Proc. Natd. Acad. Sci. USA Vol. 86, pp. 4460-4464, June 1989 Biochemistry Molecular of the human -derived neurotoxin: A member of the ribonuclease family (/cationic proteins/angiogenin) HELENE F. ROSENBERG*t, DANIEL G. TENENt, AND STEVEN J. ACKERMAN* Divisions of *Infectious Diseases and tHematology/Oncology, Department of Medicine, The Beth Israel Hospital and Harvard Medical School, Boston, MA 02215 Communicated by Seymour J. Klebanoff, March 30, 1989

ABSTRACT We have isolated a 725-base-pair cDNA clone sequence showed 67% identity with the amino-terminal se- for human eosinophil-derived neurotoxin (EDN). EDN is a quence ofthe related eosinophil protein, ECP, as well distinct cationic protein of the eosinophil's large specific gran- as 29% identity with the sequence of human pancreatic ule known primarily for its ability to induce ataxia, paralysis, ribonuclease (HPR), and was shown to be identical to the and central nervous system cellular degeneration in experi- amino-terminal sequences of both a nonsecretory ribonucle- mental animals (Gordon phenomenon). The open reading ase isolated from human urine (HNSR) (8) and human liver frame encodes a 134-amino acid mature polypeptide with a ribonuclease (HLR) (9). Slifman et al. (10) and Gullberg et al. molecular mass of 15.5 kDa and a 27-residue amino-terminal (11) have shown that both EDN and ECP have ribonuclease hydrophobic leader sequence. The sequence of the mature activity. The significance of this ribonuclease activity as well polypeptide is identical to that reported for human urinary as the physiologic role of EDN in eosinophil function remain ribonuclease [Beintema, J. J., Hofsteenge, J., Iwama, M., unknown. Morita, T., Ohgi, K., Irie, M., Sugiyama, R. H., Schieven, We report the isolation of a 725-base-pair (bp) cDNA clone G. L., Dekker, C. A. & Glitz, D. G. (1988) Biochemistry 27, for EDN and have found that the complete amino acid 4530-4538] and to the amino-terminal sequence of human liver sequence of EDN is identical to that of HNSR (8).§ Analysis ribonuclease [Sorrentino, S., Tucker, G. K. & Glitz, D. G. of EDN mRNA in various hematopoietic cells suggests that (1988) J. Biol. Chem. 263, 16125-16131]; the cDNA encodes a there may also be a protein in neutrophils that is identical or tryptophan in position 7, which was previously unidentified in very closely related to EDN. the amino acid sequences of EDN or the urinary and liver ribonucleases. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; se- METHODS quence similarities among EDN, eosinophil cationic protein, cDNA Library Screening. cDNA clones for EDN were ribonucleases from liver, urine, and pancreas, and angiogenin isolated from a Agtll library prepared from poly(A)+ RNA derme a ribonuclease multigene family. mRNA encoding EDN purified from the peripheral mononuclear cells of a patient was detected in uninduced HL-60 cells and was up-regulated in with eosinophilic (12). Screening was done with a cells induced toward eosinophilic differentiation with B-cell 17-base 32-fold-degenerate oligonucleotide (Fig. 1) synthe- growth factor 2/interleukin 5 and toward neutrophilic differ- sized on an Applied Biosystems model 381 DNA synthesizer entiation with dimethyl sulfoxide. EDN mRNA was detected in and radiolabeled on the 5' end using [y-32P]ATP and T4 mature neutrophils even though EDN-like neurotoxic activity is polynucleotide kinase (13) or on the 3' end with [a-32P]dCTP not found in extracts. These results suggest that and terminal deoxynucleotidyltransferase (International Bio- neutrophils contain a protein that is closely related or identical technologies). Prehybridization, hybridization, and washing to EDN. was done as described (15), except for a washing procedure that included two washes at room temperature in 6x SSC (1 x The human eosinophil granule contains a number of distinct, SSC = 0.015 M sodium citrate/0.15 M NaCl, pH 7.0) arginine-rich cationic proteins that have been purified and followed by a 1-min