Coding

Part:BBa_K4990008

Designed by: Dingjian Zhang, Tianle Xue   Group: iGEM23_CPU-CHINA   (2023-09-19)
Revision as of 04:50, 12 October 2023 by Norreland (Talk | contribs) (Wetlab Characterization)


Dual-Edged Harpoon

Usage in short

You can use it to target and kill both Fn and CRC!

What is it?

Here is the structure of Dual-Edged Harpoon.The name,"Harpoon",is the nickname we call it.Of course, you can call it more formally as "Dual-targeted Cytotoxic peptide".

mFada B-domain https://parts.igem.org/Part:BBa_K4990002

Auto cleaving linker A https://parts.igem.org/Part:BBa_K4990005

LL-37 Truncated Peptide https://parts.igem.org/Part:BBa_K4990001

Auto cleaving linker B https://parts.igem.org/Part:BBa_K4990006

HlpA monomer https://parts.igem.org/Part:BBa_K4990007

TO KNOW ABOUT IT!

With the advancement of molecular biology, numerous active proteins and peptides have been invented or discovered for therapeutic applications. Recombinant technology, renowned for its high expression levels and straightforward operations, has been widely employed in the biopharmaceutical sector. To achieve multi-target, multi-functional active proteins, it's imperative to link and fuse two or more proteins with known functions. This method of obtaining bifunctional or multifunctional fusion proteins has emerged as one of the novel approaches for new drug development and bioproduct research. Particularly, it has been extensively utilized in the preparation of bispecific single-chain antibodies (scFv) or antibody-drug conjugates [1-5].

Many bacteria have long chain fiber-protein complexes on their surfaces, which are called pili or fimbriae. These pili are composed of individual pilus monomers that link together end-to-end in the extracellular environment, self-assembling into long chain fibers with high physical strength.

For Fusobacterium nucleatum, its pili are referred to as FadA (Fusobacterium adhesin A). The monomers that make up these pili come in two forms: ①pre-FadA, which serves as an anchoring structure, attaching the entire pilus to the bacterial inner membrane. ②mFadA (mature FadA), which can link head-to-tail and self-assemble into a long filament.[6-7]

In our project, the aim is to achieve bacteria-bacteria targeting. To accomplish this, we intend to leverage the self-assembly property of mFadA. We have fused a bacterial pilus monomer onto a membrane protein of the engineered bacterium, which we call the "fishing rod protein" . The membrane protein acts as the fishing rod, the linker serves as the fishing line, and the bacterial pilus monomer functions as the bait. By utilizing surface display techniques to display the fishing rod protein, our engineered bacteria can essentially "fish" for target bacteria, enabling precise bacteria-bacteria targeting.

Using surface display technology, pili monomers were expressed on the surface of Bifidobacterium longum. However, we quickly realized that the direct display of pili monomers was unnecessary and would lead to a range of issues, including steric hindrance, nonspecificity, and metabolic waste. Intriguingly, the self-assembly of the pili is driven by a pivotal role of a 26 amino acid-long α-helix at the N-terminus. Therefore, we contemplated using truncated pili monomers, eliminating all structures unrelated to self-assembly, and only displaying the critical 26 amino acid-long motif. We termed this structure mFadA B-domain and designed it as a Basic Part (BBa K4990002). Consequently, the Dual-targeted cytotoxic peptide utilized mFadA B-domain to target Fn.

In 2006, Tjalsma, employing the Highly accurate tandem MS method, identified a protein named Histone-like protein A (HlpA). This discovery forged a connection between Streptococcus bovis and colorectal cancer. It was posited that S. bovis binds to the colorectal cancer cell surface's heparan sulfate-proteoglycans (HSPG) through HlpA, mediating the colonization of S. bovis in colorectal cancer [8].

By 2009, Boleij substantiated that Streptococcus gallolyticus binds to the colorectal cancer cell surface through interactions between HlpA and HSPG [9].

