Modifying the Mechanical Properties of Silk Fiber by Genetically

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Modifying the mechanical properties of silk fiber by genetically disrupting the ionic environment for silk formation Xin Wang, Ping Zhao, Yi Li, Qiying Yi, Sanyuan Ma, Kang Xie, Huifang Chen, and Qingyou Xia Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00724 • Publication Date (Web): 24 Aug 2015 Downloaded from http://pubs.acs.org on August 24, 2015

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Modifying the mechanical properties of silk fiber by

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genetically disrupting the ionic environment for silk

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formation

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Xin Wanga‡, Ping Zhaoa‡, Yi Lia, Qiying Yib, Sanyuan Maa, Kang Xiea, Huifang Chena, Qingyou

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Xiaa*

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a

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Road, Chongqing 400716, P. R. China

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b

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China.

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State Key Laboratory of Silkworm Genome Biology, Southwest University, 216 Tiansheng

Animal center, Chongqing Medical University, 1 Yixuanyuan Road, Chongqing 400016, P. R.

‡ These authors contributed equally to this work.

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ACS Paragon Plus Environment

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ABSTRACT

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Silks are widely used biomaterials, but there are still weaknesses in their mechanical properties.

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Here we report a method for improving the silk fiber mechanical properties by genetic disruption

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of the ionic environment for silk fiber formation. An anterior silk gland (ASG) specific promoter

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was identified and used for overexpressing ion-transporting protein in the ASG of silkworm.

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After isolation of the transgenic silkworms, we found that the metal ion content, conformation

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and mechanical properties of transgenic silk fibers changed accordingly. Notably, overexpressing

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endoplasmic reticulum Ca2+-ATPase in ASG decreased the calcium content of silks. As a

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consequence, silk fibers had more α-helix and β-sheet conformations and their tenacity and

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extension increased significantly. These findings represent the in vivo demonstration of a

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correlation between metal ion content in the spinning duct and the mechanical properties of silk

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fibers, thus providing a novel method for modifying silk fiber properties.

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KEYWORDS

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biomaterials, biotechnology, metal ions, silk fiber, mechanical properties.

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Biomacromolecules

INTRODUCTION

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Silk fibers from the silkworm Bombyx mori are widely used fibers because of their

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exceptional mechanical properties as well as biocompatibility and environmentally friendly

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nature.1-3 Currently, in both the commodity textile world and high-tech fields, there is an urgent

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need for silks with a growing web of applications.4-6 Thus, it has prompted researchers to aim at

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modifying the properties of silk fibers. Learning that silk fibers properties are determined by

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their conformations, and moreover, by their amino acid coding sequence7-9, tremendous efforts

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have been made to alter the genetic sequences of silk proteins.10-12

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Alternatively, a rapidly growing number of studies have shown that the physiological and

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biochemical environment in the spinning duct, can affect the conformational transitions of silk.

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In the spinning duct, liquid silk protein undergoes an α-helix to β-sheet conformational

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transition.13, 14 In vitro studies have shown that metal ions, together with shear force, pH, and

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spinning speed, are involved in this process.2, 15-18 In the silkworm spinning duct (known as the

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anterior silk gland, ASG), metal ions, such as sodium and potassium, may weaken the stable gel

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and prepare the silk fibroin (the major component of silk protein of silkworm) macromolecules

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for β-sheet formation.19 Together with a low pH value, calcium ion concentrations at a defined

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range may induce gradual water removal and promote the formation of β-sheet structures.20

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Although accumulating evidences indicate that these ions play key roles in the

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conformational transitions in vitro, it remains unclear whether they regulate silk conformations

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and textile properties in vivo. Herein, we report that genetic modification of metal ion content in

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the spinning duct changed the conformations and properties of silk fibers. In the present study,

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silkworm Na+/K+-ATPase α subunit (BmNKAα) and endoplasmic reticulum Ca2+-ATPase


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(BmSERCA) were overexpressed in the ASG of silkworm, and remarkable differences in the

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mechanical properties were evident in transgenic silks. Our work presents a new method for

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improving the silk fiber mechanical properties and may provide instructive experimental

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evidences for illustrating the mechanism for silk fiber formation.