final wash in 6-x SSC at 37°C. Positive extensively characterized (1, 2). One ofthese, the eosinophil- recombinants were purified and the inserts were subcloned derived neurotoxin (EDN), is a glycosylated, low molecular into M13 phage for dideoxynucleotide sequencing (16). Se- mass protein (1, 3) found in the matrix of the eosinophil's quence evaluation was done with the assistance of BIONET large (4). Whereas other eosinophil granule- and DNASTAR (DNASTAR, Inc., Madison, WI) sequence derived proteins, such as eosinophil cationic protein (ECP) analysis software. and , display potent cytotoxic and hel- Purification of RNA from Peripheral Blood Cells. Peripheral minthotoxic activities (2), the only known biological activity blood obtained by leukapheresis of a patient of EDN is its ability to induce ataxia and paralysis and to with the hypereosinophilic syndrome were washed twice in damage myelinated neurons when injected intrathecally or cold Hanks' balanced salt solution (HBSS) and the erythro- intracerebrally into experimental animals (3, 5, 6) [the Gor- cytes were lysed by brief suspension in ice-cold lysis buffer don phenomenon (7)]. Human EDN purified from eosinophil (100 mM potassium carbonate/150 mM ammonium chloride/ granule extracts migrates as two bands with apparent mo- 0.1 mM EDTA, pH 7.2) followed by another wash with cold lecular masses of 18.6 and 20.1 kDa when analyzed by HBSS. The resuspended cell pellet was layered over a SDS/PAGE; digestion with endoglycosidase F results in a shift to a single band with a molecular mass of approximately Abbreviations: EDN, eosinophil-derived neurotoxin; ECP, eosino- 16 kDa (6). Fifty-three amino-terminal residues of purified phil cationic protein; HPR, human pancreatic ribonuclease; HNSR, EDN have been sequenced directly (6); the amino-terminal human non-secretory ribonuclease; HLR, human liver ribonu- clease; BCGF-2, B-cell growth factor 2; IL-5, interleukin 5; DMSO, dimethyl sulfoxide. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M24157).

4460 Downloaded by guest on September 23, 2021 Biochemistry: Rosenberg et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4461 HL-60 GCTGGATCAGTTCTCACAGGAGCTACAGCGCGGAGACTGGG CATG 50 subline of committed to eosinophilic differentiation MV -2 !6 (19) grown in the absence of inducing agents or in the presence of 10o (vol/vol) B-cell growth factor 2/interleukin 10 Io P K L F T S Q I C LL L L L G L L - 9 5 (BCGF-2/IL-5) (Cellular Products) for 72 hr. 10 Northern Blotting and Hybridization. Total RNA samples TGGCTGTGGAGGGCTCACTCCATGTCAAACCTCCACAGTTTACCTGGGCT 151 (10 ,ug), extracted and purified as described above, were A V E G S L H V k f f8 p p q t W a analyzed by agarose/formaldehyde gel electrophoresis, CAATGGTTTGAAACCCAGCACATCAATATGACCTCCCAGCAATGCACCAA 20(0 ethidium-stained to ensure equivalent RNA loading, and q w f e t q h i n m t s q q c t n 2!5 #73 blotted onto nylon membranes (ICN) in lOx SSC (13). The TGCAATGCAGGTCATTAACAATTATCAACGGCGATGCAAAAACCAAAATA 25(0 EDN cDNA probe (bases 171-725) was labeled with [a- a m q v i n n y q r r c k n q n t 4 2 32P]dCTP by the random hexamer priming method (20). CTTTCCTTCTTACAACTTTTGCTAACGTAGTTAATGTTTGTGGTAACCCA 3030 Filters were prehybridized for at least 4 hr in a solution f 1 1 t t f a n v v n v c g n p 538 containing Sx SSC, 50% (vol/vol) formamide, 5x Den- AATATGACCTGTCCTAGTAACAAAACTCGCAAAAATTGTCACCACAGTGG 35Sn hardt's solution (13), 0.05 M sodium phosphate (pH 6.5), 1% n m t c p s n k t r k n c h h s g 755 glycine, 0.1% SDS, and sheared denatured salmon sperm EI DNA (250 at 420C. Hybridization was done in a AAGCCAGGTGCCTTTAATCCACTGTAACCTCACAACTCCAAGTCCACAGA 40C0 ;Lg/,.l) S q v p 1 i h C n 1 t t p S p q n 92 solution containing 50% formamide, 5 x SSC, 1 x Denhardt's solution, 0.02 M sodium phosphate (pH 6.5), and denatured ATATTTCAAACTGCAGGTATGCGCAGACACCAGCAAACATGTTCTATATA 450C s n c r a t a n m f 108 radiolabeled probe (106 cpm/ml) at 420C for at least 12 hr (13). i y q p y i Hybridized