From the Department of Entomology, University of
Wisconsin-Madison, Madison, Wisconsin 53706, the
§ Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226, the ¶ Department of Biochemistry,
University of California, Berkeley, California 94702, and the
Biochemical Laboratory, Institute of Low Temperature Science,
Hokkaido University, Sapporo 060-0819, Japan
Received for publication, January 22, 2001
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ABSTRACT |
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Plasmatocyte spreading peptide (PSP)
is a 23-amino acid cytokine that induces a class of insect immune cells
called plasmatocytes to spread on foreign surfaces. The structure of
PSP consists of a disordered N terminus (residues 1-6) and a
well-defined core (residues 7-23) stabilized by a disulfide bridge
between Cys7 and Cys19, hydrophobic
interactions, and a short In insects, the primary immune response toward parasites and other
large foreign targets that enter the hemocoel is encapsulation (1, 2).
During an encapsulation response, circulating blood cells (hemocytes)
attach to the target and one another to form a smooth capsule comprised
of overlapping layers of cells. The two types of hemocytes most often
observed in capsules formed by Lepidoptera (moths and butterflies) are
granular cells and plasmatocytes. In the moth Pseudoplusia
includens, some foreign entities are recognized by factors in
plasma, but most are recognized by surface receptors on granular cells
(3-5). After attaching to the target, granular cells and/or humoral
opsonins then induce plasmatocytes to change from non-adhesive to
strongly adhesive cells that form the capsule. Previously, we
determined that plasmatocyte activation is mediated by a 23-amino acid
cytokine named plasmatocyte spreading peptide
(PSP)12
(6). Both purified and synthetic PSP induce plasmatocytes to rapidly
adhere and spread across foreign surfaces at concentrations PSP is expressed by granular cells and fat body as a propeptide of 142 residues with the PSP sequence located at the C terminus (10). This
biologically inactive precursor is then cleaved by an unknown protease
to release the mature peptide. 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 and a short
Insects--
P. includens larvae were reared on
artificial diet at 27 °C and with a 16-h light/8-h dark photoperiod
(16). Moths were fed 20% sucrose in water and maintained under
identical environmental conditions.
Hemocyte Collection and Bioassays--
P. includens
hemolymph contains four hemocyte types (plasmatocytes, granular cells,
spherule cells, and oenocytoids) with plasmatocytes and granular cells
accounting for 30 and 65%, respectively, of the total hemocyte
population (3, 18). Hemocytes were collected by anesthetizing
36- to 48-h fifth instar larvae with CO2 and bleeding them
from an incision across the last abdominal segment. Hemolymph was
collected in a microcentrifuge tube containing anticoagulant
buffer (98 mM NaOH, 186 mM NaCl, 17 mM Na2EDTA, and 41 mM citric acid,
with pH adjusted to 4.5). The ratio of hemolymph to buffer was
approximately 1:5. Hemocytes were pelleted for 1 min at 200 × g, and the plasma-buffer supernatant was then removed.
Hemocytes were then resuspended in 1 ml of fresh anticoagulant. After a
40-min incubation at 4 °C, hemocytes were washed twice by
centrifugation in Ex-cell 400 medium (JRH Biosciences). Plasmatocytes were isolated to high purity by loading hemocytes from 8-10 larvae onto Percoll step-gradients made in 12- × 75-mm plastic tubes (Falcon
352058). Gradients were made in Ex-cell 400 in two layers: a 2-ml
bottom layer of 62.5% Percoll (100% Percoll equals 9 parts Percoll to
1 part 10× Pringle's saline (150 mM NaCl2,
2.5 mM KCl, 1 mM CaCl2, 20 mM Dextrose), and a 2-ml top layer of 47.5% Percoll. Plasmatocytes with an average purity of 93% were collected from the
47.5-62.5% Percoll interface whereas the other hemocyte types (granular cells, spherule cells, and oenocytoids) banded elsewhere in
the gradient. The number of plasmatocytes collected per gradient ranged
from 1.0 to 1.4 × 106 cells with the primary
contaminant being granular cells.
The plasmatocyte spreading activity of wild-type and mutant peptides
was assayed in 96-well culture plates (Corning) as described by Clark
et al. (6). Plates were prepared by first adding the indicated peptide solution (6 µl at 10× the desired final
concentration) to each well. Wells were then filled with 54 µl of
Ex-cell 400 medium containing 1 × 103 plasmatocytes.
