Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas)

Abstract

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.

Production of codon-optimized Factor C fragment from Tachypleus gigas in the Pichia pastoris GS115 expression system for endotoxin detection

“…By binding to the receptor’s area to create a ligand-receptor complex, three ligands diphosphoryl lipid A, core lipid A, and Kdo2 lipid A could behave as activators of the Factor C protein, according to our docking simulation. As a result, three structures (Complexes 2, 3, and 4) were chosen for additional examination [ 25 ].…”

“…Recombinant Factor C (rFC) has been expressed in different host cells, including, among others: Pichia pastoris [ 19 , 20 ], Saccharomyces cerevisiae [ 21 ], mammalian cells [ 22 , 23 ], and insect cells using the baculovirus system [ 24 ]. Factor C has also been cloned from the species L. polyphemus [ 25 ]. Our report on the analysis of docking simulations in silico and molecular dynamics of protein Factor C (Receptor) simulations with ligands of lipid A (lipopolysaccharides) shows that the active sites and binding pockets between proteins and lipopolysaccharide ligands of gram-negative bacteria, are present in Heavy Chain Fragment Factor C with Lectin Domain [ 26 ].…”

Molecular Recognition ofMoringa oleiferaActive Compounds for Stunted Growth Prevention Using Network Pharmacology and Molecular Modeling Approach

“…All docking parameters are computed by using AutoDock Tools 1.5.6 developed by Morris and co-workers . The docking procedure was carried out according to similar protocols presented in our previous studies. − …”