Specific Residues in Plasmatocyte-spreading Peptide Are Required for Receptor Binding and Functional Antagonism of Insect Immune Cells

Abstract

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions:an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg 13 , Phe 3 , Cys 7 , Cys 19 , and the N-terminal amine of Glu 1 . Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met 12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125 I-M12A generated an IC 50 of 0.71 nM and indicated that unlabeled M12A, at concentrations >100 nM, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125 I-M12A at a concentration of 100 nM. In the structured core, we found that Cys 7 and Cys 19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg 13 , in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe 3 yielded a peptide that reduced binding of 125 I-M12A 326-fold. This and all other mutants of Phe 3 we bioassayed were unable to antagonize PSP. Deletion of Glu 1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.

Plasmatocyte‐spreading peptide influences hemocyte behavior via eicosanoids

“…Rather than independently mediating hemocyte spreading, we propose a model (Fig. 8), in which PSP interacts with a hemocyte surface receptor, most likely the 190-kDa protein reported earlier (Clark et al, 2004). In our model, receptor binding activates PLA 2 , which migrates to a membranous cell fraction, where it Figure 8.…”

“…The proPSP is activated by a proteolytic cleavage to a 23 residue PSP that mediates plasmatocyte spreading (PS) (Clark et al, 1998). This is the first reported insect cytokine and it acts via an approximately 190-kDa receptor to induce PS (Clark et al, 2004). The P. includens PSP also mediates hemocyte spreading and aggregation behaviors of other lepidopteran species, including Spodoptera exigua, in a dose-dependent manner Kim et al, 2008).…”

Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection

“…Plasmatocytes are normally non-adhesive and must be activated by cytokines in hemolymph in order to bind and spread onto foreign surfaces. The most potent known activator of plasmatocytes is the plasmatocyte-spreading peptide (PSP) (Clark et al, 1997), homologs of which have been identified from a number of other moth species, including M. sexta (Clark et al, 2004). PSP and related homologs are the only molecules identified to date that activate plasmatocytes (Clark et al, 1997;Strand et al, 2000;Miura et al, 2002;Nakahara et al, 2003), although circumstantial evidence suggests that several other molecules besides PSP mediate plasmatocyte function (Lackie, 1988;Gillespie et al, 1997;Lavine and Strand, 2002).…”

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

“…Hemocytes were collected from non-parasitized fifth instars by anesthetizing larvae with CO 2 and bleeding them from an incision across the last abdominal segment. Hemolymph was collected in a microfuge tube containing anticoagulant buffer (98 mM NaOH, 186 mM NaCl, 17 mM Na 2 EDTA, and 41 mM citric acid, pH adjusted to 4.5) (Clark et al, 2004). spherule cells plus oenocytoids were isolated on Percoll gradients as previously described (Pech and Strand, 1996 (Gardiner and Strand, 1999 (Strand et al, 1992;Strand, 1994 (Strand, 1994;Trudeau et al, 2000;Beck and Strand, 2003).…”