filters were washed twice at room temperature in GTTGCATGTGACAACAGAGATCAACGACGAGACCCTCCACAGTATCCGGT 50O 2x SSC and once for 30 min at 55°C in 0.2x SSC; washed v a c d n r d q r r d p p q y p v 125 filters were exposed for 48 hr to Kodak XAR film with a single GGTTCCAGTTCACCTGGATAGAATCATCTAAGCTCCTGTATCAGCACTCC 550 intensifying screen at -80°C. RNA size standards (0.16-1.77 V p v h 1 d r i i 134 kilobases) were purchased from Bethesda Research Labora- TCATCATCACTCATCTGCCAAGCTCCTCAATCATAGCCAAGATCCCATCT 600 tories. CTCCATATACTTTGGGTATCAGCATCTGTCCTCATCAGTCTCCATACCCC 650 RESULTS Isolation and Sequencing of the cDNA Clone. Primary TTCAGCTTTCCTGAGCTGAAGTGCCTTGTGAACCCTGCAATAAACTGCTT 700 screening ofthe eosinophil leukemia Agtll cDNA library with the 32-fold-degenerate oligonucleotide 73 (Fig. 1) yielded a TGCAAATTCAAAAAAAAAAAAAAAA 725 554-bp cDNA clone (with the 5' end at nucleotide 171). Subsequent screening of400,000 recombinant phage with the FIG. 1. Nucleotide sequence ofthe 725-base coding strand ofthe EDN cDNA with translation of the open reading frame (161 amino cDNA isolate yielded 20 positive recombinants (frequency acids), potential N-linked glycosylation sites (underlined), and loca- 0.005%). Fig. 1 shows the nucleotide sequence of the cDNA tion of the 32-fold degenerate (oligonculeotide 73) 17-base oligonu- for EDN. Both coding and non-coding strands were se- cleotide probe used to isolate the cDNA are shown. The presumptive quenced to completion using four independently isolated start (ATG) and stop (TAA) codons are denoted with asterisks, the overlapping clones (a single clone containing nucleotides amino terminus (k, position 28) of the granule-derived protein is 1-725, and three initial clones, beginning at nucleotides 56, underlined, the Kozak-like initiation sequence (14) is boxed, and the 171, and 339) as templates. The complete cDNA clone 3' polyadenylylation signal (AATAAA) is double-underlined. The contains a Kozak-like initiation sequence at nucleotides 27-amino acid signal sequence is in bold-faced capital letters. The 43-49 (14), a polyadenylylation signal at nucleotides 689- single-letter amino acid code is used. 694, and a 16-base poly(A) tail. The longest open reading frame extends from nucleotide 46 through nucleotide 528, cushion of Ficoll/Hypaque (1.077 g/ml; Pharmacia) and encoding a total of 161 amino acids. The nucleotide sequence centrifuged at 400 x g for 30 min at room temperature. The confirms the identity of 52 of the 53 amino acid residues fraction found in the pellet (mature granulocytes) was sepa- determined by amino-terminal sequencing (6) and identified rated from that suspended in the Ficoll/Hypaque layer (hy- the amino acids missing from the amino-terminal sequence as podense granulocytes and monocytes) and washed in ice-cold tryptophan at position 7 and asparagine at position 17; the HBSS, and the RNA was extracted and purified by guanid- amino acid at position 54 that was previously reported as ium-isothiocyanate extraction followed by cesium chloride threonine is identified as valine. The amino-terminal residue density gradient centrifugation (17). RNA from discarded of the granule-extracted protein was identified as the lysine samples of normal bone marrow cells was purified as de- encoded by nucleotides 127-129 (6). The molecular mass of scribed, except without cell separation over Ficoll/Hypaque. the protein encoded from nucleotide 127 through nucleotide Neutrophil RNA was purified from peripheral blood neutro- 528 is 15.5 kDa, which is in agreement with the estimate (16 phils obtained from normal donors. Cells were washed and kDa) made for endoglycosidase F-treated EDN based on its separated over Ficoll/Hypaque as above; in each case, the mobility in SDS/PAGE (6). The amino acid sequence con- pellets contained less than 5% contaminating . tains five Asn-Xaa-Thr/Ser sequences, which have been Monocytes were likewise purified from peripheral blood from identified as sites ofpotential N-linked