Silk proteins were taken from the major ampullate glands found in the abdomens of Nephila clavata spiders that were collected on different days during an autumn season in Japan. The molecular weights of the silk proteins taken from 125 spiders were determined under a reduced state using electrophoresis. It was found that the molecular weight of N. clavata spider silk protein changed depending on the time period during the autumn season and showed a peak value of ~300 kDa during mating season. However, the molecular weight of N. clavata spider silk protein was, on average, ~270 kDa except during mating season. Such a peak in the molecular weight for female N. clavata may be ascribed to the necessity for building mechanically strong orb-webs, which consist of silk with relatively large proteins for the purpose of accepting many male spiders.
Spiders secrete seven kinds of silk during their lives.1 Draglines of spider silk play an important role as a mechanical lifeline for the spiders.2, 3 Many researchers have been interested in draglines spider silk because of their excellent physical properties, such as mechanical strength,2, 3, 4, 5, 6, 7 heat resistance8, 9 and UV resistance,10, 11, 12 and also because of the recently discovered potential for the application of spider silk to sutures13 and violin strings;14 in addition, silk mimetic materials have utility in genetic technology.15, 16 It is very important to know the molecular weight of spider silk when addressing its mechanical properties and its thermal and UV degradation. The molecular weight of a material is one of the most important factors for elucidating the relationship between its structure and physical properties. Previously, the molecular weights of spider silk proteins from Nephila clavipes17, 18, 19, 20, 21, 22 and Nephila clavata were determined.23, 24 These studies focused mainly on the silk obtained from the adult spiders collected at a fixed period. However, it is unknown whether reported values of the molecular weight are representative values for spider silk because studies on the molecular weight of silk during the growth of spiders have not been conducted.
Previously, Osaki reported the effects of seasonal changes on the safety coefficient,25 color26 and elastic modulus7 of N. clavata spider silk. The data changed markedly just after ecdysis. In particular, the elastic modulus of the silk showed a relatively high value in mid-autumn compared with silks obtained in early and late autumn.7 Thus, it is interesting to study whether the molecular weight of silk also changes with the growth of spiders. Here, we investigated the effects of the growth of spiders on the molecular weight of spider silk proteins contained in major ampullate glands.
The present study describes measurements of the molecular weights of silk proteins obtained from the major ampullate glands of N. clavata spiders, which were collected between early and late autumn. The results of this study indicate that the molecular weight changed depending on the season when the spiders were collected and gave a peak in mid-autumn.
In Japan, N. clavata (Japanese golden web) spiders are born from egg cocoons in the late spring or early summer and diverge by sex in late summer, accompanied by final ecdysis. Male spiders visit the orb-webs of female spiders for courtship in mid-autumn after ecdysis. After mating, pregnant spiders produce egg cocoons and die soon in late autumn. The weight of a female N. clavata spider in Japan increases exponentially from prematurity in late summer or early autumn to maturity in mid-autumn25 or late autumn; the weight of a male spider stays almost constant.25 However, a small difference exists in the speed of the spiders’ growth because of differences in the individuals and habitats. Here, we divided autumn into three seasons: early, mid- and late autumn.
Female N. clavata spiders that inhabit Nara and Osaka prefectures, were collected in different restricted periods between 29 August and 15 December 2003. The total number of collected spiders was 125.
Spider silk proteins
Spiders were dissected in physiological saline solution immediately after collection, and then a pair of major ampullate glands (see Figure 1) was taken from each abdomen. The major ampullate glands were yellow when the spiders were collected in late autumn. The major ampullate silk glands were cut into small pieces with scissors, homogenized in Tris-HCl buffer solution (50 mM, pH 7.5) and centrifuged to remove the insoluble epithelium of the silk glands. The supernatant containing silk proteins was then used for sample for electrophoresis.
Special grade chemical compounds, including Tris(hydroxymethyl)aminomethane (Tris), sucrose, sodium dodecyl sulfate (SDS), 2-mercaptoethanol and bromophenol blue (Wako Pure Chemical Industries, Ltd, Osaka, Japan), were used. The supernatant was mixed with a half volume of the sample buffer (0.1875 M Tris-HCl pH 6.8, 6.0% (wt/v) SDS, 15% (v/v) 2-mercaptoethanol, 30% (wt/v) sucrose and 0.006% (wt/v) bromophenol blue and was incubated at 100 °C for 5 min; the resulting solution containing silk proteins was then used for electrophoresis. The electrophoresis measurements of the silk protein solutions were carried out within 2 days of dissecting the spiders.
