Next Article in Journal
Impact of Cytochrome P450 Enzyme on Fruit Quality
Previous Article in Journal
Integrated Methylome and Transcriptome Analysis between Wizened and Normal Flower Buds in Pyrus pyrifolia Cultivar ‘Sucui 1’
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 13
10.3390/ijms25137179
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Overview and Evolution of Insect Fibroin Heavy Chain (FibH)

by
Tong Zhang
1,
Sanyuan Ma
1,
Ziyang Zhang
2,
Yongkang Guo
1,
Daiying Yang
1 and
Wei Lu
1,*
1
Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing 400715, China
2
College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(13), 7179; https://doi.org/10.3390/ijms25137179
Submission received: 21 May 2024 / Revised: 16 June 2024 / Accepted: 17 June 2024 / Published: 29 June 2024
(This article belongs to the Section Molecular Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2–3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39–45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

1. Introduction

Unrivaled in their adaptability and ubiquity, insects serve as a cornerstone of Earth’s biodiversity and play a crucial role in ecosystems as decomposers, pollinators, and food. Beyond their ecological significance, certain insects synthesize silk proteins to construct various marvelous structures, including a cocoon, nest, and egg case [1]. Silkworm (Bombyx mori) has the unique ability to spin silk, a sustainable biomaterial captivating human attention for millennia due to its distinctive physical and chemical properties [2].
Insects are renowned for their silk production, with moths and butterflies being the most common silk-producing insects. Other fascinating orders such as Trichoptera and Hymenoptera including bees, wasps, and ants are also known for their silk-spinning abilities [1,3,4]. Silk is an impressive and versatile biopolymer, primarily composed of proteins interwoven with metal ions, and other inorganic and organic substances. These components vary across species, resulting in silk with distinct characteristics. Silkworm cocoon silk is predominantly made up of sericin and fibroin including FibH, FibL, and P25/fibrohexamerin [5,6]. The oriental moth cocoon is composed of the most abundant asparagine-rich proteins, fibroin, mineral crystals, and organic substances [7]. The caddisfly silk contains FibH, FibL, endoplasmin, serpin, chymotrypsin-like protease, two mucin-like proteins, pseudofibrin, and three additional unannotated proteins without P25 [8].
The FibH is a fundamental component of insect silk, nearly universally present in the silk of Lepidoptera and Trichoptera. The FibH of Bombyx mori (BmorFibH) is encoded by the fibroin heavy chain gene nearly 17 kb in length, with a molecular weight of 350 kDa [5]. Based on sequence characteristics, BmorFibH is divided into N- and C-terminal non-repetitive domains (NTDs and CTDs, respectively), enclosing a repetitive core (RC). The NTD and CTD are involved in the folding, assembly, and secretion of silk proteins, and extremely conserved across silkworm strains [9,10,11,12,13,14]. The RC is composed of many tandem repetitive sequences [13,15,16]. Under the synergistic effect of many factors such as pH, metal ions, and shear force, the RC forms the crystalline region, which confers physical properties to the silk fiber [17,18,19,20]. Despite having the same repetitive motifs, such as hexapeptide GAGAGS and GAGAGY, the number and distribution of these motifs vary, leading to significant differences in the FibH sequences across silkworm strains [13,16].
The FibH is a pivotal determinant in the mechanical properties of insect silk fibers [21,22]. Deciphering and expanding the repertoire of the FibH gene promise to shed light on the intricate molecular evolution of insect silk [23]. This knowledge is critical for the synthetic replication of silk’s unique properties [24]. Furthermore, understanding the diversity of FibH can shed light on the pathways by which nature fine-tunes these proteins for a specific function. This revelation could spark innovative methodologies in the design of silk-based materials, enabling tailoring the properties of silk fiber to a specific application, such as achieving superior mechanical properties and flexibility. To date, the complete FibH sequences of about 280 mulberry silkworms (Bombyx mori and Bombyx mandarina) and six other insect species have been deciphered, revealing a vast and unexplored landscape of FibH diversity [13,16]. However, the complete FibH sequences of countless insects remain unknown, presenting a formidable challenge to our quest for a holistic grasp of silk’s evolutionary history and its functional diversity across the insect kingdom.
In this study, we undertook a comprehensive analysis of publicly available Lepidoptera and Trichoptera genomic data, focusing on extracting the complete FibH sequence through a homology analysis. We systematically examined the characteristics of all the FibH sequences, including unique amino acid composition, repetitive motifs, and evolutionary relationships. Our work can substantially enrich our grasp of the insect silk diversity. Moreover, it will establish a solid theoretical framework for the innovation and synthesis of novel artificial silk, thereby opening new avenues in the field of biomaterial science.

