Fig 1.
Molecular cloning and functional prediction of Toxocara canis mucin 1 (Tc-MUC-1).
(A) Infective larvae of T. canis hatch from eggs in vitro are used for the molecular cloning of the gene (Tc-muc-1) encoding the Tc-MUC-1 protein. (B) Schematic diagram of Tc-muc-1 and the predicted domain architecture of Tc-MUC-1. Signal peptide, low complexity region and two abutting ShKT domains are indicated. (C) The three-dimensional structure of Tc-MUC-1 (without signal peptide) modelled using AlphaFold2 [38]. Cysteine residues and disulfide bonds in each ShKT domain are indicated in red.
Fig 2.
Pull-down and mass spectrometric analyses to identify proteins in murine macrophages that bind recombinant Toxocara canis mucin 1 (rTc-MUC-1).
(A) Prokaryotic expression of His-tagged bait protein (rTc-MUC-1) and elution of proteins of RAW264.7 macrophages bound to a Ni-NTA column. (B) SDS-PAGE analysis of rTc-MUC-1 and proteins pulled down by rTc-MUC-1. M: protein marker; Lane 1: Empty His-tag peptide (~ 55 kDa); Lane 2: proteins of RAW264.7 cells targeted by the empty His-tag peptide; Lane 3: purified rTc-MUC-1 fused to His-tag peptide (~ 75 kDa); Lane 4: proteins of RAW264.7 cells targeted by purified His-tag fused rTc-MUC-1 (red box indicated). (C) Proteins of RAW264.7 cells pulled down by rTc-MUC-1, and resolved by SDS-PAGE and stained with silver stain (red box indicated). (D) LC-MS/MS analysis of proteins pulled down by rTc-MUC-1. Peptides and proteins identified as well as the predominant functional predictions (cellular component, molecular function and biological processes) are indicated.
Fig 3.
Co-immunoprecipitation analysis of proteins of murine macrophages bound to recombinant Toxocara canis mucin 1 (rTc-MUC-1).
(A) Eukaryotic expression of FLAG-tagged Tc-MUC-1 of T. canis and HA-tagged aldo_ket_red domain-containing protein (AKR1B3), rho GDP-dissociation inhibitor 1 (ARHGDIA), cofilin 1 (CFL1), fatty acid binding protein 5 (FABP5), peroxiredoxin 1 (PRDX1) and profilin (PFN1) of murine RAW264.7 macrophages in HEK 293T cells. (B) Western blot analyses of the expression of HA-tagged PRDX1 (lane 1), PFN1 (lane 2), CFL1 (lane 3), AKR1B3 (lane 4), FABP5 (lane 5), ARHGDIA (lane 6) and FLAG-tagged Tc-MUC-1 in transfected HEK 293T cells, using anti-haemagglutinin (HA)-tagged tag polyclonal antibodies (α-HA). M: Maker. Uncropped blot image can be found in S4 Fig. Western blot analyses of the expression of Tc-MUC-1, and (C) AKR1B3, (D) ARHGDIA, (E) CFL1, (F) FABP5, (G) PRDX1, and (H) PFN1 in transfected HEK 293T cells (Input), and in immunoprecipitated complex (IPC), using DYKDDDK tag monoclonal antibody (binds to FLAG tag epitope (α-Flag). Mouse IgG was used as a blank control.
Fig 4.
In silico docking of Toxocara canis mucin 1 (Tc-MUC-1) protein and two key interacting proteins.
(A) Protein complex predicted for Tc-MUC-1 and the cofilin 1 (CFL1) of murine RAW264.7 macrophage cells. Structures of Tc-MUC-1 and CFL1 are indicated in grey and red, respectively. (B) Protein complex predicted for Tc-MUC-1 and a dimer of the fatty acid binding protein 5 (FABP5) of murine RAW264.7 macrophages. Structures of Tc-MUC-1 and FABP5 are indicated in grey and red, respectively. Ionic interactions, hydrogen bonds or cation-pi binding interaction between the residues in Tc-MUC-1 (grey) and CFL1 (red) are indicated.
Fig 5.
Schematic diagram summarising the inferred functions of cofilin 1 (CFL1) and fatty acid binding protein 5 (FABP5) that interact with a prokaryotically-expressed (non-glycosylated) recombinant form of the Toxocara canis mucin 1 protein (rTc-MUC-1).
FABP5 and CFL1 physically interact with rTc-MUC-1, and are inferred to positively regulate cellular processes. FABP5 transports fatty acid ligands to activate peroxisome proliferator-activated receptors (PPAR), which then regulate the transcription of genes coding for malic enzyme 1 (ME1) and for ubiquitin C (UBC), participating in adipocytokine signalling pathway associated lipogenesis and ubiquitination, respectively. CFL interacts with actin gamma 1 (ACTG1), cyclase associated actin cytoskeleton regulatory protein 1 (CAP1), coronin actin binding protein 1A (CORO1A), heat shock protein 90 alpha family class A member 1 (HSP90AA1), lymphocyte cytosolic protein 1 (LCP1), ubiquitin-like modifier (NEDD8), PFN1, Ras homologue family member C (RHOC) and 14-3-3 protein zeta (YWHAZ), to positively regulate ME1-associated signal transduction in the adipocytokine signalling pathway.