The percentage of plasmatocytes that spread in an assay was scored
1 h after adding peptide by counting 100 cells from a randomly
selected field of view. Plasmatocytes were scored as spread if they
assumed a flattened morphology and were Peptide Synthesis and Purification--
All peptides were
synthesized using standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. The resin
peptide was cleaved and deprotected for 4 h in reagent K (19), a
mixture containing 5% phenol, 1.25% water, 2.5% thioanisole, and
2.5% dithioethane in TFA. After removing the resin from the reaction
mixture by filtration, the peptide was precipitated in cold
t-butylmethyl 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
periodically monitored 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-Aldrich) and a linear gradient of acetonitrile
(0-80 min, 20-60%) at 0.5 ml/min. Both the H2O and the
acetonitrile contained 0.05% TFA. After the conversion was complete,
the sample was purified by a series of 1-ml injections onto a
preparatory HPLC column (5-µm particle size, 10 mm × 25 cm,
Jupiter, Phenomenex Inc., Torrance, CA) using HPLC-grade
H2O and a linear gradient of acetonitrile (0-80 min,
10-50%) at 3 ml/min. Both the H2O and the acetonitrile contained 0.05% TFA. The desired peak was identified by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry, and 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.
NMR Spectroscopy--
NMR samples were prepared by dissolving
lyophilized peptide in 0.5 ml of a buffer containing 90%
H2O-10% D2O and 20 mM sodium phosphate and adjusting the final pH to 6.0. Peptide concentrations ranged from 0.5 to 1.0 mM. All NMR spectra were recorded at
10 °C on Bruker DMX600 or DMX750 spectrometers equipped with
triple-resonance (1H/13C/15N)
probes and three-axis pulsed field gradient capabilities.
One-dimensional 1H spectra were acquired with 4096 complex
points and a spectral width of 12.5 ppm. Two-dimensional TOCSY and
NOESY spectra were acquired at 600.13 MHz on the mutants C7.19A, R13A,
R18A, and F3A with spectral widths of 6944.44 Hz in both dimensions,
and time domain data sizes of 1024 and 512 complex points in the direct and indirect dimensions, respectively. Mixing periods of 75 and 300 ms
were used for the TOCSY and NOESY experiments, respectively. All
Fourier transformations of NMR data were performed with the NIH-produced software nmrPipe (20), and chemical shift
assignments were obtained using the program XEASY (11). All
1H dimensions were referenced to
2,2-dimethyl2silapentane-5-sulfonate.
Sequence Comparison of ENF Peptides and Synthesis of PSP
Mutants--
The tertiary structure of PSP reveals a disordered N
terminus (residues 1-6) and a well ordered domain (residues 7-22)
stabilized by a combination of the covalent disulfide linkage between
Cys7 and Cys19, hydrogen bonding within the
Selected Residues of the PSP Core Domain Are Critical for
Biological Activity--
The most important element for maintaining
the three-dimensional structure of PSP is the disulfide bond between
Cys7 and Cys19. Plasmatocyte spreading assays
indicated that C7.19A had no biological activity except at the highest
concentration tested (10 µM). In contrast, wild-type PSP
showed a normal dose-response with a threshold at 1 nM and
saturation at 1 µM (Fig.
2). We next considered the uncharged
hydrophilic threonines at positions 14 and 22. Thr14
resides in the
As noted above, all ENF peptides have either a phenylalanine or
tyrosine residue at positions 11 and 23. Tyr11 appears
important for proper conformation of PSP as the side chains of
Cys7, Leu8, Ala9,
Cys19, Pro21, and Phe23 form a well
defined hydrophobic core centered around the side chain of this residue
(11). Comparison of PSP to other EGF-like subdomains also revealed the
presence of an aromatic residue at a position equivalent to
Tyr11 in transforming growth factor-alpha (TGF-
We next considered the four charged residues (Arg13,
Asp16, Arg18, Lys20) surrounding
Cys19. Each of these amino acids resides within the
The Unstructured N-terminal domain of PSP Is Also Critical for
Biological Activity--
The lack of defined structure over the first
six residues of PSP originally suggested to us that this domain was
likely less important for receptor binding and plasmatocyte activation
than the highly structured C-terminal domain (Fig. 1A).