2. Results

2.1. FibH Gene Copy Number Variation Occurs Only in Skipper (Hesperiidae)

Using publicly available genomic data, we extracted 101 complete FibH gene sequences from Lepidoptera, involving 40 families. Limited by the available assembled genomic data, we only extracted 13 complete FibH gene sequences from four families of Trichoptera. The FibH gene of Lepidoptera is 8040 (AperFibH)-30,526 bp (HcomFibH 2) long. The FibH gene of Trichoptera varied between 16,133 bp (OalbFibH) and 28,991 bp (CdisFibH). All insect FibH genes consist of two exons separated by a single intron. Exon 1 is relatively short, ranging from 39 to 45 bp, while exon 2 is between 7 and 29 kb and varies greatly among different species. The introns of all FibH genes range from 73 to 5159 bp, and their guanine–cytosine (GC) content is around 50% (Figure 1A and Figure S1).
The FibH in Trichoptera and moths is a single-copy gene. Interestingly, there is copy number variation (CNV) in the FibH gene of skippers. Both Thymelicus acteon and Thymelicus sylvestris have three copies of the FibH gene, and they are located on chromosome 20. The lengths of the TactFibH (TactFibH 1, 2, and 3) and TsylFibH (TsylFibH 1, 2, and 3) genes are 21,450 bp, 27,758 bp, 27,295 bp, 15,511 bp, 23,197 bp, and 23,172 bp, respectively. Hesperia comma and Carterocephalus palaemon each have two FibH genes located on chromosomes 22 and 17, respectively. The HcomFibH 1 and 2, as well as CpalFibH 1 and 2, have 14,044, 30,526, 19,690, and 14,355 base pairs, respectively (Figure 1B, Table 1).

2.2. FibH Differs in Length, Amino Acid Composition, and Properties among Insects

The protein sequences of all FibH genes have been deduced. The length and molecular weight of FibH in moths vary significantly, ranging from 2603 (IaveFibH) to 8773 (TacrFibH) amino acids (with an average of 5006), with 216.05948–746.16907 kDa. Butterflies have FibH of equal length to moths, between 3178 and 8754 (average: 5420). Skippers with multiple copies have their FibH distributed between 4284 (HcomFibH 1) and 8754 (TactFibH 2) (Table 1). In contrast, the FibH of Trichoptera is generally longer and has a larger molecular weight than that of the Lepidoptera, with 5344–9027 amino acids (average: 7650), and 531.07887–912.29248 kDa. Ala, Gly, Ser, Pro, Gln, and Asn are the most abundant in Lepidoptera FibH, while Trichoptera FibH is rich in Gly, Ser, and Arg, with only less Ala (Figure 2A–C, Table 1). Based on the complexity of the sequence and domain, the FibH is divided into the conserved NTD, CTD, and highly variable repetitive core (RC). The NTD of BmorFibH contains 151 amino acids, with the first 14 amino acids encoded by exon 1, and the first 18 amino acids being a signal peptide, which is the same as that in skippers (Table 1). Gly 1, Ile 8, and Gly 52 are three conserved sites that are present in all NTDs. In the NTD of Trichoptera FibH, the sixth and seventh amino acids are LL (LeuLeu). The CTD of BmorFibH contains 58 amino acids, and the 20th residue (Cys-c20) from the C-terminus forms a disulfide bond with FibL, which is also conserved and present in the CTD of non-Saturniidae FibH (Figure S2).
To investigate the effects of amino acid composition on the properties of FibH, the isoelectric points (pI) and grand average of hydropathicity (GRAVY) of all FibHs were analyzed. The pI of moth FibH is distributed between 3.2 and 12.3, with 85.9155% being less than 7; the GRAVY scores range from −1.415 to 0.557, with hydrophilic FibH constituting 57.7465%. For butterfly FibH, the pI is distributed between 3.8 and 9.19, with 76.6667% of the pI values being less than 7; the GRAVY scores are between −1.256 and 0.183, with hydrophilic FibH making up 86.6667%. Compared to moths and butterflies, the pI and GRAVY of Trichoptera (caddisfly) FibH show more pronounced differences. Except for OalbFibH, which has a pI less than 7, the pI of other Trichoptera FibH ranges from 10.33 to 11.99. All Trichoptera FibH are hydrophilic proteins with an average hydrophobicity ranging from −0.827 to −0.048, which is likely closely related to their living environments (Figure 2D). In skippers, different copies of FibH exhibit varying properties. The pI values for both HcomFibH 1 and HcomFibH 2 are above 8, with GRAVY scores of −0.288 and −1.029, respectively. Conversely, the pI of CpalFibH 1 and CpalFibH 2 are both less than 7, and their GRAVY scores are −0.977 and −0.395, respectively. Interestingly, the pI values for TactFibH 1 and TsyFibH 1 are 4.89 and 5.29, respectively, with GRAVY scores of −0.323 and −0.104. The pI values for the other copies are all around 8, and both TactFibH 3 and TsyFibH 3 are highly hydrophilic (Table 1). The different chemical properties of the various FibH copies suggest that the mechanical properties of this skipper silk may also differ.