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EXPERIMENTAL SECTION

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piggyBac vectors construction

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The genomic DNA and total RNAs were isolated from B.mori strain DaZao. DNA

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fragments encoding the promoter of BmASSCP2 (named BmCP231) were produced by PCR

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with genomic DNA. BmNKAα and BmSERCA fragments were amplified by PCR with the

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cDNAs reverse transcribed by total RNAs. The sequences of the primers and restrictive

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endonucleases used are as listed in Table. S1. The following program was used for PCR: 95°C

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for 10min, 35 cycles of 95°C for 40 s, 65°C for 40s, 72°C for 3min, and then 72°C for 10min.

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Each amplification product was gel-purified, recovered, and cloned into plasmid vectors, and

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bacterial transformants containing error-free inserts were identified. The BmCP231 promoter and

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protein coding sequences were then assembled to create functional cassettes in intermediate

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plasmids (pSL1180, modified and stored in our laboratory). Finally, these cassettes were excised

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and subcloned into piggyBac[3×P3-EGFPaf] by Asc I and T4 DNA ligase (NEB, UK) to produce

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the piggyBac vectors.

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Acquisition of the transgenic silkworm

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The piggyBac vector purified by QIAprep Spin Miniprep Kit (Qiagen), together with

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pHA3PIG vector (stored in our laboratory) used as the helper plasmid for the production of


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transposase, were injected into each non-diaspause eggs collected 1 – 2 h after oviposition with a

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FemtoJet5247 microinjector (Eppendorf). The injection opening was sealed with non-toxic glue

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and the embryos were allowed to develop at 25°C. The 7-day G1 pupae were screened for EGFP

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expression in the compound eyes under a fluorescent stereomicroscope (Olympus) equipped with

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appropriate filter. The EGFP-positive individuals were reared to moths and sibling mated to

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generate offsprings. Southern blotting was used to verify the genomic insertion of piggyBac

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vector. Firstly, the genomes of transgenic and wild-type silkworm on the third day of fifth instar

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were extracted and purified by Insect DNA Kit (Omega). And then, after the cleavage of Bgl II

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(TaKaRa), the DNA was transferred and fixed into a nylon membrane (Roche) followed by the

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separation by gel electrophoresis. Lastly, the membrane contained DNA was used in

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hybridization

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DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). The EGFP-specific probes

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were generated and labeled by PCR DIG Probe Synthesis Kit (Roche). The transgenic silkworms

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were raised for five generations and their cocoon shell ratio of each generation was calculated.

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Cocoon shell ratio = (cocoon shell weight / cocoon weight) × 100%.

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RT-PCR

against

the

EGFP-specific

probes

according

to

the

procedures

of

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Larval tissues (gonad, head, integument, fat body, hemocyte, mid-gut, malpighian tubes,

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anterior silk gland, middle silk gland, posterior silk gland) on the third day of fifth instar were

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collected and rinsed with diethylpyrocarbonate (DEPC) - treated water. Total RNAs were

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extracted using TRIzol reagent (Life Technology) following manufacturer’s manual. Then, total

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RNAs were reverse transcribed with GoScript Reverse Transcription System (Promega) and with

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oligo(dT)15 as a primer, followed by the synthesis of the second cDNA strand. Then, cDNA

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amplification was achieved using primers listed in Table S1. PCR amplification was carried out


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according to the following conditions: initial heating at 94°C for 4 min, then 25 cycles of 94°C

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for 40 s, 55°C for 40 s, 72°C for 3 min, and 72°C for 10 min. BmActin3 transcript levels were

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used as an internal control.

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Real-time PCR

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Total RNAs from various tissues dissected from the wandering stage of transgenic and

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wild-type silkworm were isolated and purified using a Total RNA Kit II (Omega) and reverse

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transcribed with the GoScript Reverse Transcription System (Promega) and using oligo(dT)15 as

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a primer. The cDNAs were used for subsequent real-time PCR reactions. Amplified products

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were detected using SYBR-green supermix (TaKaRa) with a 7500Fast thermal cycler (Life

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Technology). PCR amplification was carried out according to the following conditions: 95°C for

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30 s, 40 cycles of 95°C for 3 s, 60°C for 30 s. Transcription initiation factor 4a gene (Tif4a) was

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used as an internal control. Primer sequences are listed in Table S1. All gene expression levels

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are means ± s.d. of relative values of signals, normalized to the Tif4a controls from three

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independent experiments.

