramidon) obtained from Roche to avoid degradation of proteins. For western blots, 10 µL of an extract or solubilized filament was used. The sources were as follows: gland extract (4 glands in 100 µL 8 M urea); gland secretion extract (4 gland contents, i.e., the gellike like substance after removal of the gland epithelium, in 100 µL 8M urea); thread solubilized in a solution of hexafluoro-2-propanol (ca. 10 mg/mL) followed by dialysis against 8 M urea; and a solution containing approximately 10 mg/mL in saturated lithium rhodanide. Samples were either boiled in Laemmli sample buffer and separated on 4-15% gradient (BioRad) or 5% gels, followed by Coomassie staining or transfer to Nitrocellulose (NC) filters42,43 or directly spotted to NC-filter strips. Filters were first blocked in TBS-Tween/5% dry milk powder and then probed with sera using the ECL system. In some cases, the reducing agent (10 mM DTT or 100 mM β-mercaptoethanol) was omitted from the Laemmli sample buffer. To compare the cross-reactivities of the sera to materials from different glands, extracts of 20 glands were prepared in 250 µL 8 M urea. The protein content was estimated with the BioRad Protein Assay Kit using IgG as a standard. Approximately 100 µg total protein were analyzed by western blotting and 10 µg were spotted for dot blots. Transmission Electron Microscope (TEM) Investigations. Ultrathin sections of spider dragline thread were fixed overnight at 4 °C with 0.5% glutaraldehyde in PBS and then incubated in 1% osmium tetroxide. Fixed sections were then embedded in LR White. After dehydration, the sections were mounted on nickel grids with carbon coated Formvar films (Plano). The grids were placed face down onto drops of the respective solutions in a humid chamber. The sections were first blocked with 2% BSA in PBS buffer pH 7.4 for 1 h, washed 3 times with buffer, and incubated with the respective diluted primary anti-sera (1:100 in PBS/2% BSA). After 4 h incubation at room temperature, the grids were washed 3× with PBS and incubated with secondary anti-rabbit antibodies (1:20 in PBS/2% BSA) that had been conjugated to 12 nm colloidal gold (Dianova). After washing 3 × 5 min in PBS and 3 × 5 min in distilled water, which had been filtered through 0.2 µm pore sized membranes (Millipore), sections were either stained with uranyl acetate and lead citrate or carefully dried.44 The examination was conducted with either a Zeiss 900 or a Zeiss 902 TEM at 80kV. Results Cloning, Expression and Characterization of the CTermini of Spidroin 1 and 2. We cloned the C-termini of

842

Biomacromolecules, Vol. 5, No. 3, 2004

Figure 1. Disulfide bridge formation in fusion peptides representing the C-terminal parts of Spidroins and in the high molecular protein fraction of the major ampullate gland content. Panel A: 2 µg of the indicated fusion peptide were applied to a 12.5% SDS-PAGE in the presence (lanes 2 and 4) and absence (lanes 3 and 5) of the reducing agent β-mercaptoethanol (β-MSH). A 10 kDa molecular weight marker (BD Bioscience) was run in parallel. The positions of the 10 and 50 kDa polypeptides in lane 1 are indicated. Panel B: A fraction of the major ampullate gland contents was applied to a 4-15% SDS-PAGE in the absence (lanes 2 and 4) or presence of the reducing agents β-mercaptoethanol (β-MSH; lane 3) and dithiothreitol (DTT; lane 5). The 200 kDa band of the molecular weight standard in lane 1 is indicated.