However, the conservation of the first three residues (Glu-Asn-Phe)
among all ENF peptide family members (Fig. 1B)
circumstantially argued that the unstructured N terminus may be more
important for activity than the tertiary structure of PSP suggested.
Bioassays with PSP-(7-23) clearly supported a role for the
unstructured N terminus in plasmatocyte activation as this mutant had
no activity at any concentration tested (Fig.
5A). PSP-(7-23) also lacked
spreading activity at a concentration of 100 µM (data not
presented). We then tested alanine point mutants in the unstructured N
terminus. Surprisingly, both E1A and N2A had higher activity than
wild-type PSP with threshold responses shifted to approximately a
100-fold lower level than the native peptide (Fig. 5B). In
contrast, F3A had no plasmatocyte spreading activity from 1 nM to 10 µM and only a low level of activity
at 100 µM. We thus concluded that the lack of activity for PSP-(7-23) is largely due to the critical importance of
Phe3.
Structural Analysis of PSP mutants--
The mutant peptides
C7.19A, F3A, R13A, and R18A all had reduced plasmatocyte spreading
activity when compared with wild-type PSP. To determine whether this
reduction in activity correlated with alterations in tertiary
structure, we compared these mutants to wild-type PSP by NMR
spectroscopy. Because amide proton shifts are quite sensitive to
secondary and tertiary protein structure, changes in the Implications for Receptor Binding and Activation--
The PSP
receptor has not been identified, but the strong similarity in the
structure of PSP and the C-terminal subdomains of other EGF-like
molecules suggest that PSP-receptor interactions may be analogous to
other EGF domain-receptor complexes (15). As such, we initially
hypothesized that the ordered portion of PSP (residues 7-21) would be
the region most essential for biological activity. The distinction
between residues critical for proper folding and those directly
involved in target cell binding (and biological activity) are not
always clear from mutagenesis experiments alone. However, the small
size of PSP makes this molecule especially amenable for assessing how
specific mutations affect activity and structure. Our results with
C7.19A clearly indicate that the disulfide bond is essential for
maintenance of both the structure and activity of PSP. In contrast, the
F3A and R13A mutants exhibit greatly reduced activity without a
concomitant change in tertiary structure. Arg13 and
Phe3 therefore may be especially critical residues for
receptor binding. The loss of activity associated with F3A and R13A
also indicates that both the ordered and disordered domains of PSP are
important for plasmatocyte activation.
The three charged amino acids surrounding Cys19 on
the -hairpin. Structural comparisons also
indicate that the core region of PSP adopts an epidermal growth factor
(EGF)-like fold very similar to the C-terminal subdomain of EGF-like
module 5 of thrombomodulin. To identify residues important for
plasmatocyte spreading activity, we bioassayed PSP mutants in which
amino acids were either replaced with alanine or deleted. Within the
well-defined core of PSP, alanine replacement of Cys7 and
Cys19 (C7.19A) eliminated all activity. Alanine replacement
of Arg13 reduced activity ~1000-fold in comparison to
wild-type PSP, whereas replacement of the other charged residues
(Asp16, Arg18, Lys20) surrounding
Cys19 diminished activity to a lesser degree. The point
mutants Y11A, T14A, T22A, and F23A had activity identical or only
slightly reduced to that of wild-type PSP. The mutant
PSP-(7-23) lacked the entire unstructured domain of PSP and was
found to have no plasmatocyte spreading activity. Surprisingly, E1A and
N2A had higher activity than wild-type PSP, but F3A had almost no
activity. We thus concluded that the lack of activity for PSP-(7-23)
was largely due to the critical importance of Phe3. To
determine whether reductions in activity correlated with alterations in
tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants
to wild-type PSP by NMR spectroscopy. As expected, the simultaneous
replacement of Cys7 and Cys19 profoundly
affected tertiary structure, but the R13A, R18A, and F3A mutants did
not differ from wild-type PSP. Collectively, these results indicate
that residues in both the unstructured and structured domains of PSP
are required for plasmatocyte-spreading activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 nM (6, 7). PSP homologs occur in other Lepidoptera and in
some cases have been shown to have plasmatocyte spreading activity (8,
9). Based on the consensus sequence of their N termini (Glu-Asn-Phe-X-X-Gly-Cys), these peptides are now
referred to as the ENF peptide family (9).