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Western blotting

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Total proteins from larval tissues were dissected from the wandering stage of transgenic and

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wild-type silkworms and extracted using RIPA lysis buffer. Total protein content was

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determined photometrically by BCA assay (Beyotime). Samples were normalized for equal

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protein content and 200 µg total proteins were loaded per sample. Then, proteins were separated

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on 10% SDS–PAGE gels and transferred to PVDF membranes (Roche). The RFP was detected

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using anti-RFP antibodies (Sigma). BmNKAα and BmSERCA proteins were detected using

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affinity-purified rabbit anti-BmNKAα and anti-BmSERCA antibodies. At the primary antibody

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reaction step, the affinity-purified antibody was diluted 1: 10,000 in Tris-buffered saline with


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0.1% Tween 20 and 1% non-fat dry milk, followed by 2 h incubation with goat peroxidase–

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conjugated anti-rabbit IgG (Sigma) secondary antibody diluted 1: 20,000 in Tris-buffered saline

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containing 0.1% Tween 20 and 1% non-fat dry milk. Immunoreactive proteins were visualized

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from the reaction mixture with stable peroxide solution and luminol/enhancer solution

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(Millipore). Silkworm α-tubulin was used as an internal control. Primary antibody of α-tubulin

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and goat peroxidase–conjugated anti-mouse IgG was purchased from Sigma.

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Elemental analysis by inductive coupled plasma atomic absorption spectroscopy (ICP-

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AAS)

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Tissues and cocoons were collected and cut into pieces, then were dried in an oven at 120°C

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to a constant weight. Dry samples were quantitatively dissolved in acid (HNO3 : HClO4 = 5 : 1)

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at 140 °C for 2.5 h. The resulting solution was diluted with a known volume of deionized water.

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All measurements were performed with Z-5000 (Hitachi) using the method of N2O-acetylene

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flame AAS. All results are presented as mean ± s.d. of three separate experiments performed in

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triplicate.

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FTIR microspectroscopy of single silk fibers

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Silk fibers from transgenic and wild-type cocoons were degummed by boiling in two 30

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min rounds using 0.5% (w/w) NaHCO3 solution. Then, degummed silk fibers were washed with

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distilled water and air-dried at room temperature. Experiments were performed and the data was

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processed following the procedures described previously.21 Notably, each spectrum shown in this

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study represents the mean taken from separate deconvolutions from at least 15 separate samples

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from different cocoons. This experiment was repeated for three silkworm generations.

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Mechanical properties of silk fibers


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Larvae of silkworm reared to the fifth larval stage on a diet of mulberry leaves were divided

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into weight-matched groups when silk fibers were initially observed. The wandering silkworms

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were allowed to construct cocoons naturally. The cocoons were harvested. Then, the first 50 m of

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silk was reeled for mechanical testing. For forcible silking, the silk was grasped and reeled

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artificially from the silkworm using the method developed by Khan et al.22 We attempted to

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obtain silk fibers at 60 rpm/min for more than 5 min. Subsequently, the silk fibers obtained were

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cut to initial lengths of 16 mm. The average cross-sectional diameters were measured across two

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brins by a scanning electron microscopy (JEOL). Single-fiber testing and data processing were

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performed as described previously.21 The number of silks using in the mechanical testing was 17

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and each silk came from different cocoon or silkworm. Significant differences in the mechanical

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properties of different types of silk fibers were determined by Student’s t-test using PASW

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Statistics 18 (SPSS Inc). Also, this experiment was repeated for three silkworm generations.

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RESULTS AND DISCUSSION

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Expression profiles of BmNKAα and BmSERCA in the silk gland

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Previous studies have shown that sodium, potassium and calcium are abundant in the silk

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gland.19,

23, 24

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transition of silk fibroin in vivo. To verify this hypothesis, we first investigated the expression

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pattern of two P-type ATPases (NKA and SERCA) in the silk gland of silkworms. NKA

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localizes to the cell membrane and is responsible for sodium/potassium exchange across the

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membrane in the cytoplasm.25 SERCA, which localizes to the membrane of the endoplasmic

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reticulum, is one of the key factors to maintain the calcium balance in cells.26 Based on real-time

Thus, we expect that these ions might play key roles in the conformational


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quantitative PCR (qPCR) results, we found that the BmNKAα gene expressed in all sections of

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the silk gland, while BmSERCA gene predominantly expressed in the ASG (Figure 1). Our

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findings suggested that these two proteins might participate in the ion transport in the ASG.