2.3. Insect FibH Has Developed Unique Motifs during Evolution

The fibroin heavy chain is divided into the NTD, CTD, and repetitive region (RC), which constitutes approximately 85% of FibH and is composed of numerous motifs. FibH is the core protein of insect silk, and its evolutionary direction is consistent with that of insect silk fibers. To elucidate the evolutionary history of FibH, the phylogenetic relationships of all FibH were analyzed. In the phylogenetic tree, the Trichoptera (caddisfly) FibH forms a separate branch and possesses typical motifs distinct from those of the Lepidoptera (moth and butterfly) (Figure 3 and Figure S3). The amino acids Tyr, Val, Arg, and Trp are unique to the majority of Trichoptera FibH. The phylogenetic tree indicates that MminFibH and PmusFibH are ancestral to other Lepidoptera FibH, from which many branches have evolved. There are differences in the typical motifs among different Lepidoptera insects. PolyAla, polySer, and the hexapeptide GAGSGA are ubiquitously present in Lepidoptera FibH and represent peptides that distinguish Lepidoptera FibH motifs from those of Trichoptera FibH. It is well known that GAGSGA is a typical motif of the BmorFibH [13,15]. In fact, apart from the silkworm, other species in the Bombycidae family, such as TvarFibH, and in the Notodontidae and Psychidae family, such as FfurFibH and LferFibH, also contain numerous GAGSGA motifs (Figure 3, Table S1).
Butterfly FibH is believed to have evolved from moth FibH, which is consistent with the conclusion that moths are the ancestors of butterflies. The FibH of skippers (a subfamily of butterflies) evolved from that of Papilionoidea, and multiple copies of FibH have evolved from single-copy FibH, such as PmalFibH and EtagFibH (Figure S3). Interestingly, there are significant differences in the motif of different FibH copies. While they possess different motifs, all contain the tripeptide QGP. TactFibH 1 and 2 and TsylFibH 2 have similar motifs that include polyAla and GPQ. TactFibH 3 and TsylFibH 3 share the same typical motif, QGPQGPYGPQ. Other FibH have different motifs, and all contain the tripeptide QGP (Figure 3, Table S1). These results suggest that the evolution of skipper FibH is not merely the result of a simple gene duplication event.