-hairpin turn (11). Comparison with other proteins reveals that,
despite sequence identity at only four positions, the core region of
PSP adopts a very similar structure to the C-terminal subdomain of
human epidermal growth factor (hEGF) and the anticoagulant protein
thrombomodulin (hTM5). 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 known
only among members of the ENF peptide family (9). In this study, we
used an alanine scanning mutagenesis approach to identify residues
critical for the plasmatocyte spreading activity of PSP. Our results
indicate that selected residues in both the core and unstructured
domains of PSP are required for plasmatocyte spreading activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
35 µm along their longest
axis (3, 6). Unspread plasmatocytes in contrast remained spheroidal in
shape. Each mutant peptide was bioassayed four times using an
independently collected sample of plasmatocytes. Bioassays with each
mutant peptide were always paired with bioassays of wild-type PSP to
control for any variation in spreading response that might exist
between plasmatocyte samples. The estimated maximum spreading response
was defined as the percentage of plasmatocytes that spread when
cultured in the highest concentration of peptide tested (10 µM). The lowest concentration of peptide that induced
5% of plasmatocytes to spread was referred to as the threshold
spreading response. Threshold spreading responses were determined
empirically from dose-response curves.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-hairpin, and hydrophobic packing (Fig.
1A). Fourteen other ENF
peptides have also been identified (6, 21-26). Comparing the primary
sequences of these peptides to PSP indicated that the most variable
positions were positions 4, 5, 8, 9, and 12 (Fig. 1B). Some
variation also existed at positions 11, 15, 21, and 23 (Fig.
1B). Prolines were present at positions 9, 15, and 21 in
some family members, which could potentially alter their backbone
structure in comparison to PSP. Several family members also differed
from PSP at positions 11 and 23, but these were conservative aromatic
ring substitutions. The remaining 14 residues were invariant among
family members. These included 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. In
addition to being identical at these positions, three ENF peptide
family members (PSP, Pss GBP, and Mas PP1) are also known to have
plasmatocyte spreading activity (see legend, Fig. 1B). We
therefore hypothesized that residues important for plasmatocyte spreading activity most likely reside among these 14 conserved residues
and the aromatic amino acids at positions 11 and 23. To test this
hypothesis, we synthesized PSP mutants in which amino acids were either
replaced with alanine or deleted. Alanine was selected as the
replacement residue because of its lack of charge, functional side
groups, and because it was least likely to alter the backbone structure
of the peptide (17). Cys7 and Cys19 were
simultaneously replaced with alanine in one mutant (C7.19A), whereas
the first six residues of wild-type PSP were deleted for another mutant
designated PSP-(7-23). The other 11 constructs we synthesized were all
alanine point mutants. Although Gly6, Gly10,
and Gly17 are conserved among all ENF peptide
family members (Fig. 1A), we did not synthesize alanine
mutants at these positions, because we assumed the role of these
residues was primarily for proper folding of the peptide.
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Fig. 1.
Tertiary structure of PSP and comparison of
the PSP sequence to other ENF peptides and EGF-like domains.
A, front view of PSP derived from the ensemble of PSP
conformers determined by Volkman et al. (11). The backbone,
N, C , and C' atoms are shown in green, and
side-chain carbon, nitrogen, oxygen, and sulfur atoms are shown in
gray, blue, red, and
yellow, respectively. B, amino acid content by
position for the 15 known members of the ENF peptide family. The
subscript numbers indicates the number of family members
with that particular amino acid at that position. At each position,
amino acids are listed from the most to the least prevalent
(top to bottom). The sequence of PSP is
underlined. The 15 known ENF peptides were originally named
plasmatocyte spreading peptide (PSP), growth blocking peptide (GBP),
paralytic peptide (PP), or cardioactive peptide (CAP). PSP is from
P. includens (Psi PSP) (6); the GBPs are from
Pseudaletia separata (Pss GBP), Mamestra
brassicae (Mas GBP), and Spodoptera litura (Spl GBP)
(21, 22); the PPs are from Manduca sexta (Mas PPI and II)
(23), Spodoptera exigua (Spe PPI, II, and III) (23),
Heliothis virescens (Hev PPI and II) (23),
Trichoplusia ni (Trn PPI and II) (24), and Antheraea
yamamai (Any PP) (25); and CAP is from Spodoptera
eridania (Spe CAP) (26). Thirteen of the ENF family members are 23 amino acids in length whereas two members have additional amino acids
at their C termini: Trn PPI with 24 and Pss GBP with 25. C,
comparison of the sequences of PSP, and the C-terminal subdomains of
hEGF, hTM5, and hTGF-
. Residues conserved among these factors are
indicated in red.
