Identification of Two Critical Contact Residues in a Pathogenic Epitope from Tetranectin for Monoclonal Antibody Binding and Preparation of Single-Chain Variable Fragments

Abstract

Sepsis is a fetal disease that requires a clear diagnostic biomarker for timely antibiotic treatment. Recent research has identified a pyroptosis-inducing epitope known as P5-5 in tetranectin (TN), a plasma protein produced by monocytes. Previously, we produced a 12F1 monoclonal antibody against the P5-5 and discovered that it could not only diagnose the presence but also monitor the progress of sepsis in the clinic. In the current study, we further investigated the structure site of the P5-5 and the recognition mechanism between the 12F1 mAb and the P5-5 epitope. To this end, 10 amino acids (NDALYEYLRQ) in the P5-5 were individually mutated to alanine, and their binding to the mAb was tested to confirm the most significant antigenic recognition sites. In the meanwhile, the spatial conformation of 12F1 mAb variable regions was modeled, and the molecular recognition mechanisms in detail of the mAb to the P5-5 epitope were further studied by molecular docking. Following epitope prediction and experimental verification, we demonstrated that the motif "DALYEYL" in the epitope sequence position 2-8 of TN-P5-5 is the major binding region for mAb recognition, in which two residues (4L and 8L) were essential for the interaction between the P5-5 epitope and the 12F1 mAb. Therefore, our study greatly narrowed down the previously reported motif from ten to seven amino acids and identified two Leu as critical contact residues. Finally, a single-chain variable fragment (scFv) from the 12F1 hybridoma was constructed, and it was confirmed that the identified motif and residues are prerequisites for the strong binding between P5-5 and 12F1. Altogether, the data of the present work could serve as a theoretic guide for the clinical design of biosynthetic drugs by artificial intelligence to treat sepsis.

Keywords: P5-5; epitope; scFv; sepsis; tetranectin.

Figure 1

TN-P5-5 epitope prediction and three-dimensional structural analysis. (A) The output graph of presented linear B-cell epitopes displays amino acid positions (X-axis) and Bepipred scores (Y-axis). Residues with scores above the default threshold of 0.35 (shown in yellow) are more likely to be part of the epitope. Green implies a decreased likelihood of belonging to the epitope. (B) The 3D structure of TN-P5-5 with the predicted epitope from SWISS-MODEL homology modeling. Amino acid residues 4–8 motif (Leu-Tyr-Glu-Tyr-Leu) are shown in light blue.

Figure 2

12F1 mAb homology model and molecular dock. The scFv binding location and interactions between the antibody paratope and the P5-5 epitope were revealed following docking. The conformation prediction of scFv residues (green) to the TN-P5-5 epitope (blue) was depicted as a stick model and labeled. Among them, residues of P5-5 involved in the binding pockets are Leu4, Tyr7, and Leu8.

Figure 3

TN-P5-5 pathogenic epitope sequence and mutagenesis map of the P5-5 epitope through alanine (red) scan. To find out the interaction of the 12F1 mAb and different mutants of P5-5 recognition, standard peptides of ADALYEYLRQ, NDALYEYLRQ, NAALYEYLRQ, NDAAYEYLRQ, NDALAEYLRQ, NDALYAYLRQ, NDALYEALRQ, NDALYEYARQ, NDALYEYLAQ, and NDALYEYLRA were chemically synthesized.

Figure 4

Epitope identification based on P5-5 (WT) and mutants. The indirect ELISA titer for various site mutant peptides. Error bars represent the standard deviation (SD) of six repeated experiments. Data are presented as mean ± SD. **** p < 0.0001.

Figure 5

Molecular recognition mechanism of 12F1 mAb towards TN-P5-5. Competitive reactions of P5-5 and mutagenesis. The concentrations of P5-5 (WT) and mutagenesis were diluted to 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 625 ng/mL, 312.5 ng/mL, 156.25 ng/mL, 78.13 ng/mL, 39.06 ng/mL, 19.53 ng/mL, 9.77 ng/mL, 4.88 ng/mL, 2.44 ng/mL, 1.22 ng/mL, and 0.61 ng/mL with blocking solution as competition antigen to react with 12F1 mAb, respectively.

Figure 6

Construction and production of scFv fusion proteins that target TN-P5-5. (A) The diagram illustrates the construction of MBP tag and MBP-fused scFv formats against the P5-5 epitope in this study (MBP, MBP-linker-scFv). ATG, start codon; MBP, maltose binding protein; Linker, (G₄S)₃ linker chain; scFv, single-chain variable fragment; Stop, stop codon. (B) The production and purification of fused scFv proteins were analyzed based on SDS-PAGE. The molecular weight of the expressed MBP protein was approximately 50 kDa, while the presence of distinct protein bands at 70 kDa confirms the successful purification of the scFv following E. coli lysis. M: Protein marker; Lane 1: control E. coli TB1 proteins; Lane 2: MBP tag protein (empty vector pMAL-c2x in E. coli TB1); Lane 3: Expressed total proteins of MBP-linker-scFv; Lane 4: Purified scFv protein.