3. Discussion

In this study, we obtained and characterized 114 FibH sequences, providing the most systematic description of insect FibH to date. There are four significant findings. Firstly, we have completed the FibH sequences of many insects, such as bagworms (Luffia ferchaultella). Second, it was the first time that copy number variation events of the FibH gene were found in insects, occurring only in skippers. Third, we revealed that the FibH of skippers evolved from that of Papilionoidea. Lastly, we identified GAGAGS, polyAla, or polySer as the predominant motifs in Lepidopteran FibH, whereas amino acids Tyr, Val, Arg, and Trp are unique to the majority of Trichoptera FibH.
Lepidoptera comprises approximately 130 families, with a genome of about 2279 insect species from about 46 families being sequenced. However, many insect genomes are still incomplete with only contigs; thus, we were only able to extract the FibH gene from 40 families. In contrast, there is even less whole-genome data available for the Trichoptera. Scaling up the sequencing of Lepidoptera and Trichoptera, accelerating insect whole-genome assembly, and improving genome quality will further expedite research on the FibH gene, providing a more comprehensive and in-depth understanding of insect silk proteins.
The complexity and functionality of spider silk originate from the diversity of spidroins, including MaSp, MiSp, and PySp, among others. These spidroins have various variants, each with a unique structure, which imparts different physical and chemical properties to spider silk. Additionally, multiple spidroin variants can enhance silk protein synthesis efficiency, which is particularly important in extreme environments or rapid spider web construction [25,26,27,28,29]. Previously, there have been no reports on copy number variations of the insect FibH gene. In this study, we first discovered that some butterfly species have 2–3 copies of the FibH gene, with each copy having different isoelectric points and hydrophobicity, suggesting unique mechanical properties of these butterfly silks (Table 1). Silkworm silk exhibits different mechanical properties at each instar, primarily due to the varying proportions of three silk fibroins (FibH, FibL, and P25) in different instar silks. In butterflies, the relationship between an increased FibH copy number and the silk protein yield, synthesis rate, and mechanical properties of silk fibers still needs experimental verification.
BmFibH is synthesized by the silkworm posterior silk gland (PSG). The FibH forms a silk protein complex with FibL and P25 in a 6:6:1 ratio, and its secondary structure is mainly a random coil or α-helix in the PSG cell. When the silk protein is secreted into the silk gland lumen, during the transportation process from the PSG to the anterior silk gland (ASG), FibH undergoes changes in physical and chemical environments, causing its secondary structure to transition from a random coil or α-helix to an antiparallel β-sheet [17,18,19,20]. Near the spinneret of ASG, a highly ordered anisotropic herringbone pattern gradually forms, preparing for spinning silk [30]. To understand the structural differences of FibH across insects, we used Alphafold 2 to predict the three-dimensional structure of FibH and MaSp 1. BmorFibH, LhesMaSp 1, and MiSp (A6YP79.1.A) exhibit similar structures characterized by numerous relatively uniform helices, which may be related to strong selection pressures acting on silkworms and spiders. AalFibH (moth) and OalbFibH (caddisfly) share a similar structure with fewer non-uniform helices, and their NTD contains both β-sheets and α-helices. However, the NTD of PbarFibH (butterfly) also contains β-sheets and α-helices, but no uniform helical structure (Figure S4). It is worth mentioning that these structures are only predictions and need to be further experimentally verified.
Insect FibH is composed of numerous repetitive motifs (Figure 3, Table S1). The type, number, and arrangement of motifs determine the mechanical properties of the silk fibers. Spider dragline silk is stronger than silkworm silk, mainly due to the high uniformity of repetitive sequences and polyAla in MaSp. The FibH of bagworms contains both polyAla and GAGAGS motifs, making the mechanical properties of bagworm silk comparable to that of spider silk [31]. The most abundant motifs in the BmorFibH are hexapeptide GAGAGS and GAGAGY [13]. Yoko Takasu et al. [24] assembled these two motifs into a highly ordered artificial FibH and completely replaced the original FibH gene. The resulting homozygous individuals were unable to spin silk, suggesting that the design of silk proteins must follow natural principles. Artificial intelligence, with its powerful learning and computational capabilities, has been used for protein structure prediction and the development of new Cas proteins, among other applications [32,33,34,35]. It is also an inevitable trend to predict the structure of silk proteins, design silk proteins, and customize artificial silk [36,37,38]. A large database of silk protein sequences and mechanical properties’ data of silk fibers is fundamental for artificial intelligence to design silk proteins. Currently, there are about 500 silk protein sequences available, and many insect genomes are accessible [13,16,27]. However, what is most lacking at present are data on the mechanical properties of silk fibers. Collecting numerous silk fibers and measuring their mechanical properties to clarify the relationship between mechanical performance and the FibH sequence will help deep learning to accelerate the development and utilization of novel silk fibers.
All in all, our study develops a profound understanding of insect FibH, providing a broader perspective on the possible utility of these proteins in bioengineering and other apposite applications.

4. Materials and Methods

4.1. Extract the Complete FibH Gene Sequence from Database

Search in NCBI (https://www.ncbi.nlm.nih.gov/datasets/genome/) accessed on 27 March 2024 with the insect species name as a keyword. Genomes sequenced by PACBIO_SMRT (Sequel II or Revio) and with coverage depth above 20× are downloaded, while genomes sequenced by NGS and ONT are not utilized. Meanwhile, the 40 amino acids at the N- and C-terminus of the known FibH of Bombyx mori, Ephestia kuehniella, Antheraea pernyi, Samia ricini, Antheraea yamamai, Antheraea assama, and Yponomeuta cagnagella were used as tag sequences to blast the searched genome. Use the retrieved nucleotide sequences with homology to search the genome, determine the 5′ and 3′ terminus of the FibH gene, and extract the complete FibH gene sequence. Then, refer to the FibH genes of Bombyx mori, Ephestia kuehniella, Antheraea pernyi, Samia ricini, Antheraea yamamai, Antheraea assama, and Yponomeuta cagnagella, and finally determine the exons and introns of the FibH gene of the selected insect. The complete FibH protein sequence was deduced using Bioedit 7.2.

4.2. Data Analysis and Graphing

The theoretical isoelectric point (pI) and grand average of hydropathicity (GRAVY) of FibH were calculated by protparam (https://web.expasy.org/protparam/ accessed on 27 March 2024), and the signal peptide was predicted by SignalP-5.0 [39]. Multiple sequence alignment was performed using the online software mafft version 7 (https://mafft.cbrc.jp/alignment/server/ accessed on 27 March 2024). The multiple sequence alignment results and FibH gene mapping on the genome were visualized by TBtools-II v2.086 [13]. The phylogenetic tree was generated by IQ-TREE 2 [40] with maximum likelihood (ML) and bootstrap value 1000. Use Adobe Illustrator 2023 (Adobe Inc., Mountain View, CA, USA) and the online software ITol v6 (https://itol.embl.de/itol.cgi accessed on 27 March 2024) to beautify the phylogenetic tree. OriginPro 2021 (OriginLab, Northampton, MA, USA) is used to draw curve graphs and scatter plots. We first retrieve the FibH sequences using a sliding window with a width of 6 and count the most frequently occurring motif. Then, using the left boundary of this motif sequence, we divide the entire FibH sequence into sequence subsets. Each subset is then analyzed for motifs using MEME software (v5.5.5) with parameters (-minw 6 -maxw 100 -allw -mod anr -nmotifs 20). The motifs with occurrence sites ≥3 in the results are considered the motif set for that FibH sequence.

