(Received for publication, May 31, 1994; and in revised form, January 4, 1995)
From the
Eosinophil cationic protein (ECP) is a toxin secreted by
activated human eosinophils that has anti-parasitic, antibacterial, and
neurotoxic activities; ECP also has ribonuclease activity and
structural homology to other mammalian ribonucleases. To determine the
relationship between the ribonuclease activity and cytotoxicity of ECP,
a method for producing recombinant ECP (rECP) in a prokaryotic
expression system was devised. Periplasmic isolates from induced
bacterial transfectants contained enzymatically active rECP; micromolar
concentrations of rECP were shown to be toxic for Staphylococcus
aureus (strain 502A). In contrast, recombinant eosinophil-derived
neurotoxin, with 67% amino acid sequence identity to ECP, had little to
no toxicity for S. aureus; these findings are analogous to
those obtained with purified, granule-derived ECP and
eosinophil-derived neurotoxin. Two single base pair mutations were
introduced into the coding sequence of rECP (Lys to Arg
and His
to Asp) to convert ribonuclease active-site
residues into non-functional counterparts. These mutations eliminated
the ribonuclease activity of rECP but had no discernible effect on the
antibacterial activity of this protein, demonstrating that ribonuclease
activity and cytotoxicity are, in this case, independent functions of
ECP.
Eosinophil cationic protein (ECP) ()is a small
(18-25 kDa) cationic toxin stored in the large specific granules
of human eosinophilic leukocytes(1, 2, 3) .
ECP is a potent anti-parasitic agent, with particular activity against Schistosoma(4, 5, 6) and Trichinella(7, 8) species. ECP also has
antibacterial activity(9) , and a role for ECP in host defense
against bacterial infections in vivo has been
suggested(10) . ECP is also toxic to host tissues; the
cardiovascular damage associated with chronic hypereosinophilia (1, 11, 12, 13, 14) has
been attributed in part to the cytotoxicity of secreted
ECP(15, 16) , and ECP has been shown to damage
respiratory epithelial cells in vitro(17) .
The mechanism by which ECP destroys its target cells remains unclear. In 1986, Young and colleagues (18) reported that ECP purified from eosinophil granules could disrupt artificial lipid membranes in vitro. Since then, several investigators have noted the sequence similarities between ECP and ribonucleases (19, 20, 21) and have shown that ECP purified from eosinophil granules has ribonuclease activity(22, 23) . In addition, several ribonucleases, including ECP, human eosinophil-derived neurotoxin (EDN, a protein with 67% amino acid sequence identity to ECP), and onconase, isolated from Rana pipiens, have been identified as neurotoxins(19, 24, 25, 26, 27) . Two independent groups have shown that alkylation at the ribonuclease active site simultaneously eliminated both the ribonuclease and neurotoxic activities of EDN and onconase (27) and of EDN and ECP(28) . Yet, in another study, the anti-parasitic activity of ECP appeared to be unaffected by the presence of a ribonuclease inhibitor(8) .
In the work presented here, the relationship between the ribonuclease activity and cytotoxicity of ECP is examined directly. Initial experiments with granule-derived, purified ECP and EDN suggested that ribonuclease activity might be incidental to the antibacterial activity of ECP. To consider this possibility experimentally, wild type recombinant ECP (rECP) and a mutant, ribonuclease-minus form were produced, and their respective antibacterial activities were examined.
Figure 1: A, CFUs of S. aureus 502A remaining after exposure to increasing concentrations of granule-derived, purified ECP (circles) or EDN (squares). The two curves representing the response to ECP (open and filledcircles) represent experiments that were identical except for the initial CFU/ml (see points at 0 µM ECP) B, percentage of colony-forming units remaining after exposure of either E. coli HB101 (blackcolumns) or S. aureus 502A (shadedcolumns) to increasing concentrations of purified ECP; a, the percentage of S. aureus CFU remaining after exposure to 2.3 µM ECP was 0.1%.
