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Originally published In Press as doi:10.1074/jbc.M401157200 on June 10, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33246-33252, August 6, 2004
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Specific Residues in Plasmatocyte-spreading Peptide Are Required for Receptor Binding and Functional Antagonism of Insect Immune Cells*

Kevin D. Clark{ddagger}, Stephen F. Garczynski§, Aditi Arora¶, Joe W. Crim§, and Michael R. Strand{ddagger}||

From the {ddagger}Departments of Entomology and §Cellular Biology, University of Georgia, Athens, Georgia 30602 and the Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received for publication, February 2, 2004 , and in revised form, June 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 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 125I-M12A at a concentration of 100 nM. In the structured core, we found that Cys7 and Cys19 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. Arg13, 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 Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The insect immune system consists of both cellular and humoral elements that innately recognize broad classes of foreign intruders (1-3). Insects usually kill multicellular parasites by encapsulation, which involves attachment of multiple layers of immune cells (hemocytes) to the foreign target. In Lepidoptera (moths and butterflies) like Pseudoplusia includens, many intruders are recognized as foreign by a class of hemocytes called granular cells, which release cytokines that induce a second class of hemocytes, plasmatocytes, to bind to the target and form a capsule (4, 5). The most potent known activator of plasmatocytes is plasmatocyte-spreading peptide (PSP)1 (6). Natural and synthetic PSP induce plasmatocytes to adhere and spread on foreign surfaces within minutes at concentrations ≥100 pM. PSP is expressed by granular cells and fat body as a propeptide of 142 residues with the PSP sequence located at the C terminus (7). This biologically inactive precursor is then cleaved by an unknown protease to release the mature 23-amino acid peptide (ENFNGGCLAGYMRTADGRCKPTF). PSP homologs have been identified from a number of other moth species, and based on the consensus sequence of their N termini (Glu-Asn-Phe-X-X-Gly), these molecules are collectively referred to as the ENF peptide family (8). Other ENF peptides besides PSP also function as plasmatocyte activators, suggesting these molecules may be of widespread importance as regulators of the cellular immune response in Lepidoptera (8-10).

The three-dimensional structure of PSP consists of a disordered N terminus (residues 1-6) and a well defined core (residues 7-22) stabilized by a disulfide bond between Cys7 and Cys19, hydrophobic interactions, and a short {beta}-hairpin turn (11). Comparison with other proteins reveals that the core region of PSP adopts a very similar structure to the C-terminal subdomain of human epidermal growth factor and the anticoagulant protein thrombomodulin. No consensus binding site has yet been identified for EGF domains and their receptors, but several studies have implicated the C-loop as a critical region for binding (12-15). In contrast, the consensus sequence for the N terminus of PSP is found solely in the ENF peptide family (11). Using alanine replacement and deletion mutants, we also have identified residues in both the structured and unstructured regions of PSP that are essential for plasmatocyte-spreading activity (16, 17). These include Cys7 and Cys19, which form the disulfide bond required for the proper three-dimensional structure of PSP, and the charged residue Arg13 within the {beta}-hairpin turn. Deletion mutants in the unstructured N terminus also eliminate all biological activity. Ala replacement of Phe3 (F3A) abolishes activity because of the specific requirement of the phenyl side chain (16). In contrast, Ala replacement of Glu1 (E1A) actually enhances activity (17). This is, in part, because the activity of PSP requires the presence of the N-terminal primary amine but not the side chain of Glu1 (17). Recent studies of the PSP homolog growth-blocking peptide likewise identify Phe3 as critical for plasmatocyte activation in Pseudaletia separata (10).

The preceding studies provide important insight about structure-function relationships but do not indicate whether mutations in PSP that reduce biological activity affect receptor binding. In the current study we addressed this question by conducting competitive binding and functional antagonism experiments. Our results indicate that in most instances mutant peptides that strongly bound to hemocytes also functionally antagonized plasmatocyte spreading. However, we also identified a few mutants that bound to hemocytes but were poor functional antagonists. Receptor labeling experiments showed that a photoaffinity analog of PSP bound to a single 190-kDa protein in a manner consistent with our binding and antagonism results.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Insects—P. includens larvae were reared on an artificial diet at 27 °C and with a 16-h light/8-h dark photoperiod (18). Moths were fed 20% sucrose in water and maintained under identical environmental conditions. All experiments were conducted with hemocytes collected from 36-48-h fifth instar larvae.