4.3. Three-Dimensional Structure Prediction of FibH

The 3D structure of FibH was predicted using online Alphafold 2 [41] (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb?authuser=3#scrollTo=KK7X9T44pWb7 accessed on 27 March 2024). The N-terminal 705 amino acids of FibH were used as a query sequence. Except for setting num_recycles to 12, other parameters are defaults.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25137179/s1.

Author Contributions

Conceptualization, W.L.; methodology, W.L.; validation, T.Z. and W.L.; formal analysis, T.Z. and Y.G.; investigation, T.Z. and W.L.; resources, T.Z.; writing—original draft, T.Z.; writing—review and editing, W.L., S.M., Z.Z., and D.Y.; visualization, T.Z., Z.Z., and W.L.; supervision, W.L.; project administration, W.L.; funding acquisition, W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Youth Foundation [32300411] and Chongqing Natural Science Foundation [CSTB2023NSCQ-BHX0183].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Sutherland, T.D.; Young, J.H.; Weisman, S.; Hayashi, C.Y.; Merritt, D.J. Insect Silk: One Name, Many Materials. Annu. Rev. Entomol. 2010, 55, 171–188. [Google Scholar] [CrossRef] [PubMed]
  2. Xia, Q.; Li, S.; Feng, Q. Advances in Silkworm Studies Accelerated by the Genome Sequencing of Bombyx mori. Annu. Rev. Entomol. 2014, 59, 513–536. [Google Scholar] [CrossRef] [PubMed]
  3. Sutherland, T.D.; Weisman, S.; Trueman, H.E.; Sriskantha, A.; Trueman, J.W.H.; Haritos, V.S. Conservation of Essential Design Features in Coiled Coil Silks. Mol. Biol. Evol. 2007, 24, 2424–2432. [Google Scholar] [CrossRef] [PubMed]
  4. Shi, J.; Lua, S.; Du, N.; Liu, X.; Song, J. Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana. Biomaterials 2008, 29, 2820–2828. [Google Scholar] [CrossRef] [PubMed]
  5. Inoue, S.; Tanaka, K.; Arisaka, F.; Kimura, S.; Ohtomo, K.; Mizuno, S. Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J. Biol. Chem. 2000, 275, 40517–40528. [Google Scholar] [CrossRef] [PubMed]
  6. Peng, Z.; Yang, X.; Liu, C.; Dong, Z.; Wang, F.; Wang, X.; Hu, W.; Zhang, X.; Zhao, P.; Xia, Q. Structural and Mechanical Properties of Silk from Different Instars of Bombyx mori. Biomacromolecules 2019, 20, 1203–1216. [Google Scholar] [CrossRef] [PubMed]
  7. Qin, L.; Li, J.; Guo, K.; Lu, M.; Zhang, Y.; Zhang, X.; Zeng, Y.; Wang, X.; Xia, Q.; Zhao, P.; et al. Insights into the structure and composition of mineralized hard cocoons constructed by the oriental moth, Monema (Cnidocampa) flavescens Walker. Insect Biochem. Mol. Biol. 2022, 151, 103878. [Google Scholar] [CrossRef] [PubMed]
  8. Rouhová, L.; Sehadová, H.; Pauchová, L.; Hradilová, M.; Žurovcová, M.; Šerý, M.; Rindoš, M.; Žurovec, M. Using the multi-omics approach to reveal the silk composition in Plectrocnemia conspersa. Front. Mol. Biosci. 2022, 9, 945239. [Google Scholar] [CrossRef] [PubMed]
  9. Zhou, C.Z.; Confalonieri, F.; Jacquet, M.; Perasso, R.; Li, Z.G.; Janin, J. Silk fibroin: Structural implications of a remarkable amino acid sequence. Proteins 2001, 44, 119–122. [Google Scholar] [CrossRef]
  10. He, Y.-X.; Zhang, N.-N.; Li, W.-F.; Jia, N.; Chen, B.-Y.; Zhou, K.; Zhang, J.; Chen, Y.; Zhou, C.-Z. N-Terminal Domain of Bombyx mori Fibroin Mediates the Assembly of Silk in Response to pH Decrease. J. Mol. Biol. 2012, 418, 197–207. [Google Scholar] [CrossRef]
  11. Hu, W.; Lu, W.; Wei, L.; Zhang, Y.; Xia, Q. Molecular nature of dominant naked pupa mutation reveals novel insights into silk production in Bombyx mori. Insect Biochem. Mol. Biol. 2019, 109, 52–62. [Google Scholar] [CrossRef] [PubMed]