Figure 2: A, schematic of the rECP constructs, including wild type (hECP#7) and ribonuclease-minus mutant (hECP#2) in the pFLAG-CTC bacterial expression vector. The signal sequence is encoded by the ECP cDNA(20) , and the carboxyl-terminal FLAG peptide (DYKDDDDK) is encoded by the pFLAG-CTC vector. The ribonuclease catalytic residues are enclosed in the openboxes, and the mutations are enclosed in the shadedboxes. The numbersabove the boxes refer to the position of each amino acid in the mature form (without signal sequence) of ECP. B, Coomassie Blue-stained SDS-PAGE; C, corresponding Western blot probed with the M2 anti-FLAG peptide mAb. Lanes contain total cell extracts from bacteria transfected with hECP#7 prior to (lanes1) and 30 min after addition of 0.1 mM IPTG (lanes2). Lanes3 contain periplasmic proteins isolated from the induced transfectants bound to heparin-Sepharose at pH 8.0. D, amino-terminal sequencing of the upper (19 kDa) immunoreactive band at the arrow in lane2 of panelC; the anticipated sequence is shown below. E, serial dilutions of the periplasmic proteins shown in C compared with known quantities of rBAP FLAG (F). The dilutions of rECP are: lane1, undiluted; lane2, 1:2; lane3, 1:4; lane4, 1:9; lane5, 1:19; and lane6, 1:49. The concentrations of rBAP are: lane1, 6 µg/ml; lane2, 3 µg/ml; lane3, 1.2 µg/ml; lane4, 0.6 µg/ml; lane5, 0.3 µg/ml; and lane6, 0.12 µg/ml.
Figure 5: Fraction of CFUs of S. aureus 502A surviving after overnight incubation with rEDN (hEDN#1, black bars), wild type rECP (hECP#7, lightly shaded bars), or ribonuclease-minus mutant rECP (deeply shaded bars). Each bar represents the average of duplicate samples; errorbars are as shown. wt, wild type.
Growth of bacteria treated with increasing concentrations of purified, granule-derived ECP or EDN was measured is shown in Fig. 1. Growth of both E. coli (strain HB101) (panelB) and S. aureus (strain SA502A) (panelsA and B) was inhibited by micromolar concentrations of ECP; these results are consistent with those reported by Lehrer and colleagues(9) . In addition, we show that EDN, at the same and at higher concentrations, had no effect on bacterial growth (panelA).
The data presented in Fig. 2describe the production of rECP and its distribution to the intracellular and periplasmic compartments. The Coomassie Blue-stained gel in panelB demonstrates the proteins loaded in the corresponding lanes of the Western blot in panelC. Two distinct protein bands were detected by the M2 mAb (directed against the carboxyl-terminal FLAG peptide as described under ``Experimental Procedures'') in total cellular extracts of IPTG-induced hECP#7 transfectants (panelC, lane2). The identical bands were detected with a polyclonal anti-ECP antiserum (data not shown). The mobility of the lower band (16 kDa) was consistent with the expected size of rECP without signal sequence as predicted by cDNA cloning(20, 21) . The mobility of the upper band (19 kDa, at arrow) was consistent with the size predicted for unprocessed rECP(20, 21) ; amino-terminal sequencing confirmed the presence of the ECP signal sequence in this protein band (panelD, at arrow, with expected sequence, including the three initial vector-encoded residues below).
Periplasmic proteins isolated from IPTG-induced hECP#7 transfectants by osmotic shock followed by heparin-Sepharose affinity isolation (see ``Experimental Procedures'') were probed with the M2 mAb as shown in the Western blot in panelC (lane3). Only the 16-kDa band (rECP) was detected in periplasmic isolates. The estimated yield of rECP in a given periplasmic extract is determined as shown in panelsE and F. The blot in panelE contains serial dilutions of the periplasmic extract, and in panelF, the blot contains serial dilutions of a commercial preparation of rBAP. Both rECP and rBAP-FLAG contain a single copy of the carboxyl-terminal FLAG peptide. The two Western blots shown were transferred, treated with antibodies, and developed simultaneously to ensure equivalent signals from equivalent molar amounts of recombinant protein. The diminishing signals in lanes1-3 of panelE (rECP) match those of lanes2-4 of panelF (rBAP), permitting an estimated yield of 3 µg/ml in the undiluted sample of rECP.
In Fig. 3, the ribonuclease activity of rECP is examined. Periplasmic protein isolates were prepared as described from IPTG-induced hECP#7 transfectants and from induced bacteria transfected with the pFCTC vector alone. A Coomassie Blue-stained gel displaying both isolates is shown in panelB, and the corresponding Western blot probed with M2 mAb is shown in panelC. These isolates were evaluated for their ability to solubilize a yeast tRNA substrate (panelA). All measurements shown were within the linear portion of the reaction curve, permitting calculation of initial rates. The rate calculated for the ribonuclease activity of the control isolate was 0.60 OD/ng/min, while that of the isolate that included rECP (hECP#7) was 6.0 OD/ng/min, or 10-fold over the control rate, indicating that rECP isolated from the bacterial periplasm was enzymatically active.