glycosylation (21-23). normal donors by Ficoll/Hypaque density centrifugation and The molecular mass ofglycosylated EDN has been estimated adherence to plastic. at 18.6 and 20.1 kDa (6); however, the distribution of the HL-60 Inductions. RNA was extracted as described above glycosyl groups among the five potential attachment sites is from cells of the promyelocytic leukemia HL-60 cell line not known. (ATCC CCL 240) grown in RPMI 1640 medium (supple- From nucleotide 46 through nucleotide 126, the open mented with 10% fetal bovine serum) both with and without reading frame encodes a 27-residue segment that was not inducing agents, which included 48 hr in 5 x 10-7 M vitamin previously identified in the amino-terminal sequence ofgran- D3 or 48 hr in 1.1% (vol/vol) dimethyl sulfoxide (DMSO). The ule-derived EDN. This segment is markedly hydrophobic; 20 monocytic or granulocytic induction of HL-60 cells was of the 27 residues are either hydrophobic or aliphatic, in- verified by morphology as well as mRNA and surface expres- cluding an uninterrupted segment of 10 sequential hydropho- sion of CD11b (18). RNA was also extracted from a cloned bic/aliphatic residues. Downloaded by guest on September 23, 2021 4462 Biochemistry: Rosenberg et al. Proc. Natl. Acad. Sci. USA 86 (1989)

Secondary structure was evaluated by the method of Chou En-:'M and Fasman (24); two regions (12% of total sequence) with 28S- a-helical potential were identified. The first potential helix .||,iRtE :..'. extends from leucine (position -16) through valine (position -7) within the amino-terminal hydrophobic segment de- 18S- scribed above. The second potential helix extends from :: 4 phenylalanine (position 5) through glutamine (position 14); the remainder ofthe protein is assigned an extended (73%) or 970bp_ 9.. /3-turn conformation (39%o). Sequence Comparisons. Fig. 2 shows the alignment of the cDNA-derived amino acid sequence of EDN [beginning at the lysine at position 1 of the amino terminus of the granule- extracted protein (6)] with the sequences of HNSR (8), ECP The com- FIG. 3. Northern blot analysis of mRNA for EDN. Total RNA (7), HLR (9), HPR (25), and angiogenin (26, 27). from hematopoietic cells and cell lines were probed with a 554-bp plete amino acid sequences of EDN and HNSR are identical; cDNA for EDN. HL-60 (ATCC CCL 240) cells grown in the absence the tryptophan residue encircled at position 7 of EDN, which ofinducing agents (lane 1); HL-60 cells grown for 48 hr with 5 x 10-7 was not identified in the amino acid sequence of HNSR (8), M vitamin D3 (lane 2) or for 48 hr with 1.1% DMSO (lane 3); cloned is, in all likelihood, also tryptophan in HNSR; the tryptophan HL-60 EOS subline committed to eosinophilic differentiation (19) residue at position 7 was also not identified in the amino- grown in the absence of inducing agents (lane 4) and for 48 hr with terminal sequence of HLR (9) or in the original amino- 10% (vol/vol) BCGF-2/IL-5 (lane 5); peripheral blood monocytes terminal sequence of EDN (6). Furthermore, the sequence of (lane 6); hypodense leukocytes (density, approximately 1.077 g/ml) 25 amino-terminal residues of HLR is identical to that of from a patient with the hypereosinophilic syndrome (68% eosino- 70% identity with phils, 30% neutrophils, and 2% mononuclear cells; lane 7); normal EDN/HNSR. The sequence ofEDN shows human bone marrow cells (lane 8); mature peripheral blood neutro- the 60 known amino-terminal residues of ECP. In contrast, phils (>95% neutrophils; lane 9); more normal density (>1.077 g/ml) EDN and HPR show 32% identity, and the homology be- granulocytes from a patient with the hypereosinophilic syndrome tween EDN and angiogenin is 29%. (73% eosinophils and 27% neutrophils; lane 10). The label 970 bp Northern Blot Analyses. The presence of mRNA encoding indicates the size estimate of the EDN mRNA as determined by EDN was assessed in a number of hematopoietic cells and comparison with RNA standards; the positions of the 28S and 18S