Structural analysis of ASCUT‐1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides

Abstract

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20°C, both as insoluble inclusion bodies or in soluble form, contains about 50% β structure, 14% A-helix and 25% turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.Keywords : nematode cuticle; inclusion bodies ; circular dichroism ; Fourier-transform infrared spectroscopy; recombinant protein.The nematode cuticle is a multilayered elastic extracellular structure which surrounds the animal and functions as an exoskeleton, determining the body shape of the worm. Its major components are collagen-like proteins which can be extracted by a combination of strong ionic detergents and disulphide reducing agents. Cuticlin, the insoluble residue left after extraction, is resistant to collagenase and consists of a highly cross-linked mixture of different proteins.We have previously identified two genes, cut-1 and cut-2, coding for components of the insoluble cuticlin residue of the free living nematode Caenorhabditis elegans [1,2]. The cloned genes have provided the opportunity to obtain recombinant cuticlin proteins. In particular, it has been possible for the first time to study some aspects of the mechanism of CUT-2 cross-linking [3]. In addition, specific antibodies have been raised against the recombinant proteins and have been used to localise the corresponding proteins within the cuticle layers [4,5]. While CUT-2 is a component of the cuticle of all stages of C. elegans [2], CUT-1 is present only in the cuticle of dauer larvae where it forms two ribbons running the whole length of the worm, underneath the lateral alae The two genes represent two different families of cuticlin components. CUT-2 shares similarity with a group of insect proteins which also participate in the formation of extracellular protective structures such as the egg-shell layers [...

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“…Moreover, the temperature increases do not result in any shift of the distribution center, suggesting that the presence of SDS increases the protein stability. These data are in good agreement with our previous investigation on the stability of ASCUT-1 by using circular dichroism and infrared spectroscopy [17]. In particular, we showed that ASCUT-1 protein structure was quite stable up to 85°C and that its stability was enhanced by the presence of 3% SDS [17].…”

“…To this end we have produced, in Escherichia coli, the mature version of CUT-1 from Ascaris lumbricoides (ASCUT-1). We have recently reported studies with circular dichroism and Fourier Transform Infrared spectroscopy on this recombinant protein [17] that indicate that the protein structure is uncommonly stable at high temperatures and that this stability is increased by the addition of the detergent SDS. Here we report conformational dynamics studies on ASCUT-1 in the presence and in the absence of SDS using frequency-domain fluorometry and anisotropy decays which may be particularly informative since the protein contains a single tryptophan residue.…”