Figure 3:
A, ribonuclease activity of periplasmic
proteins isolated from IPTG-induced bacterial transfectants. 3 µg
of periplasmic proteins containing wild type rECP (from hECP#7
transfectants, opencircles), containing
ribonuclease-minus rECP (from hECP#2 transfectants, filledcircles), and without rECP (from vector control
transfectants, opensquares). Initial rates are as
follows: wild type rECP, A/ng/min;
ribonuclease-minus rECP, A
/ng/min; no rECP
control, A
/ng/min. The Western blots in panelsC and E probed with the M2 anti-FLAG
peptide mAb document the presence of 16-kDa rECP in the hECP#7 (panelC, lane1) and hECP#2 (panelE, lane2) transfectant
preparations and its absence in the vector control preparation (panelC, lane2). PanelsB and D are Coomassie Blue-stained gels with lanes corresponding to those shown in panelC and E, respectively.
The
data in Fig. 3also describe the production, distribution, and
ribonuclease activity of a mutant form of rECP. The hECP#2 construct
(see Fig. 1, panelA) contains two single base
pair mutations that were intended to convert two known ribonuclease
active-site residues into non-functional counterparts (Lys to Arg, His
to Asp)(34) . The Coomassie
Blue-stained gel (panelD) and the corresponding
Western blot probed with the M2 mAb (panelE) contain
total cell extracts (lanes1) and periplasmic
isolates (lanes2) from IPTG-induced hECP#2
transfectants. The results indicate that these mutations do not alter
the production of rECP nor do they inhibit its transfer to the
periplasmic space. In panelA, the ribonuclease
activity of periplasmic isolates containing the mutant rECP (hECP#2) is
examined; the periplasmic protein preparations were adjusted as
necessary so that each rECP (mutant or wild type) represented the same
proportion of total periplasmic protein. The ribonuclease activity
calculated for the preparation containing mutant rECP (hECP#2) was 0.40
OD/ng/min, which was indistinguishable from the rate determined for the
control isolate. The results indicate that, as anticipated, the two
base pair mutations introduced destroyed the ribonuclease activity of
rECP.
The toxicity of wild type rECP, ribonuclease-minus rECP, and
wild type rEDN for their respective host bacterial transfectants (E. coli) is examined in Fig. 4. Growth (A) ceased immediately upon addition of IPTG
to bacterial transfectants producing either wild type rECP (hECP#7, panelA) or the ribonuclease-minus mutant rECP
(hECP#2, panelB). The growth of bacteria transfected
with the vector alone (panelC) was unaffected by the
addition of IPTG. Interestingly, transfectants that produce rECP from
constructs that do not include a signal sequence do not transfer rECP
to the periplasm and exhibit no growth cessation in response to IPTG. (
)Growth of transfectants producing wild type rEDN (67%
amino acid sequence identity with ECP, panelD) also
remained unaffected by the addition of IPTG. In panelE, the Western blot probed with the M2 mAb contains total
cell extracts (lane1) and periplasmic isolates (lane2) from IPTG-induced hEDN#1 transfectants,
indicating that the production and distribution of rEDN is identical to
that shown for rECP; rEDN also has ribonuclease activity. (
)
Figure 4:
Growth of bacterial transfectant cultures. A, hECP#7 (wild type rECP); B, hECP#2 (Lys to Arg and His
to Asp); C, vector alone; D, hEDN#1 (rEDN). IPTG (0.1 mM) was added to cultures
in exponential phase growth at times indicated by the arrows.
Growth of the uninduced bacterial cultures is indicated by opensymbols; growth of the IPTG-induced bacterial cultures is
indicated by filledsymbols. E, Western blot
probed with M2 mAb containing a total cell extract (lane1) and periplasmic isolate (lane2)
from the IPTG-induced hEDN#1 transfectants.