cell lines. Fig. 3 shows a Northern blot analysis of various rRNA bands, visualized by ethidium bromide staining, are indicated. sources of RNA probed with a 554-bp cDNA for EDN. Comparison with RNA standards gives an estimate of the normal human bone marrow cells, presumably reflecting the molecular size of the EDN mRNA as 970 bases, compatible relatively small number of appropriate precursors. The most with a short untranslated region on the 5' side ofand a poly(A) surprising finding was the significant increase in EDN mRNA tail on the 3' side ofthe sequence of the 725-bp cDNA clone. detected in HL-60 cells induced toward neutrophilic differ- mRNA for EDN was detected in uninduced cells of the entiation with DMSO. An mRNA of identical size was also promyelocytic leukemia line, HL-60, and in cells of an detected in normal peripheral blood neutrophils (lane 9, uninduced cloned HL-60 subline committed to eosinophilic >95% neutrophils) and also in two additional neutrophil differentiation (19). EDN mRNA was down-regulated in preparations containing 97% and 98% neutrophils, 3% and HL-60 cells induced toward monocytic differentiation with 2% eosinophils, respectively, from normal donors (data not vitamin D3, and no EDN mRNA was detected in RNA from shown). The signal was less intense than that detected in purified peripheral blood monocytes. In contrast, there was RNA prepared from Ficoll/Hypaque fractions of hypodense a significant increase in EDN mRNA detected in the HL-60 peripheral blood eosinophil-rich granulocytes (lane 7; 68% eosinophilic subline induced toward eosinophilic differenti- eosinophils, 30% neutrophils, and 2% mononuclear cells) or ation with BCGF-2/IL-5. No EDN mRNA was detected in from peripheral blood eosinophil-rich granulocytes of more

Q C:

- _ D FIG. 2. Alignment of the amino acid sequences of EDN *I D [identical to HNSR (8)], HLR (9), ECP (7), HPR (25), and an- 2.'rD.N.-DD_ giogenin (ANG) (26, 27). Re- bPC"-K P V-TT L gions of conserved sequence are enclosed in boxes; conserved cysteine residues are heavily shaded, and the putative cata- ," Te ZI1C T T . r l Hr-7 m vw lytic histidine and lysine resi- dues are lightly shaded. The res- idue encircled at position 7 was not identified in the published amino acid sequence of HNSR (8), HLR (9), or EDN (6). - E T v o ~ Z v s P P st1 kE - El Dashes represent gaps intro- duced to permit alignment of cysteines and of the catalytic

L residues. Numbers above and to the right are as for the sequence of EDN. The single-letter amino acid code is used. Downloaded by guest on September 23, 2021 Biochemistry: Rosenberg et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4463

normal density (lane 10; 73% eosinophils and 27% neutro- by cloacin DF13, a 59-kDa plasmid-encoded bacteriocin that phils), both obtained from a patient with the hypereosino- also has ribonuclease activity. The cloacin DF13 protein philic syndrome. binds to an outer membrane receptor in sensitive bacterial strains (38-40) and inactivates protein synthesis by specific DISCUSSION cleavage of 16S rRNA near its 3' end (41-43). St. Clair et al. (44) have reported that angiogenin, another member of the EDN is one of several distinct cationic proteins found in the ribonuclease multigene family, abolishes protein synthesis in eosinophil's large specific granule and is known primarily for vitro by specific cleavage of 28S and 18S rRNA; it is possible its toxicity for myelinated neurons in association with the that EDN and/or ECP may participate in similar reactions. production of the Gordon phenomenon (3, 5-7, 11). Despite Molina et al. (45) reported that at high (10-5 M) concentra- its sequence similarity to another granule protein, ECP (6), tions, EDN killed trypomastigotes of Trypanosoma cruzi in EDN shares little to none of the cytotoxic or helminthotoxic vitro; the addition of ribonuclease inhibitor or excess RNA activities of ECP (2). Both EDN and ECP have ribonuclease inhibited EDN's toxicity. Young et