Hemocyte Collection and Bioassays—Hemocytes were collected by anesthetizing larvae with CO2 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 Na2EDTA, and 41 mM citric acid, pH adjusted to 4.5). The ratio of hemolymph to buffer was ~1:5. Hemocytes were pelleted for 1 min at 200 x g, and the plasma-buffer supernatant was then removed. Hemocytes were resuspended in 1 ml of fresh anticoagulant. After a 40-min incubation at 4 °C, hemocytes were washed twice by centrifugation in Excell 400 insect cell culture medium (JRH Biosciences). These unseparated hemocytes were then used in binding assays (see below). Plasmatocytes and granular cells account for 30 and 65%, respectively, of the total hemocyte population in P. includens (19-20). Plasmatocytes were isolated to high purity on Percoll step gradients as previously described (16). On average, 1.2 x 106 plasmatocytes were collected per gradient. Purity was 93%, with the primary contaminant being granular cells. These plasmatocytes were washed once in Excell 400 before use in antagonism assays (see below).

Peptide Synthesis and Purification—All peptides were synthesized on an Applied Biosystems 433 synthesizer using standard Fmoc chemistry as previously described (16, 17). The resin-peptide was cleaved and deprotected for 4 h in reagent K (21), a mixture containing 5% phenol, 1.25% water, 2.5% thioanisole, and 2.5% dithioethane in trifluoroacetic acid. After removing the resin from the reaction mixture by filtration, the peptide was precipitated in cold t-butyl methyl ether, followed by repeated ether washes and air drying. Peptides were resuspended at a concentration of 1 mg/ml in 10 mM Tris-HCl, pH 8. Disulfide bond formation was monitored periodically by 5-µl injections onto an HPLC (Rheodyne 9725i manual injector, Hitachi L-6220 pump, Hitachi L-4500A photodiode array detector, and Hitachi D-7000 chromatography software) where the reduced and oxidized peptides eluted in separate peaks on a C18 column (5-µm particle size, 4.6 mm x 25 cm, Supelco 58298) using HPLC-grade H2O (Sigma) and a linear gradient of acetonitrile (0-80 min, 20-60%) at 0.5 ml/min. Both the H2O and acetonitrile contained 0.05% trifluoroacetic acid. After the conversion was complete, the sample was purified by a series of 4- to 7-ml injections onto a preparatory HPLC column (10-µm particle size, 21.2 mm x 25 cm, Jupiter C18; Phenomenex Inc., Torrance, CA) using HPLC-grade H2O and a linear gradient of acetonitrile (0-70 min, 10-80%) at 9 ml/min. Both the H2O and acetonitrile contained 0.05% trifluoroacetic acid. The desired peak was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; disulfide bond formation was confirmed by NMR. After numerous purification runs, peaks were pooled, lyophilized, and resuspended in HPLC-grade H2O for the determination of amino acid composition and concentration. Mutant peptides were named according to the residue that was modified. For example, alanine replacement of Met12 was named M12A. Wild-type PSP, which lacks any modifications, is referred to as PSP throughout this report.

F23Bpa (AAFNGGCLAGYARTADGRCKPTBpa) was synthesized as described above except that the C-terminal Phe was replaced with the photoaffinity reagent benzoylphenylalanine (Bpa), incorporated during the synthesis as the derivative Fmoc-p-Bz-Phe-OH (Bachem). Cleavage and deprotection were carried out as described for the other peptides except that dithioethane was replaced with dithiothreitol to avoid degradation of the benzoylphenylalanine ketone (26). Iodination of 125IF23Bpa was carried out as described below.

Iodination of the PSP Mutant M12A—Preliminary studies indicated that M12A had plasmatocyte-spreading activity indistinguishable from PSP.2 We then used M12A in hemocyte binding experiments because it avoided the possibility of methionine sulfoxide formation during iodination of Tyr11 using the chloramine T method (22). To iodinate M12A, 5.6 µg of synthetic peptide was added to 500 µCi of Na125I (Amersham Biosciences) and 2.5 ng of chloramine T in 35 µl of 200 mM sodium phosphate buffer, pH 7.4. After incubation at room temperature for 20 s, the reaction was terminated by addition of an equal volume of 50% acetic acid. The radiolabeled mixture was fractionated by high performance liquid chromatography on a reverse phase C8 column (Vydac, 300 Å, 4.6 x 150 mm): solvent A, water with 0.1% trifluoroacetic acid, and solvent B, 80% acetonitrile and 20% water with 0.1% trifluoroacetic acid (gradient program: 0-29% B, 29 min, 29-31% B, 40 min; 1 ml/min). 125I-labeled M12A was detected with an inline radioisotope detector. Fractions containing the radiolabeled peptide were collected, and bovine serum albumin (Fraction V; Sigma) was added to 1%. Radioactivity in fractions was quantified by {gamma} counting. Final concentrations were determined based on specific activity (~2000 Ci/mmol) of 125I in the product, which was free of the unlabeled peptide.