The toxicity of wild type rECP, mutant rECP, and wild type rEDN introduced externally to S. aureus strain 502A is examined in Fig. 5. After an overnight incubation with 1 µM wild type rECP (hECP#7, lightlyshadedbar), only 33% of the colony-forming units remained when compared with the control cultures. In contrast, the presence of rEDN (hEDN#1, blackbars) had little to no effect on bacterial survival (90-95% remaining after overnight incubation). These results are analogous to those determined for the granule-derived proteins shown in Fig. 1. The results obtained with mutant ribonuclease-minus rECP (hECP#2, deeplyshadedbars) were similar to those obtained with the wild type; 25% of the colony-forming units survived an overnight exposure to 1 µM mutant (ribonuclease-minus) rECP.
In this work, I have shown that micromolar quantities of granule-derived, purified ECP are toxic to both S. aureus 502A and E. coli HB101 bacterial strains, consistent with results initially described by Lehrer and colleagues(9) . I have extended these observations by demonstrating that purified EDN, at the same and higher concentrations, has no antibacterial activity. To examine further the relationship between ribonuclease activity and cytotoxicity, I have produced recombinant ECP in an inducible prokaryotic expression system and have shown that the rECP isolated from the bacterial periplasm has ribonuclease activity. I have shown that specific mutations introduced into the coding sequence of rECP eliminated its ribonuclease activity. Finally, I have examined the toxicity of these recombinant proteins for S. aureus strain 502A and determined that both wild type as well as ribonuclease-minus mutant forms of rECP are toxic for these bacteria, while rEDN had little to no effect. The results of this work suggest that ribonuclease activity and cytotoxicity are, in this case, independent activities of ECP.
The method devised for the production of biologically active rECP is based on the observation that secretory proteins from both prokaryotes and eukaryotes are synthesized with amino-terminal hydrophobic signal sequences; these sequences facilitate transfer of nascent polypeptides across cellular membranes(35) . As the results presented here and elsewhere (36, 37) indicate, the secretory apparatus of E. coli can recognize and process signal sequences encoded by eukaryotic cDNAs. The other addition to the coding sequence was the inclusion of the peptide FLAG (DYKDDDDK) at the carboxyl terminus of the rECP. My results indicated that FLAG peptide aided in the detection of small amounts of rECP and did not interfere with its transport, folding, or function.
ECP is an extremely cationic protein; it has 19 arginine residues (14% of the total amino acids) and a calculated isoelectric point of 10.8 (20) . Cationic protein and peptide toxins are employed as agents of host defense at many levels of the evolutionary spectrum, and they are remarkable as a group for their diversity of sequence and mechanisms of action(38) . ECP is somewhat unique in that it also has an enzymatic activity whose role in the cytotoxic activities of ECP is unclear; while one study (28) suggested that the neurotoxicity of ECP requires the ribonuclease activity, another study (8) reported the opposite for ECP and its anti-parasitic activity. Both of the aforementioned studies utilized exogenous inhibitors (alkylation, RNasin), which may alter protein conformation and/or availability. By introducing specific single base pair mutations, the ribonuclease activity of rECP could be eliminated without significant structural change. The elimination of ribonuclease activity did not result in the elimination of toxicity, suggesting that these two activities of ECP are not inextricably linked. This finding is analogous to that reported for the neutrophil primary granule protein, cathepsin G, whose function as an antibacterial agent was shown to be independent of its serine protease activity(29, 39) . The anti-parasitic activity and neurotoxicity of rECP and its ribonuclease-minus mutant forms remain to be tested.
The observation that the cytotoxicity of ECP was not necessarily linked to its ribonuclease activity is somewhat peculiar from an evolutionary perspective. If the ribonuclease activity perse is unnecessary for function, what then is the evolutionary pressure permitting the strong homology to the ribonuclease gene family to be retained? It is possible that the pressure to maintain this homology is not directed toward the retention of the catalytic activity but rather toward the retention of the disulfide bond structure that is unique to this gene family. Interestingly, ECP has significantly less ribonuclease activity against standard substrates than either EDN or ribonuclease A(22, 23) . The toxicity of ECP may depend on the three-dimensional configuration of its cationic residues as determined by the unique disulfide bond structure. The experimental system presented here will permit direct testing of this hypothesis.
Alternatively, the ribonuclease activity of ECP may be necessary for some functions but not for others, and ECP may have physiological functions unrelated to cytotoxicity that do depend on its ribonuclease activity. ECP may have multiple functional domains. Large scale production of recombinant ECP will facilitate the investigation of these possibilities.