©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Human Eosinophil Cationic Protein
RIBONUCLEASE ACTIVITY IS NOT ESSENTIAL FOR CYTOTOXICITY (*)

(Received for publication, May 31, 1994; and in revised form, January 4, 1995)

Helene F. Rosenberg (§)

From the Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Eosinophil cationic protein (ECP) (^1)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.


EXPERIMENTAL PROCEDURES

Antibacterial Assay with Granule-derived Proteins

ECP and EDN were purified from eosinophil granules by size selection and heparin-Sepharose chromatography as described (19) . Antibacterial assay was performed essentially as described by Lehrer and colleagues(9) . Overnight cultures of Escherichia coli strain HB101 and Staphylococcus aureus strain 502A (ATCC) were washed twice and resuspended at 1:100 or 1:1000 in 10 mM sodium phosphate, pH 7.5. 20 µl of bacterial suspension were added to solutions containing ECP or EDN in phosphate buffer at the final concentrations indicated in Fig. 1and incubated for 5 h at 37 °C. The CFU/ml remaining were determined by plating serial 10-fold dilutions of the treated bacterial suspensions in triplicate on Luria-Bertani (LB) agar, followed by overnight growth at 37 °C.


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%.



Preparation of Plasmid Constructs

A diagram of the rECP constructs used in this work is shown in Fig. 2, panelA. The hECP#7 construct (wild type ECP) contains the complete 479-base pair open reading frame generated by polymerase chain reaction (PCR) from the full-length cDNA template (20) and ligated in-frame into the HindIII/EcoRI cloning sites in the pFLAG-CTC bacterial expression vector (International Biotechnologies, Inc., New Haven, CT). The rEDN construct (hEDN#1) was also generated by in-frame ligation of the open reading frame generated by PCR from its full-length cDNA template(30) . The hECP#2 construct has a single base pair mutation (Cys to Gly), which results in a conversion of histidine 128 to aspartate, which was introduced via a 3`-5` ECP-specific PCR primer, as well as a single base pair mutation (Ala to Gly), which results in a conversion of lysine 38 to arginine. This latter mutation was introduced by inclusion of this base pair change in both 5`-3` and complementary 3`-5` primer extending from bases 220-255 and 255-220, respectively. Two overlapping portions of the open reading frame of ECP were created by PCR; after cleavage at a natural ClaI site and ligation, the desired product was amplified and ligated into the HindIII-EcoRI cloning sites of the pFLAG-CTC expression vector as described. All constructs were confirmed by DNA sequencing.


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.



Protein Preparations

5 ml of overnight culture of each bacterial transfectant described above were inoculated into 3 liters of Terrific Broth (31) with 50 µg/ml ampicillin, which was then grown overnight at 37 °C. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) (Bio-Rad) was added to 1 mM (or 0.1 mM) final concentration, and bacteria were harvested after a 30-min induction period. SDS-PAGE and Western blot analysis of proteins were performed on pellets from 1-ml samples of bacterial culture resuspended directly in 2 times reducing sample buffer(32) .

Amino-terminal Sequencing

The cell pellet from 3 liters of IPTG-induced hECP#7 bacterial transfectants was snap-frozen at -80 °C. The pelleted material was resuspended in phosphate-buffered saline without calcium or magnesium (PBS) with 1 mM phenylmethylsulfonyl fluoride (Sigma) and sonicated with a Branson model 450 sonifier. After removal of debris by centrifugation, the clarified supernatant was equilibrated for 12 h with pre-washed M2 anti-FLAG peptide mAb resin (IBI). The equilibrated resin was washed with 100 volumes of PBS, and specifically bound material was eluted with 100 mM glycine, pH 3.0, and neutralized immediately with 2 M Tris, pH 8.0. The sample was concentrated, subjected to SDS-PAGE, and transferred to an Immobilon P membrane as per manufacturer's instructions (Millipore). The 19-kDa immunoreactive band was located by Coomassie Blue staining, and the band was cut from the membrane for amino-terminal sequencing (performed at National Biological Resources Branch of NIAID, National Institutes of Health).