Binding Assays—Binding experiments were conducted by adding 5 x 105 hemocytes to 1.5-ml microfuge tubes containing 100 pM 125I-labeled M12A in medium (Excell 400 plus 3% bovine serum albumin and 1x protease mixture; Roche Applied Science) plus increasing concentrations of unlabeled M12A or other PSP mutants (1 pM-10 µM). Total reaction volumes were 200 µl. Tubes were incubated at room temperature for 3 h and vortexed every 30 min. Reactions were stopped by centrifuging tubes at 14,000 x g for 5 min at 4°. Following removal of the supernatant, pellets were washed three times with ice-cold Excell 400. After the final wash, the bottoms of the microfuge tubes were cut off and counted on a Packard GammaII counter. 100 pM 125I-labeled M12A resulted in ~82,000 cpm/tube; ~12% of this 125I-M12A bound to hemocytes in the absence of unlabeled M12A. Nonspecific binding values, determined by addition of 1 µM M12A, ranged from 0.38-0.64% of the total 125I-M12A and were subtracted from the raw cpm obtained from the binding assays. The resulting data were converted to percent total binding and then analyzed by non-linear regression using GraphPad Prism, v3.0 software (GraphPad Software Inc., San Diego, CA). The concentrations of peptide that reduced specific binding of 125I-labeled M12A by 50% (IC50), R2, and S.E. for each binding assay were likewise determined using GraphPad Prism.

Antagonism of Plasmatocyte-spreading Activity by Mutant Peptides—Previously optimized in vitro assays indicated that 1-10 µM PSP induces 30-35% of plasmatocytes to spread after 30 min and a maximum of 70% of plasmatocytes to spread after 1 h (6, 23). Antagonism experiments in the current study were conducted similarly in 96-well culture plates (Corning). Plates were prepared by adding increasing concentrations of the mutant peptide and a fixed concentration of PSP/well. Wells were then filled with Excell 400 medium containing 1 x 103 plasmatocytes for a total volume of 60 µl. This yielded working concentrations of 100 pM-100 µM for the mutant peptide being assayed and 3 µM of PSP. The percentage of plasmatocytes that spread in an assay was scored 30 min later by counting 100 cells from a randomly selected field of view. Plasmatocytes were scored as spread if they assumed a flattened morphology and were ≥35 µm along their longest axis (6, 19). Unspread plasmatocytes remained no adhesive and spherical in shape. Each mutant peptide was bioassayed four times using an independently collected sample of plasmatocytes. The proportion of plasmatocytes spread in the presence of PSP alone was normalized to a value of 100%. The reduction in the number of spread plasmatocytes in the presence of the antagonist was then expressed as a percentage of this value.

Photoaffinity Cross-linking—Mixed hemocytes were plated in the presence of 125I-F23Bpa at final concentrations of 50 pM, 0.5 nM, and 5 nM. Each concentration of 125I-F23Bpa was tested alone or in combination with either N2A (17) or R13A at a final concentration of 1 µM. These hemocytes were incubated for 2 h at room temperature, washed two times at 4 °C, plated (40 µl/well), and exposed to UV light at 366 nm (BlakRay) for 1 h on ice. Cells were lysed and prepared for SDS-PAGE by adding 10 µl of 5x sample buffer to each well and mixing rapidly. After boiling, 30 µl of each sample was applied to a gradient gel (8-16%, Gradipore). The gel was run at constant current (20 mA) and transferred overnight onto nitrocellulose. The dried blot was exposed to film (X-omat) for 2 days and then developed and digitally scanned for display.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Binding Constants of Representative Peptides—Prior analysis indicated that several residues are invariant among ENF peptide family members (8, 16). These include Cys7 and Cys19, Thr14 and Thr22, all of the charged amino acids in the structured C terminus (Arg13, Asp16, Arg18, and Lys20), and the three N-terminal residues Glu1, Asn2, and Phe3. As previously noted, mutagenesis studies indicate that several of these residues are essential for biological activity (16, 17). The first step of the current study was to conduct full dose-response, competitive binding experiments using M12A and a subset of these mutant peptides. This allowed us to establish parameters for M12A binding that could then be used to optimize experiments with other mutants. For these studies, we used the alanine replacement mutants R13A and F3A, and AcPSP-(2-23), which lacked Glu1 and is acetylated at Asn2 to block the terminal amine. Each of these peptides has little or no biological activity and targets essential residues in the structured and unstructured regions of PSP. Our results indicated that M12A had an IC50 value of 0.71 nM and fully inhibited binding of 125I-M12A to hemocytes at concentrations ≥100 nM (Fig. 1). IC50 values for AcPSP-(2-23) (4 nM) and F3A (232.8 nM) were 5- and 326-fold higher, respectively, than M12A. R13A had an IC50 of 289.4 nM and did not fully inhibit binding of M12A at any concentration tested (Fig. 1).



