Structure/function studies of the insect cytokine PSP1
The insect immune response to parasites and pathogens involves the action of different classes of blood cells (hemocytes), which adhere to and spread across the surface of foreign targets. In phylogenetically advanced insects like the Lepidoptera (moths and butterflies), plasmatocytes and granular cells are the two most important classes of hemocytes involved in cellular defense responses like encapsulation and clotting. However, relatively little is known about the factors mediating the movement and action of these immune cells. A peptide that induces the adhesion and spreading of plasmatocytes on foreign surfaces was purified from the moth Pseudoplusia includens by Dr. Kevin Clark in the laboratory of Prof. Michael Strand (Clark et al, 1997). This plasmatocyte-spreading peptide (PSP1) is expressed as a preproprotein of 142 residues (Clark et al, 1998), but the mature peptide consists of only the C-terminal 23 amino acids containing one disulfide bond. Although the existence of cytokine-like factors to regulate hemocyte activity has been proposed previously, PSP1 is the first soluble mediator of the insect immune response to be purified and functionally characterized.
We have determined the solution structure of the plasmatocyte-spreading peptide (Volkman et al, 1999). This novel insect cytokine consists of 23 amino acid residues and a single disulfide bond. Torsion angle dynamics calculations utilizing a total of 337 distance constraints yielded an ensemble of 30 structures with an average backbone RMSD for residues 7-22 of 0.18 A from the mean structure. The structure consists of a disordered N-terminal region and a well-defined core which is stabilized by numerous hydrophobic interactions and a short beta-hairpin.
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Comparison of the backbone conformations of PSP1 (green), C-loop of hTM5 (blue) and the C-loop of mouse EGF (red).
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The amino acid sequence of PSP1 shows no significant similarity to vertebrate cytokines, but is related to two classes of insect peptides previously identified. The growth-blocking peptide (GBP) of Pseudaletia separata and the paralytic peptides from Manduca sexta, Heliothis virescens and Spodoptera exigua have high (>70 %) sequence identity with PSP1, including the two cysteine residues and a highly conserved glycine. High sequence variabilty within this group is found only at positions 4 and 8. Some similarity between the sequences of GBP and human epidermal growth factor has been recently suggested, and this would extend to PSP1 and the paralytic peptides. The structure of PSP1 shows clear homology to the C-terminal subdomain of the EGF domain family. The backbone of PSP1 is superimposed with those from portions of two EGF family proteins, epidermal growth factor (mEGF) and the fifth EGF-like domain of the human anticoagulant protein thrombomodulin (hTM5). The backbone conformation of PSP1 is particularly similar to that of the hTM5, despite sharing sequence identity at only 4 positions, including the two cysteines of the conserved disulfide.
Our recent studies have identified specific residues of PSP1 that are critical for its biological activity (Clark et al, 2001), revealing important roles for the sidechain of Phe3 and the N-terminal amino group in receptor activation. Current efforts are focused on the identification of the PSP1 receptor in order to pursue structural studies of this cell-signalling complex, as well as the identifcation and characterization of other proteins involved the host defense of this organism.
References
Clark, K. D., Pech L. L., and Strand M. R. Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens (1997) J. Biol. Chem. 272, 23440-7.
Clark, K. D., Witherell, A., and Strand, M. R. Plasmatocyte spreading peptide is encoded by an mRNA differentially expressed in tissues of the moth Pseudoplusia includens, (1998) Biochem. Biophys. Res. Commun., 250, 479-85.
Volkman, B. F., Anderson, M. E., Clark, K. D., Hayakawa, Y., Strand, M. R., and Markley, J. L. Structure of the Insect Cytokine Peptide PSP-1 from Pseudoplusia includens, (1999) J. Biol. Chem., 274, 4493-4496.
Clark, K. D., Volkman, B. F., Thoetkiattikul, King, D. S., Hayakawa, Y., and Strand, M. R., Alanine-scanning Mutagenesis of Plasmatocyte Spreading Peptide Identifies Critical Residues for Biological Activity, (2001) J. Biol. Chem., 276, 18491-18496.
Clark K. D., Volkman, B. F., Thoetkiattiku, H., Hayakawa, Y., and Strand, M. R., N-terminal residues of plasmatocyte spreading peptide possess specific determinants required for biological activity, (2001) J. Biol. Chem., 276, 37431-37435.
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