Electrophoresis was performed on a 5% polyacrylamide mini-slab gel using an NA-1010 mini agarose gel electrophoresis apparatus (Nihon Eido Co., Tokyo, Japan)23 since the molecular weight was relatively small in the present study. A 3% polyacrylamide gel was used to measure the high molecular weight proteins (~600 kDa) in a previous study.24
Proteins on the polyacrylamide gels were stained with Coomassie brilliant blue (Quick CBB, Wako Pure Chemical Industries, Ltd.) and the intensities of the bands obtained by electrophoresis were measured using an EPSON GT-X970 scanner (Seiko Epson Co., Nagano, Japan). The retention factor (Rf) of a sample was calculated from the position of bands related to the migration distance of the proteins. A calibration curve between the Rf values and molecular weights was calculated from Rf values obtained for marker proteins with molecular weights of 76, 116, 170 and 220 kDa (Amersham HMW Calibration Kit for SDS Electrophoresis, GE Healthcare Ltd., Little Chalfont Buckinghamshire, UK). The calibration curve was also reinforced using silkworm silk protein with a molecular weight of 350 kDa (Fibroin H)19 as a marker (Supplementary Information 1).
Results and Discussion
Figure 2 shows the typical band patterns in a 5% SDS PAGE gel for silk proteins contained in the major ampullate glands of N. clavata spiders collected on three different days (a–c). Here, a, b and c correspond to the band patterns obtained for silk protein obtained from spiders collected on 5 September (early autumn; a1, a2, a3, a4, a5, a6, a7, a8 and a9), 3 October (mid-autumn; b1, b2, b3, b4, b5, b6, b7, b8 and b9) and 2 December (late autumn; c1, c2, c3, c4, c5, c6, c7, c8 and c9), respectively. Each lane M shows a pattern for protein markers with molecular weights of 116, 170 and 220 kDa.
Several bands were observed at ~270 kDa in each lane, as shown in Figure 2. Here, we defined the strong band at ~270 kDa in each lane as the major band and the relatively weak band as the minor band. Major and minor bands in lane a9 of Figure 2a, lane b6 of Figure 2b and lane c7 of Figure 2c are also shown as enlargements on the right side of Figure 2. Here, we mainly focused on the major bands for discussion because the intensities of these major band were generally 1.5 times greater than those of the minor bands.
Most bands were observed at positions corresponding to molecular weights between 250 and 330 kDa for different spiders, as shown in Figure 2. Similar patterns were observed for the same period of collection. The molecular weight observed in Figure 2b was a little larger than the molecular weights observed in Figures 2a and c. However, no distinguishing bands were observed in the a1 band in Figure 2a. The sample from spider corresponding to a1 in Figure 2a was extracted just after ecdysis, indicating the absence of silk proteins and corresponding to the main band of samples obtained from major ampullate glands. Lanes with no bands at ~270 kDa were also observed for the other two spiders just after ecdysis.
The molecular weights of silk protein obtained from the major ampullate glands are shown in Table 1. The molecular weights corresponding to the major bands were larger than that of the minor band in most of the lanes. The average value of the molecular weight corresponding to the major band was determined to be 267±21 kDa for Figure 2a (5 September), 299±8 kDa for Figure 2b (3 October) and 268±11 kDa for Figure 2c (2 December). The average molecular weight corresponding to the minor bands was also determined to be 256±13 kDa for Figure 2a (5 September), 288±22 kDa for Figure 2b (3 October) and 257±20 kDa for Figure 2c (2 December). The P-value was 9.6 × 10−5 between Figures 2a and b and 3.8 × 10−6 between Figures 2b and c, suggesting the existence that the differences in the molecular weights of these samples were statistically significant.
Figure 3 shows the number of N. clavata spiders for which the major bands indicating silk proteins with different molecular weights were observed. These silk proteins had been obtained from the major ampullate glands of the abdomens of 125 spiders. The molecular weights range between 240 and 320 kDa. The distribution of the histogram is similar to a Gaussian one where the maximum is observed at ~275 kDa. The molecular weight disperses over a wide range even though the maximum value is ~275 kDa. The maximum value of molecular weight almost agrees with those of 270 or 272 kDa that were determined in our previous studies.23, 24
Figure 4 shows the molecular weight determined for the major bands that were ascribed to spider silk proteins from the major ampullate glands of spiders collected on different days. The molecular weight was ~270 kDa for silk proteins of premature spiders in early autumn. A maximum molecular weight of ~300 kDa is observed for mature spiders collected in mid-autumn, and the molecular weight is found to decrease for samples obtained from old spiders collected in late autumn; the samples maintained a value of ~270 kDa up to early winter. The molecular weights of the samples range between 250 and 285 kDa throughout the autumn season, except for a period in mid-autumn; this indicates that the average molecular weight is ~274 kDa. However, the molecular weight of spider silk proteins increases in mid-autumn and approaches up to 300 kDa.