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  		FIG. 1.
Displacement of 125I-M12A by unlabeled M12A, R13A, AcPSP-(2-23), and F3A. Hemocytes were incubated with 100 pM 125I-M12A for 3 h along with unlabeled peptides ranging from 10 pM to 10 µM. Values indicate means + S.E. (n = 6). IC50 values are presented in the legend to the right of each competitor tested. Cell binding assays are described under "Experimental Procedures."

 
PSP Mutants of the Core Region Exhibit Congruent Patterns of Competitive Binding and Functional Antagonism—We next conducted competitive binding and functional antagonism experiments using R13A and other alanine replacement mutants targeting the structured core region of PSP. Antagonism experiments were conducted over a range of concentrations (100 pM-100 µM). Binding experiments, in contrast, were conducted using only 100 nM of the unlabeled competitor because the data in Fig. 1 clearly indicate that unlabeled M12A at this concentration fully displaced binding of 100 pM 125I-M12A. Alanine replacement of the two cysteine residues (C7A,C19A) results in a linearized peptide lacking the {beta}-hairpin turn and >1000-fold loss of spreading activity (16). Commensurately, we found in the current study that C7A,C19A was an even poorer binding competitor than R13A (Fig. 2A). C7A,C19A and R13A were also poor functional antagonists that reduced the percentage of spread plasmatocytes only slightly at the highest concentrations tested (Fig. 2B). The other mutants we tested (D16A, R18A, and K20A) each strongly inhibited binding of 125I-M12A relative to R13A or C7A,C19A (Fig. 2A). Functional antagonism experiments were not conducted with these mutants because each has plasmatocyte-spreading activity only 10-100 times lower than PSP (16).



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  		FIG. 2.
Displacement and functional antagonism experiments using alanine replacement mutants of the PSP structured core. A, displacement of 125I-M12A by C7A,C19A, R13A, D16A, R18A, and K20A. Hemocytes were incubated with 100 pM 125I-M12A for 3 h along with 100 nM unlabeled peptides. Values indicate means + S.E. (n = 6). B, functional antagonism of PSP by C7A,C19A and R13A. Plasmatocytes were incubated with 3 µM PSP along with 100 pM-100 µM of indicated competitors and then assayed for spreading after 30 min in culture. Each data point is the mean percentage ± S.E. of spread plasmatocytes from four independent collections of cells. Cell binding and functional antagonism assays are described under "Experimental Procedures."

 
N-terminal-truncated and -acetylated Peptides Also Exhibit Congruent Patterns of Binding and Functional Antagonism—No PSP mutant with an N-terminal truncation has any plasmatocyte-spreading activity (17). However, deletion mutants varied considerably in their ability to compete for binding of 125I-M12A and to antagonize the functional activity of PSP (Fig. 3). PSP-(7-23), which lacks the unstructured N terminus, reduced binding of 125I-M12A only 17%, whereas PSP-(3-23) and PSP-(2-23) reduced binding 65 and 99%, respectively (Fig. 3A). We observed the same rank order in functional antagonism assays. At 100 µM, PSP-(7-23) reduced plasmatocyte spreading by 41% relative to PSP, whereas PSP-(3-23) and PSP-(2-23) reduced spreading by 56 and 67%, respectively (Fig. 3B). Acetylation of PSP (AcPSP) blocks the N-terminal amine and elongates the peptide, whereas AcPSP-(2-23), described above, also has a blocked N-terminal amine but is shorter than PSP. Neither of these mutants has any plasmatocyte-spreading activity (17). AcPSP and AcPSP-(2-23) reduced binding of 125I-M12A 40 and 95%, respectively. 100 µM AcPSP likewise reduced plasmatocyte spreading 64% relative to PSP alone, whereas AcPSP-(2-23) reduced spreading 83% (Fig. 3C). AcPSP-(2-23) was the most potent functional antagonist identified in the study.