Spidroin 1 and 2 into expression systems allowing the N-terminal addition of a histidine tag. The tag allowed for the convenient purification of the fusions, termed H6-S1C and H6-S2C, respectively, by metal affinity chromatography. The purified fusion proteins showed a molecular weight of approximately 12 kDa in SDS-PAGE as would have been predicted from their amino acid composition (Figure 1A lanes 2 and 4). However, if the reducing agent was omitted, the intensity of this band was reduced and a second band with approximately double the molecular weight was observed (lanes 3 and 5). This suggested the involvement of disulfide bridges in the creation of the higher molecular weight species. Indeed, the sequences of H6-S1C and H6-S2C contain 1 and 2 cysteines, respectively, that might be involved in the process. Intriguingly, a reduction in size is also observed for the proteins of the gland content in the presence of a reducing agent (Figure 1B, compare lanes 2 and 4 with lanes 3 and 5, respectively). However, in this case, the difference in the molecular weight (220 kDa versus 320 kDa) was less conclusive since it seems to be smaller than 2-fold. Generation and Characterization of Polyclonal Antisera against C-Terminal Fusion Proteins. The purified C-terminal fusion proteins were subsequently used to raise polyclonal antibodies in rabbits. The serum derived against H6-S1C strongly cross-reacted with H6-S2C and vice versa (Figure 2B and D). These cross-reactivities were not due to the common histidine tag, since both sera did not react with an unrelated fusion protein bearing the same tag (data not shown). Both fusions contained N-terminally encoded polyalanine stretches. Using appropriate controls harboring polyalanine stretches fused to unrelated sequences, we did not find any evidence for antibody reactivities against these parts. The highly conserved sequences of the C-termini10,11 must therefore account for the cross-reactivities we observed. The presence of less conserved regions led us to assume that it might be possible to obtain monospecific antibodies to either of the C-termini, if the sera were cross-adsorbed against the other fusion peptide. To this end, the anti-S1C serum was

Sponner et al.

Figure 2. Characterization of polyclonal antibodies raised against the C-terminal parts of Spidroins. Panel A shows a Coomassie stained gel (12.5%) of the histidine tagged fusions H6-S1C (lane 2) and H6-S2C (lane 3) along with the 10 kDa marker ladder of which the 10 and 20 kDa bands are indicated. Panels B-E show the reactivities of different sera against the fusion proteins. In panels B and D, the original anti S1C and anti S2C sera were used in a 1:1000 dilution. In panel C, the anti-S1C sera cross-adsorbed to H6-S2C and in panel E the anti-S2C sera cross adsorbed to H6-S1C were used in a 1:10 dilution. Detection was by ECL. Note that only the relevant parts of the gels and blots are shown.

Figure 3. Reactivities of antisera against gland extracts and solubilized silk thread. Panel A shows a Coomassie stained 5% SDSPAG to which has been applied solubilized thread (lane 1), total gland extract (lane 3), and gland secretion extract (lane 4) along with the 10 kDa marker (lane 2) of which the 90, 100, and 200 kDa bands are visible. Panel B shows the blots of soluble silk thread (a), gland secretion extract (b), and total gland extract (c) probed with different antisera. Untreated anti S2C (lane 3) and anti S1C (lane 5) sera were employed at a 1:1000 dilution, whereas cross-adsorbed anti S2Cx (lane 4) and anti S1Cx (lane 6) sera were used in a 1:10 dilution. Bound antibodies were visualized by ECL. A Coomassie stain of the material used for blotting is shown in lane 2 and the 10 kDa marker in lane 1. The positions of the 50 kDa and 200 kDa marker polypeptides are indicated.

adsorbed against immobilized H6-S2C protein and likewise the anti S2C serum against immobilized H6-S1C fusion peptide. Indeed, albeit the titer was reduced by more than a 100 fold, we were able to obtain sera (anti S1Cx and anti S2Cx) that were reactive exclusively with the fusion protein that was used in their production (Figure 2 C and E). Presence of the C-Terminal Sequences in the Major Ampullate Gland Extracts and Major Ampullate Filament. To demonstrate the presence or absence of the Spidroin specific C-terminal sequences in the gland and the gland secretion, urea extracts were prepared of both samples (Figure 3A, lanes 3 and 4, respectively). To analyze threads, they were solubilized in hexafluoro-2-propanol (lane 1). As