The molecular weights of the samples were determined under reduction with 2-mercaptoethanol. Therefore, the values were approximately half of the values obtained for samples under naturally unreduced conditions.24 The several bands observed at ~270 kDa in each lane (Figure 2) may be ascribed to differences in the cleavage of protein molecules by reduction or to differences in the covalent attachment of silk proteins by the phosphorylation or glycosylation.
The peak value of ~300 kDa for the major bands was observed only in samples obtained in mid-autumn. A peak at ~290 kDa was also observed for minor bands. These indicate that N. clavata spiders in mid-autumn secrete relatively long protein chains with slightly larger molecular weights. This high molecular weight may be related to the silk proteins obtained from N. clavata spiders after ecdysis in early autumn. The period after ecdysis corresponds to the mating season when female spiders are most active in their lives. Thus, they may be required to build strong orb-webs such that many male spiders can visit in order to mate and then impregnate the female spiders.
Many studies have been published on the molecular weight of spider silk.17, 18, 19, 20, 21, 22, 23, 24 However, no studies have investigated the effects of seasonal changes on the molecular weight of silk proteins obtained from major ampullate glands or solid silk. Previously, the effects of seasonal changes on the safety coefficient,25 color26 and elastic modulus7 of N. clavata spider silk have been reported. The safety coefficient2, 25 and the color26 of solid spider silk changed markedly from 3 to 2 and from white to yellow during mating season in mid-autumn, respectively. In particular, the elastic modulus, which may be closely related to the molecular weight of a sample, changed from 10.3 GPa in early autumn to 12.9 GPa in mid-autumn and to 10.0 GPa in late autumn.7 The mating season that occurs after ecdysis results in same rapid increase in the molecular weight of silk as observed with changes in the season.
Here, we consider the following reason for the observed rapid increase in the molecular weight of silk proteins of N. clavata spiders after ecdysis. Spider silk consists of crystalline27 and amorphous regions. The former consists of a β-sheet structure with alanine-rich regions and contributes to the mechanical strength of silk. Male and female spiders become very active during mating season after ecdysis. In particular, female spiders build mechanically strong orb-webs so that many male spiders can visit these webs for courtship and to impregnate the female spiders. The ratio of alanine in silk proteins from N. clavata spiders were found to increase in mid-autumn (see Table 1 of Osaki8). Thus, spiders are required to secrete mechanically stronger silk by increasing the ratio of alanine-rich crystalline regions.
It is known that the gene responsible for silk production generally consists of several exons even though the DNA sequence of silk proteins in the major ampullate of N. clavata has not been completely determined. Alternative splicing28 gives different sizes of mRNA that are able to synthesize several proteins29 with different lengths, which results in proteins with different molecular weights. However, the length of the expressed gene is known to depend on the stage of growth.30 Thus, the molecular weight of spider silk protein during mating season is considered to slightly lengthen compared with silk produced in other seasons because of the presence of alternative RNA splicing. A similar alternative splicing mechanism is actually observed in the sericin mRNA in the cells of the Bombyx mori silk glands.31 After mating season, the size of RNA is believed to return to its usual length through ordinary splicing. However, the split or dispersion in molecular weight at ~270 kDa is also considered to be ascribed to the difference in the length of gene expression of mRNA or to the difference in the covalent attachment to silk proteins through phosphorylation or glycosylation.
Even though we propose the mechanism of DNA splicing, it is very difficult to explain the observed steep increase in molecular weight during mating season just after ecdysis. Further studies are required to clarify the reasons for the observed peak in molecular weight during mating season.
In conclusion, we determined the effects of seasonal changes on the molecular weight of N. clavata spider silk and confirmed the presence of a molecular weight peak at ~300 kDa during the mating season. The molecular weight obtained in this study was, on average, 270 kDa except for during mating season and is in agreement with previously reported data.24, 25 We should consider the season for spider collection when determining the molecular weight of spider silk. These results will be applicable to the extraction and use of other biological materials.
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This work was supported by the JSPS KAKENHI grant numbers 24655105 and 25288102. The authors are grateful to Professor Hajime Mori of the Kyoto Institute of Technology for kind supply of silkwoms.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Polymer Journal website
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Osaki, S., Yamamoto, K., Matsuhira, T. et al. The effects of seasonal changes on the molecular weight of Nephila clavata spider silk. Polym J 48, 659–663 (2016). https://doi.org/10.1038/pj.2015.138
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