Periplasmic Isolates

The cell pellet from 3 liters of IPTG-induced bacterial transfectants was washed twice with 10 mM Tris, pH 8.0, at room temperature followed by one wash with 0.5 M sucrose in 30 mM Tris, pH 8.0, and 1 mM EDTA. The sucrose-washed pellet was resuspended in 20 ml of ice-cold distilled water, and the cellular material remaining was pelleted by centrifugation. Tris, pH 8.0, and sodium azide were added to the clarified supernatant to concentrations of 10 mM and 0.1%, respectively. The buffered supernatant was equilibrated for at least 4 h at 4 °C with heparin-Sepharose CL-6B (Pharmacia Biotech Inc.). The equilibrated resin was washed with 300 volumes of 10 mM Tris, pH 8.0; the bound proteins were either eluted directly into 2 times reducing sample buffer (for gel analysis) or were eluted into 0.3 ml of 10 mM sodium phosphate, pH 7.5, and 500 mM sodium chloride (for ribonuclease assay). The concentration of periplasmic proteins eluted into the phosphate buffer was determined by BCA protein assay (Pierce) with spectrophotometric comparison (562 nm) to bovine serum albumin standards. Quantitation was confirmed by gel electrophoresis followed by silver staining (Integrated Separation Systems, Natick, MA). Quantitation of recombinant proteins was performed by comparison of serial dilutions of the periplasmic isolates to serial dilutions of a FLAG-containing protein standard (recombinant bacterial alkaline phosphatase (rBAP) (IBI) by Western blotting with the M2 anti-FLAG monoclonal antibody (IBI) as described below.

SDS-PAGE and Western Blotting

The samples described in the text were subjected to gel electrophoresis on 14% Tris-glycine gels (Novel Experimental Technologies, San Diego, CA). Proteins were transferred to nitrocellulose membranes (Schliecher and Schuell) and probed with antibodies as per published procedures(32) . Briefly, nonspecific binding was blocked with 5% non-fat dry milk in T-TBS (50 mM Tris, pH 8.0, 150 mM sodium chloride, 0.05% Tween 20). The M2 anti-FLAG peptide mAb (IBI) was used at a 1:200 dilution (14.5 µg/ml) in T-TBS plus 1% gelatin. Secondary antibody, alkaline phosphatase-linked goat anti-mouse IgG, was used at a 1:1000 dilution in T-TBS plus gelatin. Blots were developed in TM (200 mM Tris, pH 9.5, plus 10 mM MgS0(4)) with 300 µg/ml nitro blue tetrazolium and 100 µg/ml 5-bromo-4-chloro-3-indolylphosphate.

Ribonuclease Assay

The assay used was adapted from the procedure described by Anfinsen and colleagues (33) and Slifman and colleagues(22) . To start a reaction, 5 µl (20 µg) of a 4 mg/ml solution of yeast tRNA (Sigma) was added to 0.8 ml of 40 mM sodium phosphate, pH 7.5, containing 3 µg of periplasmic proteins eluted from heparin-Sepharose as described. At the given time points, the reaction was stopped by addition of 0.5 ml of an ice-cold fresh solution of 20 mM lanthanum nitrate plus 3% perchloric acid. For the t = 0 control, the stop solution was added to the periplasmic proteins in phosphate buffer before the addition of the yeast tRNA. Stopped reactions were held on ice for at least 15 min, and the insoluble tRNA was removed by centrifugation for 5 min at 10,000 times g. Solubilized tRNA was determined as UV absorbance (260 nm) of the remaining supernatants, with the t = 0 control used as the blank. The solvent control included an equal volume of heparin-Sepharose elution buffer (phosphate plus sodium chloride as described above) without periplasmic proteins. All points shown are an average of triplicate samples with an error of ±5%.

Growth Curves

Overnight cultures of bacteria transfected with hECP#7 (wild type), hECP#2 (Lys to Arg and His to Asp), hEDN#1, or pFCTC vector alone were diluted 1:40 in LB broth (Biofluids, Inc., Rockville, MD) with 50 µg/ml ampicillin. Optical densities (600 nm, recorded on a Beckmann DU640 spectrophotometer) were recorded at t = 0 and hourly thereafter. When exponential phase growth was achieved (A between 0.15 and 0.45), 0.1 mM freshly prepared IPTG was added to one-half of the culture. Optical densities were recorded hourly thereafter for a minimum of 4 h.