-hairpin of PSP but faces away from the charged residues, whereas Thr22 resides in the less-ordered C
terminus (see Fig. 1A). The T14A mutant exhibited slightly
higher plasmatocyte spreading activity than wild-type PSP at all
concentrations tested, whereas T22A exhibited activity that was reduced
by one-half to one-third at concentrations from 1 nM to 1 µM (Fig.
3A).
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Fig. 2.
In vitro spreading responses of
plasmatocytes to the mutant C7.19A and wild-type PSP. Spreading
was assayed by observing cells after 1 h in culture with each
peptide. Each data point is the mean percentage ± S.D. of
plasmatocytes spread from four independent collections of
plasmatocytes. A stock solution (10 mM) of each peptide in
phosphate-buffered saline was diluted serially into Ex-cell 400 medium
for use in each bioassay.
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Fig. 3.
In vitro spreading responses of
plasmatocytes to: A, the mutants T14A and T22A and
B, the mutants Y11A and F23A. The activity of
each mutant peptide was compared with wild-type PSP as described in
Fig. 2.
(Tyr38)), the fifth subdomain of human thrombomodulin (hTM5
(Tyr413)), and human epidermal growth factor (hEGF
(Tyr37)) (Fig. 1C). Mutagenesis experiments
indicate these Tyr11 equivalents are essential for activity
of TGF-
and TM, but not EGF (12, 15). Our bioassays indicated that
plasmatocyte spreading activity of the Y11A mutant was only slightly
reduced to that of wild-type PSP (Fig. 3B). The activity of
F23A was virtually the same as wild-type PSP (Fig. 3B).
-hairpin turn with Asp16, Arg18, and
Lys20 in opposite orientation to Arg13 (Fig.
1A). Alanine replacements at each of these positions reduced plasmatocyte spreading activity compared with wild-type PSP (Fig. 4). The mutants that least affected
activity were K20A and R18A, which showed a threshold response at 10 nM and saturation in the 1-10 µM range. D16A
showed a greater loss of activity with a response threshold that was
two orders of magnitude lower than wild-type PSP. However, the maximal
response of D16A did not differ from intact PSP. R13A had the lowest
plasmatocyte spreading activity of the charged residue replacements
(Fig. 4). The threshold response for this mutant was 10 µM, making it ~1000-fold less active than wild-type
PSP. Although Arg13 is of prime importance for the activity
of PSP, data base searches revealed no homologous residue among other
EGF-like domains.
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Fig. 4.
In vitro spreading responses of
plasmatocytes to the mutants R13A, D16A, R18A, and K20A and wild-type
PSP. Peptides were bioassayed as described in Fig. 2.
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Fig. 5.
In vitro spreading responses of
plasmatocytes to: A, the mutant PSP-(7-23) and
B, the mutants E1A, N2A, and F3A. The activity of
each mutant peptide was compared with wild-type PSP as described in
Fig. 2.
-hairpin or
hydrophobic core of PSP can be expected to produce dramatic changes in
chemical shift (>0.5 ppm). The 1H NMR spectra of PSP and
the aforementioned mutants are presented in Fig.
6. As expected, the simultaneous
replacement of Cys7 and Cys19 with alanine
profoundly affected tertiary structure as evidenced by the collapse in
chemical shift dispersion for this peptide in comparison to wild-type
PSP (Fig. 6, A and B). This collapse is
consistent with the absence of a unique folded peptide structure. Signals from Lys20 (K20) and Met12
(M12) at ~9.5 ppm in wild-type PSP were also shifted far
upfield in the C7.19A mutant (Fig. 6B), indicating that the
PSP
-hairpin structure is not formed in the mutant. In contrast, the
R13A, R18A, and F3A replacements had no significant affect on the
1H NMR spectrum, indicating that the reduced biological
activity of these peptides is not due to significant alterations in
structure (Fig. 6, C-E). NMR spectra were also obtained on
all of the other single amino acid replacements we synthesized, and
they too had no significant effect on the tertiary structure of the
peptide (data not presented). As a further check on the structural
integrity of the PSP mutants, two-dimensional NMR spectra were used to
obtain complete backbone 1H chemical shift assignments for
the PSP-(7-23), R13A, D16A, R18A, and K20A mutants.