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  		FIG. 3.
Displacement and functional antagonism experiments using deletion and acetylated mutants of the PSP N terminus. A, displacement of 125I-M12A by PSP-(7-23), PSP-(3-23), PSP-(2-23), AcPSP, and AcPSP-(2-23). Hemocytes were incubated with 100 pM 125I-M12A for 3 h along with 100 nM unlabeled peptides. Values indicate means + S.E. (n = 6). B, functional antagonism of PSP by PSP-(2-23), PSP-(3-23), and PSP-7-23). C, functional antagonism of PSP by AcPSP and AcPSP-(2-23). Plasmatocytes were incubated with 3 µM PSP along with 100 pM-100 µM of indicated competitors and then assayed for spreading after 30 min in culture. Each data point is the mean percentage ± S.E. of spread plasmatocytes from four independent collections of cells. Cell binding and functional antagonism assays are described under "Experimental Procedures."

 
Some Phe3 Mutants Bind to Hemocytes but None Functionally Antagonizes PSP—Most alterations to Phe3 eliminate or greatly reduce plasmatocyte-spreading activity (17). These include substituting the D-isomer (F3F(D)), removing the phenyl group (F3A), removing the methylene spacer between the phenyl group and the peptide backbone (F3phenylG), and substituting a branched chain aliphatic (valine) that lacks a methylene spacer (F3V). Inserting a methylene spacer into F3V, making leucine (F3L), restores some activity. Additions at the paraposition of the phenyl ring have varying effects on activity. Adding a hydroxyl group, making tyrosine (F3Y), results in almost complete loss of activity, but replacing the hydroxyl group with a fluorine (F3p-fluoro) restores activity to levels almost identical to PSP. In the current study, we found that the F3A, F3F(D), F3V, and F3phenylG mutants each reduced binding of 125I-M12A to hemocytes by 40-50% (Fig. 4A) but none antagonized the plasmatocyte-spreading activity of PSP (Fig. 4B). F3Y reduced binding of 125I-M12A by 70%, whereas F3L and F3F(p-fluoro) reduced binding more than 95% (Fig. 4A). However, in bioassays using F3V, F3Y, F3A, F3F(D), and F3phenylG, no functional antagonism of PSP was observed at any concentration tested (Fig. 4, B and C).



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  		FIG. 4.
Displacement and functional antagonism experiments using replacement mutants of Phe3. A, displacement of 125I-M12A by F3A, F3F(D), F3V, F3L, F3Y, F3phenylG, and F3p-fluoro. Hemocytes were incubated with 100 pM 125I-M12A for 3 h along with 100 nM unlabeled peptides. Values indicate means + S.E. (n = 6). B, functional antagonism of PSP by F3V, F3Y, and F3A. C, functional antagonism of PSP by F3F and F3phenylG. Plasmatocytes were incubated with 3 µM PSP along with 100 pM-100 µM of indicated competitors and then assayed for spreading after 30 min in culture. Each data point is the mean percentage ± S.E. of spread plasmatocytes from four independent collections of cells. Cell binding and functional antagonism assays are described under "Experimental Procedures."

 
ENFN neither Binds to Hemocytes nor Functionally Antagonizes PSP—Despite the presence of the primary amine of Glu1 and phenyl group of Phe3, bioassays using the tripeptide ENF and PSP (7-23) do not induce plasmatocyte spreading (17). This strongly suggests that a covalent linkage between the unstructured and structured regions of PSP is required for function. Consistent with this interpretation, we found here that ENFN alone or in combination with PSP-(7-23) had little or no effect on binding of 125I-M12A or the functional activity of PSP (Fig. 5). We also previously reported that alanine replacement of both Glu1 and Asn2 resulted in a mutant peptide 100-fold more active than PSP (16). However, the tetrapeptide AAFN was as poor a functional antagonist of PSP as ENFN (Fig. 5B).