Antibacterial Assay with Recombinant Proteins

5 liters of overnight cultures of bacterial transfectants (pFCTC (vector alone), hECP#7, hECP#2, or hEDN#1) in Terrific Broth with 50 µg/ml ampicillin were induced with IPTG (0.1-1 mM) and periplasmic isolates were prepared by osmotic shock as described above. PBS and sodium azide were added to the supernatants to final concentrations of 1 and 0.1%, respectively. 75 µl of M2-agarose (IBI) was added to each isolate, which was then rotated end-over-end at 4 °C overnight. M2-agarose with bound recombinant protein was washed with 200 volumes of cold PBS; washed resin was resuspended in an equal volume of fresh PBS. A small aliquot of each was evaluated by SDS-PAGE and Western blotting for the presence and quantitation of recombinant protein as described above and in Fig. 2, panelsE and F. S. aureus strain 502A was grown overnight, washed, and diluted 1:1000 as described above. 5 µl (5000 CFU) of bacteria were incubated with varying concentrations of resin-bound recombinant protein overnight at 37 °C. Serial dilutions of each protein-bacteria incubation were prepared and plated as described above, and colony-forming units remaining after each treatment were determined. The denominator of the fractions shown in Fig. 5was determined as the number of CFUs remaining after overnight incubation with resin that had been subjected to a sham isolation (equilibrated at 4 °C with periplasmic proteins from induced cultures transfected with the pFCTC vector alone). Bacterial survival was not compromised by the presence of increasing amounts of M2-agarose resin from the sham isolation, remaining at 95% (±5%). Each bar in Fig. 5was determined as the average of duplicate samples, with errorbars as shown.


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.




RESULTS

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. (^2)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. (^3)


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.


DISCUSSION

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Host Defenses, Bldg. 10, Rm. 11N104, NIAID, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-2877; Fax: 301-402-0789.

(^1)
The abbreviations used are: ECP, eosinophil cationic protein; rECP, recombinant eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; CFU, colony-forming unit; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; rBAP, recombinant bacterial alkaline phosphatase.

(^2)
H. F. Rosenberg, unpublished results.

(^3)
H. F. Rosenberg, manuscript in preparation.


ACKNOWLEDGEMENTS

I thank Dr. John Coligan, NBRB, NIAID, for the amino acid sequence data reported in this work and Dr. Steven Ackerman for providing the granule-derived proteins used in the initial experiments. I also thank Dr. John I. Gallin for helpful discussions and continuing support of my work.


