Predicted Structure and Potential Binding of the Nematode Tetraspanin 7 Protein (tsp-7) With Human Tissue Inhibitor Metalloprotease 1 (TIMP-1): a Possible Role In Parasite- Host Interactions

Abstract

Human parasitic nematodes evade the host immune system through cross communication by unknown mechanisms. The proteins of the parasitic nematodes are still poorly annotated. CD63 is a human tetraspanin and binds to TIMP-1 a metalloproteinase inhibitor, which has recently been found to play an active role in inflammation by acting as a cytokine when bound to CD63. Caenorhabditis elegans is a free-living nematode, a model organism for both human and parasitic nematodes. C. elegans tetraspanin protein -7 (tsp-7) is an orthologue of human CD63. The model organism used can provide information about potential tetraspanin proteins from parasitic nematodes, which are believed to play a role in the cross communication between parasite and host. The tsp-7 protein sequence from C. elegans shares a high percentage similarity, identity, and homology with a variety of human parasitic nematode proteins. Using a number of computer-based programmes including ‘Alpha-Fold,’ which is currently the most powerful Artificial Intelligence protein structure prediction programme, the structure of the C. elegans tsp-7 was determined. From the predicted structure, the tsp-7 protein has one highly likely, membrane exposed tyrosine phosphorylation site, with implications of its potential involvement in signalling pathways and immune responses. Using multiple protein docking databases and programmes, tsp-7 has been shown to theoretically bind to human TIMP-1. Two mutant strains of C. elegans were obtained from Caenorhabditis Genetics Centre, University of Minnesota, USA, with amino acid deletions within the tsp-7 protein (mutant tm5046 and mutant tm5761). These tsp-7 mutant strains were shown to have an overall longer life span than the wild type C. elegans (N2). The mutant tm5761 showed a greater stress response compared to the N2 wild-type, when exposed to various chemical stimuli, however, mutant tm5046 was more similar to wild type C. elegans. Under heat stress, mutant tm5761 and wild type N2, were less tolerant to heat compared to the tm5046 mutant. In general, mutant tm5046 was more stable and displayed a reduced stress behavioural response compared to mutant tm5761. A primary polyclonal antibody was produced against the tsp-7 large extracellular loop domain, believed to be the key active site in protein-protein interactions. The expression of tsp-7 in mutant tm5761 was detected at lower concentrations than the wild type and mutant tm5046 in an ELISA assay and it also could not be detected by western blotting. Mutant tm5046 was detected weakly by western blotting. Cos-7 cells transfected with the tsp-7 protein-GFP tagged plasmid were incubated with active human TIMP-1. The tsp-7 tagged cells showed co-localisation with TIMP-1, using immuno-fluorescence microscopy, thus adding to the evidence that tsp-7 could interact with human TIMP-1. Such an interaction may well play a role in parasite-host interactions and its effects on the immune response. As C. elegans tsp-7 shares high similarity with many parasitic nematodes that are known to infect humans, it is a good protein candidate to further explore the potential interactions of parasitic nematodes with humans, in terms of cross communication and interaction with the host immune system.