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  		FIG. 5.
Displacement and functional antagonism experiments using tetrapeptides of the N terminus and PSP-(7-23). A, displacement of 125I-M12A by ENFN, PSP-(7-23), and ENFN plus PSP-(7-23). Hemocytes were incubated with 100 pM 125I-M12A for 3 h along with 100 nM unlabeled peptides. Values indicate means + S.E. (n = 6). B, functional antagonism of PSP by ENFN or AAFN. Plasmatocytes were incubated with 3 µM PSP along with 100 pM-100 µM of indicated competitors and then assayed for spreading after 30 min in culture. Cell binding and functional antagonism assays are described under "Experimental Procedures."

 
PSP Binds with High Affinity to a 190-kDa Protein—To determine whether PSP binds to a specific receptor on hemocytes, we used data presented both here and in previous studies (17) to design a high affinity analog that could be photochemically cross-linked. This ligand, AAFNGGCLAGYARTADGRCKPTBpa (F23Bpa), contained alanine substitutions at both the one and two positions, which produces a peptide with 100-fold greater activity than PSP (17). This peptide also had the photochemical cross-linker Bpa incorporated at the C terminus. To visualize the labeled receptor, Tyr11 was iodinated to produce 125I-F23Bpa. Binding assays indicated that 125I-F23Bpa exhibited saturation at concentrations ≥1µM and produced an IC50 value of 0.3 nM. Other mutant peptides were then tested for their ability to compete with 125I-F23Bpa. N2A, an excellent agonist (17), was found to completely compete away labeled peptide, whereas R13A, which binds very poorly (Fig. 2), was unable to remove labeled peptide from the receptor (data not shown). Cross-linking experiments using 50 pM radiolabeled peptide followed by SDS-PAGE and autoradiography detected a single ~190-kDa protein (Fig. 6). Unlabeled excess N2A blocked binding of 125I-F23Bpa, whereas R13A did not. When using higher concentrations of 125I-F23Bpa (0.5 and 5 nM), the same pattern was observed. Although there is an obvious increase in the amount of labeled protein between 50 pm and 0.5 nM, there was little or no change between 0.5 and 5 nM, suggesting that the receptor was saturated above 0.5 nM. Collectively, these results indicate that PSP binds to a specific protein on the surface of hemocytes and that the binding of PSP to this protein closely parallels the data obtained with our binding experiments.



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  		FIG. 6.
Photo cross-linking of 125I F23Bpa to PSPR. Mixed hemocytes from P. includens (400,000 cells/lane) were incubated with 50 pM-5 nM 125I F23Bpa for 2 h at room temperature. After washing and UV cross-linking, cells were lysed and prepared for SDS-PAGE. After separation, proteins were transferred to nitrocellulose and exposed to film for 48 h. 125I-F23Bpa concentrations are indicated above each set of three lanes. -, no addition of unlabeled peptide; +N, addition of 1 µM N2A; +R, addition of 1 µM R13A.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The distinction between residues critical for proper folding of a molecule versus those required for receptor binding or activation are not always clear from mutagenesis experiments alone. However, the small size of PSP makes this molecule especially amenable for using a mutagenesis approach in characterizing the structural and functional importance of different residues. In the ordered C-terminal region, our results with C7A,C19A indicate this mutant binds poorly to hemocytes and cannot functionally antagonize PSP. However, these effects are clearly because of the missing disulfide bond normally formed by Cys7-Cys19, which is required for proper folding of PSP (11). R13A also binds poorly and has little biological activity, yet this residue plays no apparent role in maintenance of structure (11). These findings suggest that Arg13 is required for receptor binding. The experiments using 125I-F23Bpa confirm this directly by showing that R13A cannot block labeling of the putative receptor. Residues in the N terminus are also nonessential for maintenance of structure, but results reported here suggest that Phe3 and the terminal amine of Glu1 have different functional roles in the plasmatocyte activation cascade.