In 2016, O'Neil elucidated the crystal structure of Hlp, revealing its crab-claw-like configuration. The claw section, rich in basic amino acids, can engage with DNA and also binds to heparin [10].

In 2018, Chun Loong Ho leveraged the binding affinity between HlpA and HSPG to engineer an Escherichia coli strain that specifically targets colorectal cancer, marking the inception of the HlpA targeting system [11].

By 2022, the iGEM22_LZU-CHINA team, harnessing synthetic biology, devised an Escherichia coli strain that targets colorectal cancer, employing the HlpA-mediated targeting of tumor cell surface HSPG [12].

In 2023, Tang designed a probiotic strain that uses HlpA for targeting and Azurin for cytotoxicity. This exhibited promising therapeutic outcomes in mouse models of colorectal cancer [13].


control
Figure A:Crystal structure of HlpA(O'Neil 2016) Figure B:Engineered EcN treat CRC(Chun 2018)

HlpA, as its name "Histone-like protein A" indicates, can bind to DNA like histones. Its overall shape resembles a crab claw, which allows it to non-specifically bind tightly to the minor groove of DNA using its ribbon-like β-fold region, leading to significant DNA bending, DNA compaction, and negative supercoiling. S. bovis, through an enigmatic mechanism, secretes it extracellularly, positioning it as an anchorless protein and a target for the humoral immune system during infections. By bridging bacterial lipoteichoic acid (LTA) and HSPG on colonic epithelial cells, it facilitates bacterial adherence to colon tumor cells.

Therefore, HlpA possesses dual capabilities: 1. Targeting colorectal cancer and 2. Non-specifically binding to DNA chains.

Antimicrobial peptides are widely sourced, first discovered in the immune systems of insects. Subsequently, similar peptides have been extracted from bacteria, fungi, amphibians, higher plants, and mammals. These active peptides possess natural immune functions and serve as essential molecular barriers for host defense against invading pathogens, hence referred to as antimicrobial peptides.[14-15]

The tissue proteinase inhibitor family is unique to mammals and constitutes a type of antimicrobial peptide.LL37 is derived from one of the tissue proteinase inhibitors, human cationic antimicrobial protein 18 (hCAP18). Among them, LL-37 is the sole antimicrobial peptide found in the human body. It possesses various functions such as antimicrobial, antitoxin, immunomodulatory, and wound healing effects. Additionally, LL-37 holds potential for treating viral diseases and neoplastic conditions. Numerous research teams have investigated the activity of truncated peptides to identify the minimal antimicrobial region of LL-37. The results indicate that the smallest region of LL-37 is within amino acid positions 17 to 29 or 18 to 29. [16-17] This suggests that the C-terminal helix, consisting of 12 or 13 amino acids of LL-37, plays a role in antimicrobial, antiviral, and antitumor activities.

What can it do?

However, not the entire structure of mFadA is involved in self-assembly. Thus, we considered removing unnecessary domains. Upon closer examination of mFadA's structure, we divided it into two domains: Domain A and Domain B. Domain A comprises two anti-parallel α-helical structures, while Domain B consists of a single anti-parallel α-helix. We believe that Domain B is the most crucial. On a microscale, it possesses the function of binding with Domain A, and on a macroscale, it exhibits the capability to target Fn (Fusobacterium nucleatum).

Therefore, by displaying the engineered bacteria with the mFadA B-domain on their surface, specific adhesion to Fn can be achieved, enabling bacteria-to-bacteria targeting.

Here is the structure of HlpA monomer and homodimer.Two helical segments from each monomeric subunit constitute an α-helical ‘body’ with two protruding β-ribbon ‘arms’ , which extend to bind the DNA helix. These DNA-binding β-ribbons are largely disordered in the absence of DNA.[12]

Monomeric HlpA can be used in the design of fusion proteins due to its capability to bind with the HSPG present on the surface of colorectal cancer cells. Thus, if you aim to target colorectal cancer cells using proteins, you can engineer a fusion protein with HlpA tethered at one end. This bestows the fusion protein, termed as Dual-Edged Harpoon, with the ability to specifically target colorectal cancer cells [11-13].