  12. Hao, Z.; Long, D.; Zhang, Y.; Umuhoza, D.; Dai, J.; Xu, Z.; Zhang, G.; Meng, W.; Xiang, Z.; Zhao, A. New insight into the mechanism of in vivo fibroin self-assembly and secretion in the silkworm, Bombyx mori. Int. J. Biol. Macromol. 2021, 169, 473–479. [Google Scholar] [CrossRef] [PubMed]
  13. Lu, W.; Ma, S.; Sun, L.; Zhang, T.; Wang, X.; Feng, M.; Wang, A.; Shi, R.; Jia, L.; Xia, Q. Combined CRISPR toolkits reveal the domestication landscape and function of the ultra-long and highly repetitive silk genes. Acta Biomater. 2023, 158, 190–202. [Google Scholar] [CrossRef] [PubMed]
  14. Moreno-Tortolero, R.O.; Luo, Y.; Parmeggiani, F.; Skaer, N.; Walker, R.; Serpell, L.; Holland, C.; Davis, S.A. Silk Road Revealed: Mechanism of silk fibre formation in Bombyx mori. bioRxiv 2023. [Google Scholar] [CrossRef]
  15. Zhou, C.Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; et al. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000, 28, 2413–2419. [Google Scholar] [CrossRef] [PubMed]
  16. Lu, W.; Zhang, T.; Zhang, Q.; Zhang, N.; Jia, L.; Ma, S.; Xia, Q. FibH Gene Complete Sequences (FibHome) Revealed Silkworm Pedigree. Insects 2023, 14, 244. [Google Scholar] [CrossRef] [PubMed]
  17. Zhou, L.; Chen, X.; Shao, Z.; Huang, Y.; Knight, D.P. Effect of Metallic Ions on Silk Formation in the Mulberry Silkworm, Bombyx mori. J. Phys. Chem. B 2005, 109, 16937–16945. [Google Scholar] [CrossRef] [PubMed]
  18. Greving, I.; Cai, M.; Vollrath, F.; Schniepp, H.C. Shear-Induced Self-Assembly of Native Silk Proteins into Fibrils Studied by Atomic Force Microscopy. Biomacromolecules 2012, 13, 676–682. [Google Scholar] [CrossRef] [PubMed]
  19. Hu, L.; Han, Y.; Ling, S.; Huang, Y.; Yao, J.; Shao, Z.; Chen, X. Direct Observation of Native Silk Fibroin Conformation in Silk Gland of Bombyx mori Silkworm. ACS Biomater. Sci. Eng. 2020, 6, 1874–1879. [Google Scholar] [CrossRef]
  20. Suzuki, Y.; Morie, S.; Okamura, H.; Asakura, T.; Naito, A. Real-Time Monitoring of the Structural Transition of Bombyx mori Liquid Silk under Pressure by Solid-State NMR. J. Am. Chem. Soc. 2023, 145, 22925–22933. [Google Scholar] [CrossRef]
  21. Ma, S.; Shi, R.; Wang, X.; Liu, Y.; Chang, J.; Gao, J.; Lu, W.; Zhang, J.; Zhao, P.; Xia, Q. Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor. Sci. Rep. 2014, 4, 6867. [Google Scholar] [CrossRef]
  22. Malay, A.D.; Sato, R.; Yazawa, K.; Watanabe, H.; Ifuku, N.; Masunaga, H.; Hikima, T.; Guan, J.; Mandal, B.B.; Damrongsakkul, S.; et al. Relationships between physical properties and sequence in silkworm silks. Sci. Rep. 2016, 6, 27573. [Google Scholar] [CrossRef] [PubMed]
  23. Frandsen, P.B.; Hotaling, S.; Powell, A.; Heckenhauer, J.; Kawahara, A.Y.; Baker, R.H.; Hayashi, C.Y.; Ríos-Touma, B.; Holzenthal, R.; Pauls, S.U.; et al. Allelic resolution of insect and spider silk genes reveals hidden genetic diversity. Proc. Natl. Acad. Sci. USA 2023, 120, e2221528120. [Google Scholar] [CrossRef] [PubMed]
  24. Takasu, Y.; Yamada, N.; Kojima, K.; Iga, M.; Yukuhiro, F.; Iizuka, T.; Yoshioka, T. Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori. Insect Biochem. Mol. Biol. 2023, 161, 104002. [Google Scholar] [CrossRef] [PubMed]
  25. Malay, A.D.; Craig, H.C.; Chen, J.; Oktaviani, N.A.; Numata, K. Complexity of Spider Dragline Silk. Biomacromolecules 2022, 23, 1827–1840. [Google Scholar] [CrossRef] [PubMed]
  26. Nakamura, H.; Kono, N.; Mori, M.; Masunaga, H.; Numata, K.; Arakawa, K. Composition of Minor Ampullate Silk Makes Its Properties Different from Those of Major Ampullate Silk. Biomacromolecules 2023, 24, 2042–2051. [Google Scholar] [CrossRef] [PubMed]
  27. Lu, W.; Shi, R.; Li, X.; Ma, S.; Yang, D.; Shang, D.; Xia, Q. A review on complete silk gene sequencing and de novo assembly of artificial silk. Int. J. Biol. Macromol. 2024, 264, 130444. [Google Scholar] [CrossRef] [PubMed]
