A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern

Abstract

Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum

“…4D shows a titration curve (corrected for the background fluorescence of DAUDA), which predicts a K d of 5.05 ×10 −6 for the rAceFAR-1:DAUDA interaction. This value is within the general range of dissociation constants reported for other soluble lipid transporters (Thumser et al, 1994) and FAR proteins (Prior et al, 2001; Basavaraju et al, 2003). We also demonstrated binding of rAceFAR-1 to cis-parinaric acid ( K d of 1.11 ×10 −6 ) (Fig.…”

“…This blue shift was greater than the blue shift seen with serum albumin (from 543 nm in buffer alone to 500 nm) (Wilton, 1990) (Fig. 4B) and is within the range of shifts seen with other FAR proteins (Prior et al, 2001; Garofalo et al, 2002, 2003a). …”

“…Computer-based secondary structure analysis (Jones, 1999; Rost, 2004) predicts a rich α-helix and strong coiled coil structure (Lupas et al, 1991) (Fig. 1), similar to other FAR proteins (Kennedy et al, 1997; Prior et al, 2001; Garofalo et al, 2003b). …”

“…FAR proteins are single domain proteins of approximately 20 kDa that exhibit a unique α-helix rich coiled coil structure compared with mammalian fatty acid binding proteins. To date, FAR proteins have been identified in the free living nematode Caenorhabditis elegans (Garofalo et al, 2003b), mammalian parasites Brugia malayi , Onchocerca volvulus (Kennedy et al, 1997), and Ostertagia ostertagi , and the plant pathogen Globodera pallida (Prior et al, 2001). Two closely related FAR proteins (AcFAR-1 and AcFAR-2) have been identified from the dog hookworm Ancylostoma caninum (Basavaraju et al, 2003), although none from species for which humans are naturally permissive hosts.…”

Investigating hookworm genomes by comparative analysis of two Ancylostoma species

“…FAR proteins are novel fatty acid and retinol binding proteins described in nematodes including A. caninum , other Strongylida, filarial, and plant parasitic species [49-51]. In C. elegans 8 FAR members are divided into 3 groups.…”