REFERENCES

  1. Spry, C. J. F. (1988) Eosinophils: A Comprehensive Review and Guide to the Scientific and Medical Literature , Oxford University Press, Oxford
  2. Makino, S., and T. Fukuda. (1993) Eosinophils: Biological and Clinical Aspects , CRC Press, Boca Raton, FL
  3. Gleich G. J., Adolphson, C. R., and Leiferman, K. M. (1992) in Inflammation (Gallin, J. I., Goldstein, M. I., and Snyderman, R., eds) pp. 663-700, Raven Press, Ltd., New York
  4. Ackerman, S. J., Gleich, G. J., Loegering, D. A., Richardson, B. A., and Butterworth, A. E. (1985) Am. J. Trop. Med. Hyg. 34, 735-745 [Medline] [Order article via Infotrieve]
  5. McLaren, D. J., McKean, J. R., Olsson, I., Venge, P., and Kay, A. B. (1981) ParasiteImmunol. (Oxf.) 3, 359-373
  6. Yazdanbakhsh, M., Tai, P.-C., Spry, C. J., Gleich, G. J., and Roos, D. (1987) J. Immunol. 138, 3443-3447 [Abstract/Free Full Text]
  7. Hamann, K. J., Barker, R. L., Loegering, D. A., and Gleich, G. J. (1987) J. Parasitol. 73, 523-529 [Medline] [Order article via Infotrieve]
  8. Molina, H. A., Kierszenbaum, F., Hamann, K. J., and Gleich, G. J. (1988) Am. J. Trop. Hyg. Med. 38, 327-334
  9. Lehrer, R. I., Szklarek, D., Barton, A., Ganz, T., and Hamann, K. J., and Gleich, G. J. (1989) J. Immunol. 142, 4428-4434 [Abstract/Free Full Text]
  10. Venge, P., Stromberg, A., Braconier, J. H., Roxin, L. E., and Olsson, I. (1978) Br. J. Haematol. 38, 475-483 [Medline] [Order article via Infotrieve]
  11. Fauci, A. S., Harley, J. B., Roberts, W. C., Ferrans, V. J., Gralnick, H. R., and Bjornson, B. H. (1982) Ann. Intern. Med. 97, 78-92 [Medline] [Order article via Infotrieve]
  12. Spry, C. J., Davies, J., and Tai, P.-C. (1983) Contrib. Microbiol. Immunol. 7, 212-217 [Medline] [Order article via Infotrieve]
  13. Spry, C. J. (1986) Postgrad. Med. J. 62, 609-613 [Medline] [Order article via Infotrieve]
  14. Parrillo, J. E., Borer, S., Henry, W. L., Wolff, S. M., and Fauci, A. S. (1979) Am. J. Med. 67, 572-582 [Medline] [Order article via Infotrieve]
  15. Tai, P.-C., Ackerman, S. J., Spry, C. J., Dunnette, S., Olsen, E. G., and Gleich, G. J. (1987) Lancet 1, 643-647 [Medline] [Order article via Infotrieve]
  16. Molina, H. A., and Kierszenbaum, F. (1989) Immunology 66, 289-295 [Medline] [Order article via Infotrieve]
  17. Motojima, S., Frigas, E., Loegering, D. A., and Gleich, G. J. (1989) Am. Rev. Respir. Dis. 139, 801-805 [Medline] [Order article via Infotrieve]
  18. Young, J. D., Peterson, C. G., Venge, P., and Cohn, Z. A. (1986) Nature 321, 613-616 [Medline] [Order article via Infotrieve]
  19. Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., Ackerman, S. J., and McKean, D. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3146-3150 [Abstract]
  20. Rosenberg, H. F., Ackerman, S. J., and Tenen, D. G. (1989) J. Exp. Med. 170, 163-176 [Abstract]
  21. Barker, R. L., Loegering, D. A., Ten, R. M., Hamann, K. J., Pease, L. R., and Gleich, G. J. (1989) J. Immunol. 143, 952-955 [Abstract/Free Full Text]
  22. Slifman, N. R., Loegering, D. A., McKean, D. J., and Gleich, G. J. (1986) J. Immunol. 137, 2913-2917 [Abstract/Free Full Text]
  23. Gullberg, U., Widegren, B., Arnason, U., Egesten, A., and Olsson, I. (1986) Biochem. Biophys Res. Commun. 139, 1239-1242 [Medline] [Order article via Infotrieve]
  24. Durack, D. T., Ackerman, S. J., Loegering, D. A., and Gleich, G. J. (1981) Proc. Natl. Acad. Sci U. S. A. 78, 5165-5169 [Abstract]
  25. Fredens, K., Dahl, R., and Venge, P. (1982) J. Allergy Clin. Immunol. 70, 361-366 [Medline] [Order article via Infotrieve]
  26. Ardelt, W., Mikulski, S. M., and Shogen, K. (1991) J. Biol. Chem. 266, 245-251 [Abstract/Free Full Text]
  27. Newton, D. L., Walbridge, S., Mikulski, S. M., Ardelt, W., Shogen, K., Ackerman, S. J., Rybak, S. M., and Youle, R. J. (1994) J. Neurosci. 14, 538-544 [Abstract]
  28. Sorrentino, S., Glitz, D. G., Hamann, K. J., Loegering, D. A., Checkel, J. L., and Gleich, G. J. (1992) J. Biol. Chem. 267, 14859-14865 [Abstract/Free Full Text]
  29. Shafer, W. M., Pohl, J., Onunka, V. C., Bangalore, N., and Travis, J. (1991) J. Biol. Chem. 266, 112-116 [Abstract/Free Full Text]
  30. Rosenberg, H. F., Tenen, D. G., and Ackerman, S. J. (1989) Proc. Natl. Acad. Sci., U. S. A. 86, 4460-4464 [Abstract]
  31. Sambrook, J., Fritsch, E. F., and Maniatis, R. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  32. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith J. A., and Struhl, K. (1991) Current Protocols in Molecular Biology , Vol. 2, pp. 10.1-10.4, John Wiley, Inc., New York
  33. Anfinsen, C. B., Redfield, R. R., Choate, W. L., Page, J., and Carroll, W. R. (1954) J. Biol. Chem. 207, 201-210 [Free Full Text]
  34. Blackburn, P., and Moore, S. (1982) in The Enyzmes (Boyer, P. D., ed) 3rd Ed., pp. 317-433, Academic Press, New York
  35. Blobel, G., Walter, P., Chang, C. N., Goldman, B. M., Erickson, A. H., and Lingappa, V. R. (1979) Symp. Soc. Exp. Biol. 33, 9-36 [Medline] [Order article via Infotrieve]
  36. Gray, G. L., Baldridge, J. S., McKeown, K. S., Heyneker, H. L., and Chang, C. N. (1985) Gene (Amst.) 39, 247-254 [CrossRef][Medline] [Order article via Infotrieve]
  37. Schein, C. H., Boix, E., Haugg, M., Holliger, K. P., Hemmi, S., Frank, G., and Schwalbe, H. (1992) Biochem. J. 283, 137-144 [Medline] [Order article via Infotrieve]
  38. Gabay, J. E. (1994) Science 264, 373-374 [Medline] [Order article via Infotrieve]
  39. Bangalore, N, Travis, J., Onunka, V. C., Pohl, J., and Shafer, W. M. (1990) J. Biol. Chem. 265, 13584-13588 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.