al. (46) have shown that activity (10, 11) whose biological significance for eosinophil ECP can form channels in both artificial and cellular mem- function is unknown. Similarities among the amino-terminal branes; we hypothesize a mechanism for eosinophil- sequences of HPR, and granule-derived EDN and ECP were mediated cyto- and/or helminthotoxicity in which ECP re- first reported by Gleich et al. (6), who suggested that this leased from the eosinophil granule inserts itselfinto the target group formed a multigene family. HPR belongs to the secre- cell membrane and assists in the transfer of EDN from the tory group of mammalian ribonucleases, which are charac- extracellular to the intracellular space. EDN might then terized by alkaline pH optima and immunological crossreac- catalyze specific cleavage of rRNA and thereby halt cellular tivity (28); secretory ribonucleases have also been found in protein synthesis, similar to the mechanism described for kidney, stomach, and saliva (29-33). This group is immuno- angiogenin in vitro. logically distinct from the neutral, nonsecretory ribonucle- The process by which the eosinophil granule cationic ases, which have pH optima near 6.5 and have been found in spleen, lung, liver, and leukocytes (29-30, 33, 34). Our results proteins are synthesized, packaged in granules, and subse- show that the EDN polypeptide, until recently believed to be quently secreted is not completely understood. The cDNA eosinophil-specific, is identical to HNSR (8) and to the for EDN encodes a 27-residue sequence preceding the amino- known 25 amino-terminal residues of HLR (9). It is possible terminal lysine residue of granule-extracted protein, whose that nonsecretory ribonucleases from other tissues share hydrophobicity suggests a role in membrane translocation similar or identical polypeptide sequences. Akagi et al. (35, (47-49). Biosynthetic labeling studies have suggested that 36) describe a ribonuclease purified from unseparated human ECP, a related protein, may also be synthesized as a pre- leukocytes with properties similar to EDN and HNSR. Thus protein (50, 51). As this 27-amino acid segment is not found with our results from Northern blot analysis (see below) it is in EDN extracted from the eosinophil granule, its removal by possible that neutrophils contain a ribonucleolytic protein a signal peptidase may occur during granule formation; it is that is very similar or identical to EDN. Spleen ribonuclease, possible, however, that loss of the peptide is an artifact of however, has been distinguished from liver ribonuclease on granule extraction and that the hydrophobic sequence may be the basis of differing interactions with antisera prepared necessary for secretion of EDN. The EDN cDNA does not against HPR (33). code for an amino-terminal acidic segment such as that The complete amino acid sequences of EDN, HNSR, and described for another eosinophil granule cationic protein, the HPR, and the partial sequences of ECP and HLR show major basic protein (52, 53). conservation for all eight cysteine residues; angiogenin has mRNA encoding EDN was detected in uninduced cells of only six cysteines. By analogy with characterized ribonucle- the HL-60 promyelocytic leukemia cell line. A clonal subline ases (37), three-dimensional structure is likely maintained by of HL-60 committed to eosinophilic differentiation (19) disulfide bonds between cysteines at positions 23 and 83, 37 showed up-regulation of EDN mRNA when induced with and 96, 55 and 111, and 62 and 71 (numbered as if from the BCGF-2/IL-5. The biosynthesis of another eosinophil pro- EDN sequence in Fig. 2). There is also conservation of the tein, the Charcot-Leyden crystal protein, was likewise up- residues at the catalytic site: in alkaline pancreatic ribonu- regulated; the number of cells staining positively for the cleases, these are histidine-12, lysine-41, and histidine-119 Charcot-Leyden crystal protein by indirect immunofluores- (37). In HLR, the initial