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Figure 1. Real-time qPCR analysis of BmNKAα and BmSERCA in different parts of the silk glands at the

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wandering stage. PSG, posterior silk gland. PMSG, posterior section of middle silk gland. MMSG, middle

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section of middle silk gland. AMSG, anterior section of middle silk gland. ASG, anterior silk gland. Error bars,

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s.d.

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BmCP231 is an ASG-specific promoter

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To study whether ion-transporting proteins are involved in the formation and mechanical

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properties of silk fibers, our purpose is to overexpress ion-transporting proteins in the ASG of

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silkworms. Because there were no reports focused on the ASG-specific promoter, we initially

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sought to identify an ASG-specific promoter. Previously, we found an ASG-specific cuticle

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protein (BmASSCP2) and we cloned its promoter (BmCP231).27 To verify the activity of this

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promoter, we constructed a piggyBac vector, in which the dsRed gene was driven by the

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BmCP231 promoter and a marker gene EGFP was driven by a nervous system- and eye-specific

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promoter (Figure 2A). After injecting the transgenic vectors into the eggs of silkworms, we

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generated the BmCP231 transgenic silkworms (Figure 2B). Fluorescent observation showed that

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the red fluorescent protein (RFP) only expressed in the ASG (Figure 2C). Southern blotting

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showed that the piggyBac vector was inserted into the silkworm genome (Figure 2D).


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Figure 2. The BmCP231 promoter can specifically induce the DsRed expression in the ASG. (A) piggyBac

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vector design. 3×P3 promoter is a nervous system- and eyes-specific promoter. (B) BmCP231 transgenic

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silkworm. Fluorescence was excited from the ASG part of the transgenic silkworm under an RFP-excitation

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wavelength light. (C) Red fluorescence could be only detected in ASG of BmCP231 transgenic silkworm

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under an RFP-excitation wavelength light. (D) Southern blotting analysis of the silkworm genome using

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EGFP-specific probes. Left and right lines represent the genome of WT and BmCP231 transgenic silkworms,

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respectively.

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In order to detect whether the DsRed gene expressed in the BmCP231 transgenic silkworms,

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the reverse transcription PCR (RT-PCR) and western blotting were introduced. The results

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shown in Figure 3 illustrated that the RFP expressed specifically in the ASG both in mRNA level

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and protein level. These findings indicated that the BmCP231 promoter could highly and

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specifically induce the transcription of downstream genes in the ASG.


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Figure 3. Molecular analysis of BmCP231 transgenic silkworms. (A) RT-PCR analysis of dsRed (arrow)

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expression in various tissues of BmCP231 transgenic silkworms by DsRed-specific primers. Transcript levels

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of Bmactin3 were used as an internal control. (B) Western blotting analysis using RFP-specific antibodies.

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Expression levels of silkworm α-tubulin were used as an internal control.

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Overexpression of the P-type ATPase in ASG altered the ion content of silk

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Using this ASG-specific promoter, we constructed piggyBac vector, in which the BmNKAα

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or BmSERCA gene was each driven by the BmCP231 promoter (Figure 4A). Next, by detecting

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the EGFP expression in the compound eyes, we isolated the transgenic silkworms, termed Over-

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NKA and Over-SERCA (Figure 4B). Southern blotting results confirmed that these two

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piggyBac vectors were integrated into the genome of B. mori (Figure 4C).

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After propogation for five generations, we investigated the biological features of transgenic

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silkworm and found the unchanged silk production ability (cocoon shell ratio). However, the

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cocoons and body size of Over-SERCA were significantly larger than the wild-type (WT; Figure


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S1). We also examined the morphological differences of cocoons and fibers between transgenic

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silkworms and WT. Still, no remarkable differences were found (Figure S2).

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Figure 4. Isolation of the Over-NKA and Over-SERCA transgenic silkworms. (A) piggyBac vector designs.