It's worth mentioning that these HlpA monomers can spontaneously self-assemble into homodimers, exhibiting a substantial affinity between them. Consequently, in our experiments associated with the dual-targeted peptide, we observed a pronounced dimerization phenomenon.

Wetlab Characterization

Dimerazation and Cleavage Test


SDS-PAGE of total protein. A. The total protein SDS-PAGE results. B. The protein fragment that may form due to the linkers’ cleavage and the dimerization of HlpA.

During the experiment, it was discovered that proteins containing HlpA such as DEH, TTR, and TTP consistently display characteristic bands across specific molecular weight regions. This cannot be attributed to chance occurrences. After thorough investigation and experimental simulations, it is believed that this is caused by the linkers’ being cleaved and the dimerization ability of HlpA.

Reference

[1] Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.

[2] Gustavsson M,Lehtio J,Denman S,et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris[J]. Protein Eng,2001,14(9):711-715.

[3] Wang SH,Zheng CJ,Liu Y,et al. Construction of multiform scFv antibodies using linker peptide[J]. J Genetics and Genomics,2008,35:313-316.

[4] Zhang JH,Yun J,Shang ZG,et al. Design and optimization of a linker for fusion protein construction[J]. Progr Natur Sci,2009,19: 1197-1200.

[5] Shan D,Press OW,Tsu TT,et al. Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths[J].J Immunology,2014,162:6589-6595.

[6]Nithianantham, S., Xu, M., Yamada, M., Ikegami, A., Shoham, M., & Han, Y. W. (2009). Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. Journal of Biological Chemistry, 284(6), 3865-3872.

[7]Témoin, S., Wu, K. L., Wu, V., Shoham, M., & Han, Y. W. (2012). Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament’s length and width. FEBS letters, 586(1), 1-6.

[8]Tjalsma H, Schöller‐Guinard M, Lasonder E, et al. Profiling the humoral immune response in colon cancer patients: diagnostic antigens from Streptococcus bovis[J]. International journal of cancer, 2006, 119(9): 2127-2135.

[9]Boleij A, Schaeps R M J, de Kleijn S, et al. Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells[J]. Infection and immunity, 2009, 77(12): 5519-5527.

[10]O'Neil P, Lovell S, Mehzabeen N, et al. Crystal structure of histone-like protein from Streptococcus mutans refined to 1.9 Å resolution[J]. Acta Crystallographica Section F: Structural Biology Communications, 2016, 72(4): 257-262.

[11]Ho C L, Tan H Q, Chua K J, et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention[J]. Nature biomedical engineering, 2018, 2(1): 27-37.

[12]https://2022.igem.wiki/lzu-china/

[13]Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. Life Sciences, 324, 121709.

[14]de Breij, A., Riool, M., Cordfunke, R. A., Malanovic, N., de Boer, L., Koning, R. I., ... & Nibbering, P. H. (2018). The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms. Science translational medicine, 10(423), eaan4044.

[15]Zhang, M., Liang, W., Gong, W., Yoshimura, T., Chen, K., & Wang, J. M. (2019). The critical role of the antimicrobial peptide LL-37/CRAMP in protection of colon microbiota balance, mucosal homeostasis, anti-inflammatory responses, and resistance to carcinogenesis. Critical Reviews™ in Immunology, 39(2).

[16]Fabisiak, A., Murawska, N., & Fichna, J. (2016). LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity. Pharmacological Reports, 68(4), 802-808.

[17]Chieosilapatham, P., Ikeda, S., Ogawa, H., & Niyonsaba, F. (2018). Tissue-specific regulation of innate immune responses by human cathelicidin LL-37. Current Pharmaceutical Design, 24(10), 1079-1091. Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 31
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 31
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 31
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 31
    Illegal NgoMIV site found at 378
  • 1000
    COMPATIBLE WITH RFC[1000]

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