histidine is found at position 15, and, cence increased 7-fold over the 72-hr induction period in EDN/HNSR, the corresponding histidine residues are at (S.J.A., unpublished observations). The most surprising find- positions 15 and 129, and the lysine is at position 37. Gaps ing was the significant increase in EDN mRNA detected in were introduced in the sequences shown in Fig. 2 to permit the "wild-type" HL-60 cells induced toward neutrophilic alignment of the cysteine and catalytic histidine and lysine differentiation with DMSO. HL-60 cells grown in DMSO residues. As EDN, HNSR, and HLR have ribonuclease show none of the staining with alkaline Biebrich scarlet activity, there must be some compensating alteration of characteristic of cationic protein-containing eosinophils and tertiary structure bringing these residues into appropriate eosinophilic myelocytes (54, 55), and show a down-regulation conformation in the catalytic site; this conformational change of the mRNA for and synthesis of the eosinophil granule may be facilitated by a lower pH, which might explain the protein, major basic protein, in comparison with uninduced lower pH optimum for catalysis by this form of ribonuclease. HL-60 cells (53, 54). An mRNA of identical size was also Although EDN, HNSR, and probably HLR have identical detected in RNA from peripheral blood neutrophils. A ribo- amino acid sequences (and the liver might be the source ofthe nuclease that is immunologically cross-reactive with HNSR enzyme found in urine), it is not known whether they share has been isolated from unseparated human leukocytes from a common function. They all may function as nonspecific both normal and leukemic donors. Similar to EDN, this ribonucleases for the degradation of extracellular ribosomal ribonuclease has a neutral pH optimum (pH 6.5), is highly material, or their ribonucleolytic activity might be modulated cationic (pI > 11.0), and has a molecular mass estimated at by other, possibly tissue-specific proteins directing them 15 kDa after carbohydrate removal (35). This neutrophil toward more specialized ribonucleolytic functions. The re- ribonuclease might show significant amino acid sequence lationship between the ribonuclease and cytotoxic activities similarity to EDN; however, neutrophil extracts injected of EDN and ECP is not known. An intriguing possibility is intracerebrally into rabbits did not produce the characteristic that the cytotoxic mechanism is analogous to that mediated ataxia, paralysis, or central nervous system cellular degen- Downloaded by guest on September 23, 2021 4464 Biochemistry: Rosenberg et al. Proc. Nad. Acad. Sci. USA 86 (1989) eration produced by eosinophil extracts or by purified EDN 20. Feinberg, A. & Vogelstein, B. (1983) Anal. Biochem. 137, (5). 266-267. We have recently isolated and sequenced a full-length 21. Pless, D. D. & Lennarz, W. J. (1977) Proc. Natl. Acad. Sci. cDNA clone for ECP USA 74, 134-138. (56). Comparison of the two complete 22. Kronquist, K. E. & Lennarz, W. J. (1978) J. Supramol. Struct. amino acid sequences and application of site-specific muta- 8, 51-65. genesis techniques will hopefully permit identification of the 23. Hart, G. W., Brew, K., Grant, G. A., Bradshaw, R. A. & specific amino acid sequence(s) present in ECP but absent in Lennarz, W. J. (1979) J. Biol. Chem. 254, 9747-9753. EDN (and vice versa) that mediate the different biological 24. Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 222-245. activities of these proteins. Results from such experiments 25. Beintema, J. J., Weitzes, P., Weickmann, J. L. & Glitz, D. G. also whether ribonuclease (1984) Anal. Biochem. 