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(B) By detecting the EGFP expression in the compound eyes, the transgenic silkworms were isolated. (C)

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Southern blotting analysis using EGFP-specific probes.

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To determine whether the expression levels of the BmNKAα and BmSERCA enhanced in

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transgenic silkworms, total RNAs and proteins were extracted from the tissues to perform

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molecular analysis. Real-time qPCR results showed that the transcripts of these genes increased

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significantly in the ASG of transgenic silkworms, whereas no expression differences were

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detected in other tissues (Figure 5A-5C). These results further confirmed that the BmCP231

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promoter was ASG-specific. By western blotting using antibodies directed against BmNKAα and

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BmSERCA, we also found that these ATPases were successfully overexpressed in the ASG

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(Figure 5D).


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Figure 5. Overexpression of BmNKAα or BmSERCA. (A) Real-time quantitative PCR (qPCR) of BmNKAα

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gene in the multiple tissues of silkworm from Over-NKA and wild-type (WT) silkworm. (B) qPCR of

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BmSERCA gene in the multiple tissues of silkworm from Over-SERCA and WT silkworm. (C) qPCR of

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BmNKAα and BmSERCA from the ASG of transgenic and WT silkworm. (D) Western blotting of BmNKAα

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and BmSERCA from the ASG of transgenic and WT silkworms using specific polyclonal antibodies. Go,

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gonad; He, head; Ep, epidermis; Mg, mid-gut; Fb, fat body; Mt, malpighii tube; Hc, hemocyte; Asg, anterior

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silk gland; Msg, middle silk gland; Psg, posterior silk gland. Error bars, s.d.; *** P < 0.001 (Student’s t-test).

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These two P-type ATPases are responsible for ion transport. Thus, we examined the sodium,

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potassium and calcium levels in the tissues and cocoons of transgenic silkworms by ICP-AAS.

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The levels of metal ions in the whole organism must be finely regulated by ionic intake, storage

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and excretion. As we expected, the ion levels changed hugely in different tissues (Figure 6). For

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Over-NKA silkworms, the sodium levels in the mid-gut and hemolymph were higher, while

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those in the silk gland and cocoons were lower than WT (Figure 6A-B). Similar results were


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observed in the potassium levels (Figure 6C-D). We also discovered that the calcium levels

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increased in the Over-SERCA hemolymph and silk gland, but decreased in the cocoon silks

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(Figure 6E-F). The data here yielded interesting clues for identifying the crosstalk between

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tissues in ionic level. Although the tissue crosstalk is beyond the scope of the current work, the

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transgenic silkworms presented here will give us the valuable materials to explore it in the future.

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NKA helps to maintain high potassium and low sodium levels in the cytoplasm by

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transporting Na+ to the extracellular environment coupled with K+ transport into the cytoplasm

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across the cytoplasmic membrane.25 Meanwhile, as a driver of ionic transportation, the

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electrostatic potential created by NKA can cause other ion-transporting proteins to mediate ionic

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transportation.28 Thus, we proposed that overexpressing NKA in ASG cells might induce Na+

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and K+ to flow away from silk protein to the cells of gland, resulting in lower sodium and

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potassium levels in the transgenic cocoons (Figure 6). Then, these ions were stored in the

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hemolymph (Figure 6). SERCA mediates calcium storage in endoplasmic reticulum.29 It was

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possible to consider that more calcium ions were delivered to the gland cells from the silks, thus

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the calcium levels decreased in silks and increased in silk gland (Figure 6). These findings

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illustrated that the ion content of silk gland and silk fiber had been successfully modified by

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overexpressing the ion-transporting proteins.


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Figure 6. Elemental analysis by ICP-AAS. (A-B) Comparisons of sodium levels in multiple tissues (A) and

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cocoons (B) from Over-NKA and wild-type (WT) silkworms. (C-D) Potassium levels in multiple tissues (C)

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and cocoons (D) from Over-NKA and WT silkworms. (E-F) Calcium levels in multiple tissues (E) and

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cocoons (F) from Over-SERCA and WT silkworms. Ep, epidermis; Mg, mid-gut; He, hemolymph; Sg, whole

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silk gland, including anterior, middle and posterior silk gland; Error bars, s.d.; * P < 0.05; ** P < 0.01; *** P

Modifying the Mechanical Properties of Silk Fiber by Genetically

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