1H
-1HN and
1HN-1HN regions
of NOESY spectra were also compared with wild-type PSP to identify the
patterns of NOEs characteristic of its tertiary structure. Overall,
analysis of the one- and two-dimensional NMR data indicated that, with
the exception of C7-19A, all of the other mutants retained the
tertiary fold determined for wild-type PSP (data not presented).
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Fig. 6.
1H NMR spectra of wild-type PSP
and the mutant peptides C7.19A, R13A, R18A, and F3A. A,
backbone amide resonances are shown and labeled by residue for
wild-type PSP. B, the reduction in chemical shift dispersion
for the C7.19A spectrum reflects the lack of tertiary structure as a
result of removing the disulfide bridge. C-E, substitution
of alanine for Arg13, Arg18, and
Phe3 results in only minor perturbations to the amide
1H shifts, and no apparent change to the structure
determined for wild-type PSP. Other variations in baseline noise or
line widths are due to differences in experimental acquisition
parameters.
-hairpin (Asp16, Arg18, and
Lys20) all align on one side of PSP, presenting a
structural motif that may be important for receptor binding. Of these
three charged residues, we originally were most interested in
Arg18, because arginines exist in analogous positions in
the receptor binding domains of both EGF (Arg41) and
TGF-
(Arg42) that are essential for binding and
activity. However, R18A had only slightly reduced activity compared
with wild-type PSP. K20A similarly exhibited a small loss, whereas D20A
had a more moderate loss of activity. Based on these results, we
anticipate that PSP requires the complete charge sequence (
+ +)
provided by these residues for normal activity, but we hypothesize that
removal of the positive charge at either Arg18 or
Lys20 is partially compensated by the other residue,
resulting in only small changes in activity. Although replacement of
Arg13 greatly reduced activity of PSP, no analogous residue
exists in EGF, TGF-
, or TM (Fig. 1C). The similarity in
structure of R13A and wild-type PSP suggests that Arg13 may
be a third residue important for receptor binding. Also supporting this
suggestion are the results of competition experiments showing that R13A
does not antagonize the plasmatocyte spreading activity of wild-type
PSP.3 We view PSP-(7-23) and
F3A as the most intriguing mutants, because they clearly indicate that
the disordered N terminus of PSP is required for biological activity.
The N terminus does not appear to be essential for receptor binding,
because both PSP-(7-23) and F3A antagonize the plasmatocyte spreading
activity of PSP.3 However, binding of the PSP core may
promote a conformational stabilization of the normally unstructured N
terminus so that Phe3 interacts with and activates the
receptor. Identification of the PSP receptor is obviously essential to
testing these hypotheses and identifying binding determinants in this
novel family of insect cytokines.
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ACKNOWLEDGEMENTS |
---|
We thank Martha Vestling for assistance in acquiring and processing of mass spectral data and members of the University of Wisconsin Biotechnology facility for assistance in synthesis of mutant peptides. We also thank the National Magnetic Resonance Facility at Madison and the University of Texas at Galveston Protein Chemistry laboratory for help during the study.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI32917 and by Wisconsin Hatch Grant WIS04436 (to M.R.S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) .
** To whom correspondence should be addressed: Dept. of Entomology, 237 Russell Laboratories, University of Wisconsin, Madison, WI 53706. Fax: (608) 262-3322; E-mail: mrstrand@facstaff.wisc.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100579200
2 The atomic coordinates and structure factors of PSP can be accessed through NCBI Protein Data base under NCBI accession 6729988 (11). The amino acid sequence of the PSP precursor protein can also be accessed through the NCBI Protein Data base under NCBI accession JE0359 or the GenBankTM data base under GenBankTM accession number AF062489 (10).
3 K. D. Clark and M. R. Strand, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
PSP, plasmatocyte
spreading peptide;
CAP, cardioactive peptide;
EGF, epidermal growth
factor;
GBP, growth-blocking peptide;
hEGF, human EGF, HPLC, high
performance liquid chromatography;
hTM5, fifth EGF-like domain of human
thrombomodulin;
hTGF-, human TGF-
;
NOE, nuclear Overhauser
effect;
TOCSY, TOC spectroscopy;
NOESY, NOE spectroscopy;
PP, paralytic peptide;
TFA, trifluoroacetic acid;
TGF-
, transforming
growth factor-
;
TM, thrombomodulin.
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REFERENCES |
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