  28. Kono, N.; Ohtoshi, R.; Malay, A.D.; Mori, M.; Masunaga, H.; Yoshida, Y.; Nakamura, H.; Numata, K.; Arakawa, K. Darwin’s bark spider shares a spidroin repertoire with Caerostris extrusa but achieves extraordinary silk toughness through gene expression. Open Biol. 2021, 11, 210242. [Google Scholar] [CrossRef] [PubMed]
  29. Kono, N.; Nakamura, H.; Mori, M.; Yoshida, Y.; Ohtoshi, R.; Malay, A.D.; Moran, D.A.P.; Tomita, M.; Numata, K.; Arakawa, K. Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk. Proc. Natl. Acad. Sci. USA 2021, 118, e2107065118. [Google Scholar] [CrossRef]
  30. Song, K.; Wang, Y.; Dong, W.; Li, Z.; Xia, Q.; Zhu, P.; He, H. Decoding silkworm spinning programmed by pH and metal ions. Sci. Bull. 2024, 69, 792–802. [Google Scholar] [CrossRef]
  31. Yoshioka, T.; Tsubota, T.; Tashiro, K.; Jouraku, A.; Kameda, T. A study of the extraordinarily strong and tough silk produced by bagworms. Nat. Commun. 2019, 10, 1469. [Google Scholar] [CrossRef]
  32. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  33. Peng, D.; Zheng, L.; Liu, D.; Han, C.; Wang, X.; Yang, Y.; Song, L.; Zhao, M.; Wei, Y.; Li, J.; et al. Large-language models facilitate discovery of the molecular signatures regulating sleep and activity. Nat. Commun. 2024, 15, 3685. [Google Scholar] [CrossRef]
  34. Lin, Z.; Akin, H.; Rao, R.; Hie, B.; Zhu, Z.; Lu, W.; Smetanin, N.; Verkuil, R.; Kabeli, O.; Shmueli, Y.; et al. Evolutionary-scale prediction of atomic-level protein structure with a language model. Science 2023, 379, 1123–1130. [Google Scholar] [CrossRef] [PubMed]
  35. Ruffolo, J.A.; Nayfach, S.; Gallagher, J.; Bhatnagar, A.; Beazer, J.; Hussain, R.; Russ, J.; Yip, J.; Hill, E.; Pacesa, M.; et al. Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences. bioRxiv 2024. [Google Scholar] [CrossRef]
  36. Jiang, M.; Shu, T.; Ye, C.; Ren, J.; Ling, S. Predicting the conformations of the silk protein through deep learning. Analyst 2021, 146, 2490–2498. [Google Scholar] [CrossRef]
  37. Kim, Y.; Yoon, T.; Park, W.B.; Na, S. Predicting mechanical properties of silk from its amino acid sequences via machine learning. J. Mech. Behav. Biomed. Mater. 2023, 140, 105739. [Google Scholar] [CrossRef]
  38. Fazio, V.; Pugno, N.M.; Giustolisi, O.; Puglisi, G. Hierarchical physically based machine learning in material science: The case study of spider silk. arXiv 2023. [Google Scholar] [CrossRef]
  39. Almagro Armenteros, J.J.; Tsirigos, K.D.; Sønderby, C.K.; Petersen, T.N.; Winther, O.; Brunak, S.; Von Heijne, G.; Nielsen, H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019, 37, 420–423. [Google Scholar] [CrossRef]
  40. Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; Von Haeseler, A.; Lanfear, R. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef]
  41. Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making protein folding accessible to all. Nat. Methods 2022, 19, 679–682. [Google Scholar] [CrossRef] [PubMed]
Ijms 25 07179 g001
Figure 1. The overview and copy number variation of the insect FibH gene. (A) The FibH genes of Trichoptera, moths, and butterflies. (B) The copy number variation of the FibH gene in skippers.
Figure 1. The overview and copy number variation of the insect FibH gene. (A) The FibH genes of Trichoptera, moths, and butterflies. (B) The copy number variation of the FibH gene in skippers.
Ijms 25 07179 g001
Ijms 25 07179 g002
Figure 2. The length, molecular weight, composition, and properties of FibH. (A) The length and molecular weight of FibH. (B) The amino acid composition of each insect FibH. The red, black, and orange colors represent the FibH of Trichoptera, moth, and butterfly, respectively. (C) The amino acid composition of moth, butterfly, and Trichoptera FibH. (D) The theoretical isoelectric point (pI) and grand average of hydropathicity (GRAVY) of FibH.
