Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms

Abstract

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein ( apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as and, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

Clonorchis sinensis MF6p/HDM (CsMF6p/HDM) induces pro-inflammatory immune response in RAW 264.7 macrophage cells via NF-κB-dependent MAPK pathways

“…MF6p/HDMs are a broad family of small proteins secreted by helminth parasites [9]. Although the physiological roles of MF6p/HDMs in trematode parasites have not been clearly established, several studies suggested two interesting biological functions of this family of proteins.…”

“…Although the physiological roles of MF6p/HDMs in trematode parasites have not been clearly established, several studies suggested two interesting biological functions of this family of proteins. The molecules act as heme scavengers and transporters to maintain heme homeostasis in trematode parasites, indicating an essential role in parasite survival by detoxifying the toxic free form [9,14]. Their ability to bind to LPS and prevent LPS-induced inflammation in mice as well as their ability to suppress antigen processing and presentation in macrophages also suggested an immunomodulatory role in suppressing host immune response, which facilitated prolonged parasite survival in the hosts [10-13, 36, 37].…”

“…1). The C-terminal conserved motif, 65 LGxKIxxVxxILxxRLTxRxExY 87 (x = any), which was reported as potential heme-and LPS-binding site [9], was conserved in all MF6p/HDM homologues. A putative signal peptide sequence was identified in the N-terminal region of CsMF6p/HDM.…”

“…The MF6p/host defense molecule (HDM) of C. sinensis (CsMF6p/HDM) is a small protein consisting of 90 amino acids and was initially reported as a 7 kDa antigen of unknown function [7,8]. However, it has recently been classified under a new family of heme-binding proteins (MF6p/HDMs) with homologues from the other flukes including Fasciola hepatica (FhMF6p/HDM), Opisthorchis viverrini (OvMF6p/HDM) and Paragonimus westermani (PwMF6p/HDM) [9]. The physiological role of MF6p/HDMs in trematode parasites has yet to be elucidated.…”

“…However, several studies investigating FhMF6p/ HDM suggested a role in host immune modulation [10][11][12][13]. Moreover, the increased interest in this molecule has been attributed to its heme-binding ability, and its role as a heme scavenger and transporter to maintain heme homeostasis in trematode parasites [9,14]. The hemescavenging ability is essential for the survival of bloodfeeding trematodes since large amount of heme, which is toxic as a free form, are released from the catabolism of host erythrocytes [15].…”