Conserved C-Termini of Spidroins

Figure 4. Crossreactivities of Spidroin 1 and 2 C-terminal specific sera to various gland extracts. Glands were extracted in urea and fractions of equal protein concentrations either subjected to 4-15% SDS-PAGE followed by western blotting (panel A) or spotted directly on NC-filters (panel B). The gel was Coomassie stained and the blots were treated with the anti S1C serum. Bound antibodies were visualized by ECL. Abbreviations are as follows: AC, aciniform gland (lane 1); AG, aggregate gland (lane 2); FL, flagelliform or Coronate gland (lane 3); MA, major ampullate gland (lane 4); MI, minor ampullate gland (lane 5); PI, piriform gland (lane 6); TU, tubuliform or cylindrical gland (lane 7).

is evident, the solubilized thread and the gland secretion extract show polypeptides approximately the same size (compare lanes 1 and 4), whereas an abundance of lower molecular weight bands are additionally observed in the case of the total gland extract (lane 3). The different preparations were then subjected to western blotting, using the original and cross-adsorbed antisera (Figure 3B). Either of the sera were able to stain high molecular weight bands in all preparations, i.e., dissolved thread (panel a), gland secretion extracts (panel b), and total gland extracts (panel c). In the latter case some of the lower molecular weight materials also stained. Due to their lower titer, the cross-adsorbed sera reacted less well than the untreated sera and the lowest molecular weight bands are not visible in lanes 4 an 6 at the exposure shown (Figure 3B panel c). Cross-Reactivity of the Anti-Sera to Other Spidroins. To test the cross-reactivities of the C-termini specific sera to other silk fibroins, they were reacted against urea extracts of various glands (Figure 4). The Coomassie stained gel shows high molecular weight products contained within each of the seven glands dissected from N. claVipes (panel A). Employing the anti S1C serum, the lane of the major ampullate gland showed strong reactivity in the western blot and less staining was observed for the minor ampullate extract, an observation that also held true for the dot blot experiment (panel B). None of the other extracts showed any significant reactivities. The anti S2C serum had the same outcome (data not shown). The cross-reactivities of the different antisera to the major and minor ampullate filaments were additionally tested in dot blot experiments after solubilizing them in lithium rhodanide (Figure 5A). In contrast to hexafluoro-2-propanol,

Biomacromolecules, Vol. 5, No. 3, 2004 843

Figure 5. Cross-reactivities of Spidroin 1 and 2 C-terminal specific sera to the major and minor ampullate filaments. Panel A shows dot blots in which filaments derived from the major and minor ampullate glands or a mixture of both were solubilized in lithium rhodanide (LiCN) and spotted onto nitrocellulose strips. The strips were reacted against the converted (CS) untreated anti S1C and anti S2C sera in a 1:1000 dilution (lanes 1 and 3) or to the cross-adsorbed anti S1Cx and anti S2Cx sera in a 1:10 dilution (lanes 2 and 4). In parallel, the preimmune serum (PIS) was tested in a 1:1000 dilution against the mixture of both threads. Visualization of bound antibodies was performed with the ECL system using a peroxidase labeled secondary antibody. In panel B, cross-sections of the major ampullate filament were stained with the converted (CS) anti S1C serum or with preimmune serum (PIS). Bound antibodies were visualized with a gold labeled secondary antibody by electron microscopy. The extension of the skin and the core51 are indicated.

the treatment of the threads with lithium rhodanide led to the generation of small polypeptides below 100 kDa (data not shown). Both major and minor ampullate filaments were reactive to the unpurified sera (lanes 1 and 3), whereas the latter reactivity was not observed for the cross-adsorbed sera (lanes 2 and 4). We also unambiguously demonstrate the presence of the C-terminal peptide sequences within thread cross sections (Figure 5B). The anti S1C serum stained specifically the core51 of the major ampullate filaments leaving the skin unstained, whereas the preimmune serum control showed no reaction. The same outcome was obtained with the anti S2C serum (data not shown). The cross-adsorbed sera did not yield consistent results, probably due to their low titer (data not shown). Discussion Despite their conservation, no function has been conclusively attributed to C-terminal sequences nor is it known if they are present in the gland lumen or are part of the spun filament.3,4,10,11,45,46 In this report, we give evidence that the C-termini are bona fide parts of the mature Spidroins and give an indication of a possible structural function that resides in the C-termini. C-Termini Are Bona Fide Parts of Major Ampullate Filament. Our original sera against Spidroin 1 and 2 were strongly cross-reactive to the other class of Spidroins and to Spidroins produced in the minor ampullate gland (Figure 2). The cross-reactivities do not come as a surprise with respect to the high sequence conservation of the C-termini between Spidroin 1, 2 and those produced in the minor ampullate