Figure 2. The length, molecular weight, composition, and properties of FibH. (A) The length and molecular weight of FibH. (B) The amino acid composition of each insect FibH. The red, black, and orange colors represent the FibH of Trichoptera, moth, and butterfly, respectively. (C) The amino acid composition of moth, butterfly, and Trichoptera FibH. (D) The theoretical isoelectric point (pI) and grand average of hydropathicity (GRAVY) of FibH.
Ijms 25 07179 g002
Ijms 25 07179 g003
Figure 3. The phylogenetic tree of insect FibH and their motifs. d1 represents the proportion of all motifs to the total sequence length, with a median of 0.89; d2 indicates the proportion of the most prevalent motif (the typical motif) to the total sequence length, with a median of 0.50; d3 denotes the number of amino acid residues in the typical motif, with a median of 35.64. The amino acid sequence on the right represents the motif sequence with the highest percentage in each insect FibH. The red, black, and orange colors in the phylogenetic tree represent the FibH of Trichoptera, moth, and butterfly, respectively.
Figure 3. The phylogenetic tree of insect FibH and their motifs. d1 represents the proportion of all motifs to the total sequence length, with a median of 0.89; d2 indicates the proportion of the most prevalent motif (the typical motif) to the total sequence length, with a median of 0.50; d3 denotes the number of amino acid residues in the typical motif, with a median of 35.64. The amino acid sequence on the right represents the motif sequence with the highest percentage in each insect FibH. The red, black, and orange colors in the phylogenetic tree represent the FibH of Trichoptera, moth, and butterfly, respectively.
Ijms 25 07179 g003
Table 1. The skipper FibH genes with multiple copies and their encoded protein.
Table 1. The skipper FibH genes with multiple copies and their encoded protein.
GeneExon 1 (bp)Intron (bp)CDS (bp)Protein (aa)Signal Peptide (aa)PIGrand Average of Hydropathicity (GRAVY)Top 5 Amino Acids
BmorFibH4297115,7925263184.390.216Gly, Ala, Ser, Tyr, Val
HcomFibH 142118912,8554284189.19−0.288Ala, Gly, Pro, Gln, Ser
HcomFibH 242490625,6208539188.85−1.029Gly, Pro, Gln, Ala, Ser
TactFibH 142133820,1126703184.89−0.323Gly, Ala, Pro, Ser, Gln
TactFibH 242149326,2658754187.87−0.153Gly, Ala, Pro, Ser, Gln
TactFibH 342287824,4178138187.85−1.256Gly, Pro, Gln, Ala, Ser
TsylFibH 142164813,8634620185.29−0.104Gly, Ala, Ser, Pro, Gln
TsylFibH 242249120,7066901189.14−0.155Ala, Gly, Pro, Ser, Gln
TsylFibH 342271820,4546817188.14−1.178Gly, Gln, Pro, Ala, Ser
CpalFibH 142334916,3415446186.74−0.977Gly, Pro, Gln, Ala, Ser
CpalFibH 24293313,4224473185.02−0.395Ala, Gly, Ser, Pro, Tyr
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhang, T.; Ma, S.; Zhang, Z.; Guo, Y.; Yang, D.; Lu, W. Overview and Evolution of Insect Fibroin Heavy Chain (FibH). Int. J. Mol. Sci. 2024, 25, 7179. https://doi.org/10.3390/ijms25137179

AMA Style

Zhang T, Ma S, Zhang Z, Guo Y, Yang D, Lu W. Overview and Evolution of Insect Fibroin Heavy Chain (FibH). International Journal of Molecular Sciences. 2024; 25(13):7179. https://doi.org/10.3390/ijms25137179

Chicago/Turabian Style

Zhang, Tong, Sanyuan Ma, Ziyang Zhang, Yongkang Guo, Daiying Yang, and Wei Lu. 2024. "Overview and Evolution of Insect Fibroin Heavy Chain (FibH)" International Journal of Molecular Sciences 25, no. 13: 7179. https://doi.org/10.3390/ijms25137179

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop