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Molecular cloning and sequence analysis of Factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda

Authors:
Jeak L. Ding at National University of Singapore
Jeak L. Ding
Tony Navas at AbbVie
Tony Navas
Bow Ho
Bow Ho
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Abstract

Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.
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... LAL and the proclotting pathway also detect fungi, specifically (1-3)-β-D-glucans, through Factor G, which has also garnered scientific interest for recombinant technology (Yamamoto et al. 2023). Although Factors C and G have been sequenced via cloning and cDNA isolation (Muta et al. 1991;Ding et al. 1995;Yamamoto et al. 2023), it is unclear if we are missing duplicates of genes in this pathway, particularly Factors C and G, and what role they may play in the proclotting pathway, which can be influential in optimizing or creating additional effective synthetic alternatives. In addition to understanding the syntenic relationships among horseshoe crab species and how WGDs have shaped horseshoe crab genomes, deeper analyses of high-quality genome assemblies can reveal important, yet unknown, components governing the genome architecture of horseshoe crabs. ...
... Both L. polyphemus and C. rotundicauda genomes exhibit a full-length tandem duplication of canonical Factor C, which are more similar within (94% to 96% sequence identity) than between species (88% to 90% identity), leading to the interpretation that these are species-specific duplications of Factor C ( Fig. 3; supplementary table S7, Supplementary Material online). It has been previously reported that single-chain copies of Factor C persist in the presence of LPS in C. rotundicauda, in addition to the cleaved enzymes (Ding et al. 1995). One of the copies from each species exhibits a mutation of the proline immediately preceding the heavy-light chain cleavage site (RS) (Fig. 3). ...
Genome Assembly of a Living Fossil, the Atlantic Horseshoe Crab Limulus polyphemus , Reveals Lineage-Specific Whole-Genome Duplications, Transposable Element-Based Centromeres, and a ZW Sex Chromosome System
Article
Full-text available
  • Feb 2025
  • MOL BIOL EVOL
Horseshoe crabs, considered living fossils with a stable morphotype spanning ∼445 million years, are evolutionarily, ecologically and biomedically important species experiencing rapid population decline. Of the four extant species of horseshoe crabs, the Atlantic horseshoe crab, Limulus polyphemus, has become an essential component of the modern medicine toolkit. Here, we present the first chromosome-level genome assembly, and most contiguous and complete assembly to date, for Limulus polyphemus using nanopore long-read sequencing and chromatin conformation analysis. We find support for three horseshoe crab-specific whole genome duplications, but none shared with Arachnopulmonata (spiders and scorpions). Moreover, we discovered tandem duplicates of endotoxin-detection pathway components Factor C and Factor G, identify candidate centromeres consisting of Gypsy retroelements, and classify the ZW sex chromosome system for this species and a sister taxon, Carcinoscorpius rotundicauda. Finally, we revealed this species has been experiencing a steep population decline over the last 5 million years, highlighting the need for international conservation interventions and fisheries-based management for this critical species.
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... The signal is proportional to the level of activated factor C and detected through photometry. 17 An advantage with recombinant lysate is that it does not contain factor G which reacts to (1,3)-β-D-glucan. Hence the lysate will not cause false-positives, in terms of reacting with glucan impurities in finished products. ...
... A further milestone development with a synthetic substitute to horseshoe crab blood was in 2001, in the form of an improved laboratory-synthesized genetically engineered rFC. 17 In 2003, the first proto-recombinant lysates emerged, at a consistency that could be produced on a commercial scale. 21 However, it remained that wider laboratory tests involving such a replacement were limited due to the availability (production to scale), uncertainty around regulatory opinion, and the market dominance of the current LAL test. ...
Historical Milestones and Industry Drivers in the Development of Recombinant Lysate for Bacterial Endotoxin Testing
Article
Full-text available
  • Nov 2020
The idea for the use of recombinant lysate is to provide a reagent that reacts in the same way as the natural cascade within the four species of horseshoe crab: (Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda). The cloning technology serves an alternative to the fishing and bleeding of horseshoe crabs (where bleeding is performed through a large dorsal blood sinus called the pericardium). In addition, the use of recombinant technology to substitute the specific pathway seeks to address the concern with some lysates that also detect beta glucans through the activation of factor G. A further advantage with recombinant lysates stems from the production of the test reagent through the use of cell culture. This may result in the supply of a more consistent product. This article attempts to show this through a discussion of the historical perspective, the current situation is a disservice to laboratory users. Much of what has been written on rFC to date has looked at things from a U.S.-centric perspective (rather than U.S. and European) and from a supplier or regulatory perspective; this article embraces the laboratory user perspective. Published in American Pharmaceutical Review, 26 (6): https://www.americanpharmaceuticalreview.com/
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... Factor C is synthesized as a single polypeptide has a molecular weight of 123 kDa. It consists of two polypeptide chains connected by disulfide bonds: a heavy chain (80 kDa) and a light chain, (43 kDa) in T. tridentatus, while in C. rotundicauda, it consists of a heavy chain (80 kDa) and a light chain (52 kDa) [13,[15][16][17]. Factor C of C. rotundicauda has been cloned and expressed in Escherichia coli [18]. ...
Production of codon‑optimized Factor C fragment from Tachypleus gigas in the Pichia pastoris GS115 expression system for endotoxin detection
Article
Full-text available
  • Oct 2023
Background Factor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. Methods and results The FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. Conclusions Protein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating
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... The signal peptides are located at amino acid positions 1-25, which consist of N-region signal peptide(1)(2)(3)(4)(5), H-region signal peptide(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and C-region signal peptide(18)(19)(20)(21)(22)(23)(24)(25). On the other hand, heavy chain has cysteine-rich regions (Cys-rich region), four complement control proteins (CCP), EGF (epidermal growth factor)-like, LCCL, and C-type lectin (CLECT). ...
Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas)
Article
Full-text available
  • Apr 2023
Background: Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab's factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary. Methods and results: Sampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156-1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex. Conclusions: Endotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C.
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... Moreover, the CES3 region immobilised onto MCC beads via the CBM3 of recombinant protein CES3:CBM3 removed a significant amount of endotoxin from the protein sample without affecting the amount of protein (Fig. 7). The N-terminal region of Factor C containing cysteine-rich, EGF-like, and sushi 1-3 domains is sufficient to bind to LPS at an extremely high affinity [23][24][25][26]39 . Factor C has been reported to detect a femtogram of endotoxins, which is much lower than the detection limit recommended for pharmaceutical products 26,40 . ...
Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples
Article
Full-text available
  • Sep 2022
Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E. coli, it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1–3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E. coli. To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.
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... Meanwhile, rFC test is accepted as a promising tool to increase reproducibility and prevent misinterpretation of results due to interference mechanisms based on β-glucans (Liebers et al. 2019). The recombinant Factor C is produced from the cDNA of the Mangrove horseshoe crab (Carcinoscorpius rotundicauda) according to Ding et al. (1995). Like the protease in the haemolymph of the horseshoe crab, the recombinant Factor C is activated by endotoxin. ...
Occupational endotoxin exposure and health effects
Article
Full-text available
  • Nov 2020
  • ARCH TOXICOL
Lipopolysaccharide (LPS), also termed endotoxin, is an integral structural component of the outer membrane of Gram-negative bacteria and has been a focus of bioaerosol research for many years. Endotoxin is nearly ubiquitous in the environment; however, exposure at specific workplaces, such as waste collecting, livestock farming, agriculture, the textile industry has been associated with adverse health effects. The aim of this review is to summarize studies published in the last 10 years on endotoxin measurement and health effects due to endotoxin in occupational settings. The search was mainly performed using MEDLINE (Pubmed), focusing on publications related to the determination of endotoxin, inhalative occupational endotoxin exposure, and health effects. The review shows that despite the well-established methods available to measure endotoxin, a global comparison of studies still remains difficult because the details of sampling strategies and exposure assessment are variable and depend on the specific workplace situation. Thus, health-based threshold limit values still cannot be derived on the basis of available data. Since endotoxin is only one component in a heterogeneous bioaerosol mixture, the question remains open on how to evaluate and record the additional effects of the other components. In particular, there is a lack of intervention studies investigating the effectiveness of protective measures with respect to health outcome. In addition, the studies selected in this review show a wide range of endotoxin exposure, even within one industry or sector. The level of exposure seems to depend more on the specific task performed and the way it was performed rather than on the profession or industry itself. The identification of hot spots of exposure, as well as methods of communication on hazards and possible protective measures, seem to remain important tasks in occupational health protection.
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Alternative Verfahren
Chapter
  • May 2021
Dieses Kapitel beschriebt drei Methoden: Methode A – quantitative Prüfung, Methode B – semiquantitative Prüfung und Methode C – Vergleichsprüfung mit Referenzchargen. Eine ganze Reihe von Versuchen wurde unternommen, den ersten Faktor der endotoxingetriggerten Aktivierungskaskade in den Amoebocyten der Schwertschwänze rekombinant herzustellen. Dieses Faktor C genannte Enzym liegt als Zymogen vor, welches durch Endotoxine aktiviert wird. Eine ganze Reihe von Versuchen wurde unternommen, den ersten Faktor der endotoxingetriggerten Aktivierungskaskade in den Amoebocyten der Schwertschwänze rekombinant herzustellen. In den Leishmaniazellen sorgen Chaperone für die genaue Faltung des rFC. Mehr als 20 Disulfidbindungen werden korrekt ausgebildet. Das Nährmedium für Leishmania ist kostengünstig, während es für Insektenzellen teuer ist. Auch ist die Kultivierung vergleichbar mit der Fermentierung von Bakterien, und die Protozoen wachsen recht schnell, da sie eine kurze Generationszeit haben. Die Entwicklung ist durch die Kooperation zwischen der Kyushu Universität und Seikagaku Corp. zustande gekommen. Verwendet werden die Sequenzen aus Tachypleus tridentatus.
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Unlocking the Use of Lipid-Based Formulations with Lipophilic Salts to Address Bioavailability Challenges in Small Molecule Drug Development
Article
Full-text available
  • Aug 2020
Drug developers have traditionally benefited from the ionizability of acidic or basic drugs to form ionic salts that increase water solubility. However, this increased water solubility does not always relate to improved oral bioavailability, and therefore specialized technologies are needed to address bioavailability challenges. Lipid-based formulations (LBFs) are widely used in drug development to improve the oral absorption of poorly water-soluble drugs, which continue to dominate the development pipeline. To render the target API more lipid-soluble and to reach the dose while unlocking the benefits of LBFs, a recent strategy has been to utilize a similar approach to ionic salts, but instead of pairing the ionized drug with a small inorganic or small organic counterion, a bulky, asymmetric non-polar organic counterion is utilized. These so-called lipophilic salts (LSs) may also be referred to as ionic liquids (ILs) or hydrophobic ion pairs (HIPs). Lipid-Based Technology Applications Lipid-based formulations using liquid-filled hard capsule or soft gel formats have been used extensively in the biopharma market to address a range of formulation challenges. Lipid-or liquid-based approaches have been used for better ensuring dose uniformity for low-dose applications, as well as to better ensure safe handling of highly potent API. LBF approaches-primarily soft gels-have also been extensively used in life cycle management strategies, especially in over-the-counter applications. Fixed-dose combinations and colonic delivery are additional areas of application. However, addressing bioavailability challenges remains the primary LBF application where lipid/solvent-based formulation approaches are especially effective in improving the solubility of highly lipophilic compounds.
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Chromosome‐level genome assembly of the coastal horseshoe crab (Tachypleus gigas)
Article
  • Jul 2020
Horseshoe crabs, represented by only four extant species, have existed for around 500 million years. However, their existence is now under threat because of anthropogenic activities. The availability of genomic resources for these species will be valuable in planning appropriate conservation measures. Whole‐genome sequences are currently available for three species. In this study, we have generated a chromosome‐level genome assembly of the fourth species, the Asian coastal horseshoe crab Tachypleus gigas (genome size 2.0 Gb). The genome assembly has a scaffold N50 value of 140 Mb with approximately 97% of the assembly mapped to 14 scaffolds representing 14 chromosomes of T. gigas. In addition, we have generated the complete mitochondrial genome sequence and deep‐coverage transcriptome assemblies for four tissues. A total of 26,159 protein‐coding genes were predicted in the genome. The T. gigas genome contains five Hox clusters similar to the mangrove horseshoe crab Carcinoscorpius rotundicauda, suggesting that the common ancestor of horseshoe crabs already possessed five Hox clusters. Phylogenomic and divergence time analysis suggested that the American and Asian horseshoe crab lineages shared a common ancestor around the Silurian period (~436 Ma). Comparison of the T. gigas genome with those of other horseshoe crab species with chromosome‐level assemblies provided insights into the chromosomal rearrangement events that occurred during the emergence of these species. The genomic resources of T. gigas will be useful for understanding their genetic diversity and population structure and would help in designing strategies for managing and conserving their stocks across Asia.
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