844

Biomacromolecules, Vol. 5, No. 3, 2004

gland. However, there are less conserved regions in the C-termini between Spidroin 1 and 2 and we have successfully made use of them to obtain sera specific for either Spidroin by cross-adsorption. With the help of these monospecific antisera, we were able to reveal the presence of the corresponding Spidroin 1 and 2 epitopes in the major ampullate gland extracts and the dissolved filaments (Figure 3). Moreover, C-terminal epitopes were detected in cross sections of the major ampullate filament (Figure 5). It should be noted that the resolution of the gels did not allow us to determine if the cross-adsorbed sera stained different bands in the SDS-PAGE blots as was reported for sera derived against the repetitive parts of Spidroin 1 and 2.45 Since the antibodies reacted with high molecular weight products of the gland content and the solubilized thread, we can rule out that this domain is cleaved off after secretion or during the process of filament formation. In total gland extracts, the C-terminal sera also detected a variety of low molecular weight polypeptides (Figure 2B). These polypeptides must therefore contain the C-terminal regions and have most likely lost N-terminal parts by degradation. Since polypeptides are synthesized from the N-terminus, these C-terminal degradation products cannot correspond to precursors produced by paused translation.21 Since they are not observed in the gland content and the final thread, it is a likely possibility that N-terminal truncated Spidroins are removed from the spinning dope by a resorption process. Our cross-reactive, unpurified sera reacted also with extracts from the minor ampullate gland, but not to any other gland demonstrating that none of the other Spidroins possess a highly homologous sequence (Figure 4). This reactivity to the Spidroins from the minor ampullate gland was lost with the cross-adsorbed sera demonstrating that within the conserved regions unique amino acid sequences specific for the major ampullate Spidroins are present. Disulfide Bridge Formation. All Spidroins produced in the major ampullate gland contain a cysteine that is followed by the charged residue aspartic acid. The cysteine is likely to be responsible for the observed dimerization of the recombinant polypeptides and might serve to cross-link the native Spidroins as well (Figure 1). The presence of a nearby highly hydrophobic Q(A/V)LLE motif and charged residues make it likely that the cysteine lies on the surface of the molecule and can take part in intermolecular disulfide bridge formation. Within several Spidroin 2 proteins, a second cysteine followed by a valine is situated in a hydrophobic environment. It is not likely to be involved in disulfide bridge formation, since no higher aggregates are found in the case of our Spidroin 2 recombinant protein that contains this additional site. Intriguingly, Mello and co-workers have previously reported different mobilities of high molecular weight proteins extracted from glands or present in the dissolved thread under reducing and nonreducing conditions,23,47 a behavior that is exemplified for the gland extracts in Figure 1. They also concluded that the formation of disulfide bridges might account for the higher molecular weight species. The difference between the molecular weights estimated under reducing

Sponner et al.

and nonreducing conditions is, however, not 2-fold as expected for a monomer and dimer, but rather smaller. Since no additional low molecular weight products of a corresponding intensity and size is observed in the gels, it is unlikely that a smaller molecule is bound to the Spidroins as is the case with the B. mori fibroin.48 Spidroin monomers and dimers may rather show unusual running behaviors that lead to an underestimation of their molecular weight as suggested by Jackson.49 Possible Role of the C-Termini. The C-termini of Spidroins might play a crucial role in the formation of the insoluble filament in analogy to the situation found in the silk worm B. mori.50 The N- and C-termini of the latter protein are highly hydrophilic. In elegant experiments, Jin and Kaplan50 were able to show that both ends contribute to the formation of micellar structures. The ends cooperate in the formation of the micelles with less extended hydrophilic regions located interspersed in the rest of the protein within larger hydrophobic stretches. These micelles can accommodate higher protein concentrations by forming larger globules and gellike states keeping the proteins in a soluble form in the gland and the duct. Upon the exertion of stress and shearing forces, structural transitions lead to the formation of fibrillar structures and finally the formation of the insoluble filament. Albeit the N-termini of Spidroin I and II are not known, the repetitive parts of Spidroins show also an alternating pattern of hydrophilic and hydrophobic regions similar to the B. mori counterpart, albeit the lengths of the hydrophilic and hydrophobic stretches do match closer in this case. The C-termini are not as hydrophilic as the ends of B. mori fibroin but they also contain alternating hydrophilic-hydrophobic stretches.50 As pointed out by Jin and Kaplan,50 withdrawal of water from the gland and the exertion of physical shear can also well account for the formation of fibrils in the case of the spider fibroin. The formation of disulfide bridges is not likely to play a major role in the formation of the insoluble filaments, since cysteines are missing from the Spidroins of the minor ampullate filament. These cross-links might, however, support the parallel alignment of the Spidroins produced in the major ampullate gland and contribute to the mechanical properties of the major ampullate filament. Acknowledgment. The authors want to thank Ursula Stephan and Kristina Hartung for excellent technical assistance. This work was supported by the Bundesministerium fu¨r Forschung und Bildung (BMBF FKZ 0311130) and the Thu¨ringer Ministerium fu¨r Wissenschaft, Forschung und Kultur (TMWFK B307-99-001). References and Notes (1) Craig, C. L. Annu. ReV. Entomol. 1997, 42, 231-267. (2) Hinman, M.; Dong, Z.; Xu, M.; Lewis, R. V. In Biopolymers; Case, S. T., Ed.; Springer-Verlag: Berlin, 1992; pp 227-254. (3) Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 71207124. (4) Hinman, M. B.; Lewis, R. V. J. Biol. Chem. 1992, 267, 1932019324. (5) Colgin, M. A.; Lewis, R. V. Protein Sci. 1998, 7, 667-672. (6) Hayashi, C. Y.; Lewis, R. V. J. Mol. Biol. 1998, 275, 773-784. (7) Gatesy, J.; Hayashi, C.; Motriuk, D.; Woods, J.; Lewis, R. Science 2001, 291, 2603-2605.

Biomacromolecules, Vol. 5, No. 3, 2004 845

Conserved C-Termini of Spidroins (8) Hayashi, C. Y.; Lewis, R. V. Science 2000, 287, 1477-1479. (9) Guerette, P. A.; Ginzinger, D. G.; Weber, B. H.; Gosline, J. M. Science 1996, 272, 112-115. (10) Beckwitt, R.; Arcidiacono, S. J. Biol. Chem. 1994, 269, 6661-6663. (11) Beckwitt, R.; Arcidiacono, S.; Stote, R. Insect Biochem. 1998, 28, 121-130 1998. (12) Hayashi, C. Y.; Shipley, N. H.; Lewis, R. V. Int. J. Bio. Macromol. 1999, 24, 271-275. (13) Hayashi, C. Y. Exs 2002, 92, 209-223. (14) Hinman, M. B.; Jones, J. A.; Lewis, R. V. Trends Biotechnol. 2000, 18, 374-379. (15) Lewis, R. V.; Colgin, M. U.S. Patent, 5,733,771, 1998. (16) Peakall, D. B. Comp. Biochem. Physio.l 1965, 15, 509-515. (17) Candelas, G.; Cintro´n, I. J. Exp. Zool. 1981, 216, 1-6. (18) Candelas, G. C.; Lopez, F. Comp. Biochem. Physiol. 1983, 74b(3), 637-641. (19) Rodriguez, R.; Candelas, G. C. J. Exp. Zool. 1995, 272, 275-280. (20) Ortiz, R.; Cespedes, W.; Nieves, L.; Robles, I. V.; Plazaola, A.; File, S.; Canelas, G. C. J. Exp. Zool. 2000, 286, 114-9. (21) Candelas, G.; Candelas, T.; Ortiz, A.; Rodriguez, O. Biochem. Biophys. Res. Commun. 1993, 116, 1033-1038. (22) Lizardi, P. M.; Mahdavi, V.; Shileds, D.; Candelas, G. C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 6211-6215. (23) Mello, C. M.; Arcidianono, S.; Beckwitt, R.; Prince, J. T.; Senecal, K.; Kaplan, D. Mater. Res. Soc. Symp. Proc. 1994, 330, 37-42. (24) Vollrath, F. J. Biotechnol. 2000, 74, 67-83. (25) Vollrath, F.; Knight, D. P. Nature 2001, 410, 541-8 2001. (26) Gosline, J. M.; DeMont, E. M.; Denny, M. W. EndeaVour 1986, 10, 37. (27) Gosline, J. M. et al. In Biomimetics; Sarikaya, M., Aksay, I., Eds.; AIP Press: Woodberry, NY, 1995; pp 237-261. (28) Gosline, J. M.; Guerette, P. A.; Ortlepp, C. S.; Savage, K. N. J. Exp. Biol. 1999, 202, 3295-3303. (29) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-7. (30) Stauffer, S. L.; Coguill, S. L.; Lewis, R. V. J. Arachnol. 1994, 22, 5-11.

(31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46)

(47) (48)

(49) (50) (51)

Denny, M. W. Symp. Soc. Exp. Biol. 1980, 34, 247-272. Winkler, S.; Kaplan, D. L. J. Biotechnol. 2000, 74, 85-93. Thiel, B.; Viney, C. Mater. Res. Bull. 1995, 20, 52-56. Gosline, J. M.; Denny, M. W.; DeMont, E. M. Nature 1984, 309, 551-552. Gosline, J. M.; Pollak, C. C.; Guerette, P. A.; Cheng, A.; Demont, M. E.; Denny, M. W. ACS Symp. Ser. 1994, 544, 328-341. Termonia, Y. Macromolecules 1994, 27, 7378-7381. Ku¨mmerlen, J.; van Beek, J. D.; Vollrath, F.; Meier, B. H. Macromolecules 1996, 29, 2920-2928. van Beek, J. D.; Ku¨mmerlen, J.; Vollrath, F.; Meier, B. H. Int. J. Biol. Macromol. 1999, 24, 173-178. Xu, G. Z.; Evans, J. S. Biopolymers 1999, 49, 303-312. Studier, F. W.; Rosenberg, A. H.; Dunn, J. J.; Dubendorff, J. W. Methods Enzymol. 1990, 185, 60-89. Harlow, E.; Lane, D. P. Antibodies: a laboratory manual; Cold Spring Harbor Laboratory: New York, 1988. Laemmli, U. K. Nature 1970, 227, 680-685. Towbin, H.; Staehelin, T.; Gorden, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350-4354. Reynolds, E. S. J. Cell Biol. 1963, 17, 208-212. Fahnestock, S. R.; Yao, Z.; Bedzyk, L A. M. J. Biotechnol. 2000, 74, 105-119. Lazaris, A.; Arcidiacono, S.; Huang, Y.; Zhou, J. F.; Duguay, F.; Chretien, N.; Welsh, E. A.; Soares, J. W.; Karatzas, C. N. Science 2002, 295, 472-476. Mello, C. M.; Senecal, K.; Yeung, R.; Vouros, P.; Kaplan, D. ACS Symp. Ser. 1994, 544, 67-79. Tanaka, K.; Kajiyama, N.; Ishikura, K.; Waga, S.; Kikuchi, A.; Ohtomo, K.; Takagi, T.; Mizuno, S. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1999, 1432, 92-103. Jackson, C.; O’Brien, J. P. Macromolecules 1995, 28, 5975-5977. Jin, H.-J.; Kaplan, D. L. Nature 2003, 424, 1057-1061. Frische, S.; Maunsbach, A. B.; Vollrath, F. J. Microsc. 1998, 189, 64-70.

BM034378B