Collectively, our results suggest that the initial binding of PSP to its putative 190-kDa receptor is mediated by the structured C-terminal region (residues 7-23 of PSP). Evidence for this derives from our experiments showing that ENFN (residues 1-4) does not inhibit binding of M12A or functionally antagonize PSP, whereas PSP-(7-23) reduces binding ~20% and is a functional antagonist. The key residue in the C-terminal region required for binding appears to be the charged residue Arg13. In contrast, alanine replacement of the other charged residues surrounding Cys19 on the {beta}-hairpin (Asp16, Arg18, and Lys20) have little effect on binding of 125I-M12A. These data collectively suggest that initial binding of the C-terminal region to the receptor allows the unstructured region of PSP (residues 1-6) to bind. Our results strongly suggest that the phenyl side chain of Phe3 is essential for binding of the N terminus and that this interaction "locks" PSP into place. The peptide backbone of the N terminus also likely contributes to binding energy, given that decreasing the length of PSP by one residue (PSP-(2-23)) binds as well as M12A whereas reducing the length two residues (PSP-(3-23)) reduces binding 35%. In contrast, deletion of the side chain from Asn2 (N2A) has no deleterious effect on either activity (17) or binding (data not presented). These results are also consistent with the ability of N2A to completely compete with 125I-F23Bpa in the receptor labeling experiments.

Prior studies indicated that the N-terminal amine of Glu1 is essential for biological activity (7), yet our current results reveal it is relatively unimportant for binding. This conclusion derives from our experiments with AcPSP and AcPSP-(2-23). AcPSP increased the overall length of the N terminus and removed the positive charge normally present at neutral pH, whereas AcPSP-(2-23) also removed the positive charge and shortened the peptide. Although AcPSP-(2-23) binds strongly to hemocytes, AcPSP does not, indicating that increasing the length of the peptide backbone hinders attachment to its activating binding pocket whereas charge removal has little effect on binding. The inability of ENFN and PSP-(7-23) together to activate the receptor further suggests the possibility that binding sites for the N- and C-terminal regions of PSP may reside on different domains or subunits of the receptor that must be "cross-linked" by full-length PSP for hemocyte activation to occur.

The relationship between the phenyl ring of Phe3 and terminal amine of Glu1 is particularly intriguing when compared with the thrombin receptor involved in platelet activation (24, 25). The thrombin receptor is activated when thrombin cleaves the amino-terminal extension of its receptor to reveal a new N terminus with a serine residue at position one and a phenylalanine at position two. This new extracellular domain then functions as a tethered ligand that self-activates the receptor. Strikingly, both the N-terminal amine of Ser1, but not the side chain, and the phenyl ring of Phe2 are required for receptor activation. Although the particular N-terminal residue and distance to the phenylalanine residue differ between this ligand and PSP, the overall similarities suggest a general mechanism for receptor activation that requires binding of an aromatic ring and a closely spaced primary amine with receptor-activating properties.

The most perplexing result of our study is that several Phe3 mutants competitively reduce 125I-M12A binding but none functionally antagonizes PSP. One possibility for this outcome is that cell binding by these mutants is too weak to impact function; we think this unlikely, given that AcPSP and F3A exhibit equivalent binding characteristics, yet AcPSP is a much stronger antagonist. Another possibility is that M12A and PSP are somehow different; this too seems unlikely, given that they are indistinguishable from one another in side-by-side binding and adhesion assays. A third possibility is that much lower concentrations of peptide (100 pM 125I-M12A) were used for binding than were needed to induce rapid spreading of plasmatocytes (3 µM PSP). As previously noted, the IC50 for binding of M12A is 0.7 nM, whereas the ED50 for PSP-induced spreading of plasmatocytes is 10-100 nM (6). The reason for this difference could be that large numbers of receptors per plasmatocyte must bind ligand before the signaling pathway regulating adhesion and spreading is activated. Recent studies suggest the sensitivity of plasmatocytes to PSP varies with the larval stage of the insect cells collected.3 It also suggests that receptor abundance may be an important factor in regulating PSP function. Molecular identification of the putative 190-kDa PSP receptor is obviously key to testing the validity of these ideas.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AI32917, USDA NRI Grant 2002-35302-11554, and funding from the Georgia Agricultural Experiment Station (to M. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 706-583-8237; Fax: 706-542-2279; E-mail: mrstrand{at}bugs.ent.uga.edu.

1 The abbreviations used are: PSP, plasmatocyte-spreading peptide; AcPSP, acetylated PSP; EGF, epidermal growth factor; Bpa, benzoylphenylalanine; Fmoc, N-(9-fluorenyl)methoxycarbonyl. Back

2 K. D. Clark and M. R. Strand, unpublished results. Back

3 K. D. Clark, L. Kappa, and M. R. Strand, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank L. Kapa for assistance with the functional antagonism experiments, J. A. Johnson for Fig. 6, D. King for assistance and advice in peptide synthesis, and D. Phillips of the University of Georgia mass spectrometry center.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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