136, 48-64. might suggest and/or perforin 26. Strydom, D. J., Fett, J. W., Lobb, R. R., Alderman, E. M., activity is or is not essential to mechanism(s) underlying Bethune, J. L. & Vallee, B. L. (1985) Biochemistry 24, 5486- neurotoxic, cytotoxic, and helminthotoxic activities of ECP 5494. and EDN. 27. Kurachi, K., Davie, E. W., Strydom, D. J., Riordan, J. F. & Vallee, B. L. (1985) Biochemistry 24, 5494-5499. We thank Drs. J. D. Griffin and J. Moore for the gift of the 28. Sierakowsa, H. & Shugar, D. (1977) Prog. Nucleic Acid Res. cryopreserved eosinophil leukemia cells. We also thank Dr. P. F. Mol. Biol. 20, 59-130. Weller for his continuing interest and support of this work and for 29. Morita, T., Niwata, Y., Ohgi, K., Ogawa, M. & Irie, M. (1986) making available granulocytes from patients with the hypereosino- J. Biochem. 99, 17-25. philic syndrome and Dr. D. Golde for providing us with the cloned 30. Ohta, T., Ogawa, M., Kurihara, M., Kitahara, T. & Kosaki, G. HL-60 subline. We are to Dr. A. Rosmarin (1982) Clin. Chim. Acta 124, 51-62. eosinophilic grateful for 31. Weickmann, J. L., Elson, M. S. & Glitz, D. G. (1981) Bio- his generous gift of induced and uninduced HL-60 RNA and to Dr. chemistry 20, 1272-1278. T. Ernst, Dr. R. Jack, and E. Newman for graciously providing us 32. Kurihara, M., Ogawa, M., Ohta, T., Kurokawa, E., Kitahara, with RNA. We also thank Drs. Linda Clayton and T., Morata, A., Matsuda, K., Kosaki, G., Watanabe, T. & Michael P. Bodger for careful reading of the manuscript; our thanks Wada, H. (1984) Cancer Res. 44, 2240-2243. as well go to M. C. Lavigne and S. E. Corrette for skilled technical 33. Weickmann, J. L. & Glitz, D. G. (1982) J. Biol. Chem. 257, assistance, and to Dr. M. L. Huang for many helpful discussions. 8705-8710. D.G.T. is a Special Fellow of the Leukemia Society ofAmerica. This 34. Niwata, Y., Ohgi, K., Sanda, A., Takizawa, Y. & Irie, M. work was supported by Grants A125230 and A122660 (to S.J.A.), (1985) J. Biochem. 97, 923-934. Grant CA41456 (to D.G.T.), and Grant RR01865-05 (to BIONET) 35. Akagi, K., Yamanaka, M., Murai, K. & Omae, T. (1978) from the National Institutes of Health. Cancer Res. 38, 2163-2167. 36. Akagi, K., Yamanaka, M., Murai, K., Niho, Y. & Omae, T. 1. Ackerman, S. J., Loegering, D. A., Venge, P., Olsson, I., (1978) Cancer Res. 38, 2168-2173. Harley, J. B., Fauci, A. S. & Gleich, G. J. (1983) J. Immunol. 37. Blackburn, P. & Moore, S. (1982) in The Enzymes, ed. Boyer, 131, 2977-2982. P. (Academic, New York), 3rd Ed., Vol. 15, pp. 317-358. 2. Gleich, G. J. & Adolphson, C. R. (1986) Adv. Immunol. 39, 38. De Graaf, F. K., Spanjaerdt-Speckman, E. A. & Stouthamer, 177-253. A. H. (1969) Antonie van Leeuwenhoek (J. Microbiol. Serol.) 3. D. S. 35, 287-290. Durack, T., Ackerman, J., Loegering, D. A. & Gleich, 39. Oudega, B., Klaasen-Boor, P., Sneeuwloper, G. & De Graaf, G. J. (1981) Proc. Natl. Acad. Sci. USA 78, 5165-5169. F. K. (1977) Eur. J. Biochem. 78, 445-453. 4. Peters, M. S., Rodrigues, M. & Gleich, G. J. (1986) Lab. 40. Oudega, B., van derMolen, J., vander Eb, J. & De Graaf, F. K. Invest. 54, 656-662. (1979) J. Bacteriol. 140, 964-970. 5. Durack, D. T., Sumi, S. M. & Klebanoff, S. J. (1979) Proc. 41. De Graaf, F. K., Niekus, H. G. D. & Klootwijk, J. (1973) Natl. Acad. Sci. USA 76, 1443-1447. FEBS Lett. 35, 161-165. 6. Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., 42. Oudega, B. & De Graaf, F. K. (1976) Biochim. Biophys. Acta Ackerman, S. J. & McKean, D. J. (1986) Proc. Natl. Acad. Sci. 392, 184-195. USA 83, 3146-3150. 43. De Graaf, F. K. & Klaassen-Boor, P. (1977) Eur. J. Biochem. 7. Gordon, M. H. (1933) Br. Med. J. 1, 641-647. 73, 107-114. 8. Beintema, J. J., Hofsteenge, J., Iwama, M., Morita, T., Ohgi, 44. St. Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. K., Irie, M., Sugiyama, R. H., Schieven, G. L., Dekker, C. A. (1988) Biochemistry 27, 7263-7271. & Glitz, D. G. (1988) Biochemistry 27, 4530-4538. 45. Molina, H. A., Kierszenbaum, F., Hamann, K. J. & Gleich, 9. Sorrentino, S., Tucker, G. K. & Glitz, D. G. (1988) J. Biol. G. J. (1988) Am. J. Trop. Med. 38, 327-334. Chem. 263, 16125-16131. 46. Young, J. D., Peterson, C. G., Venge, P. & Cohn, Z. A. (1986) 10. Slifman, N. R., Loegering, D. A., McKean, D. J. & Gleich, Nature (London) 321, 613-616. G. J. (1986) J. Immunol. 137, 2913-2917. 47. Blobel, G. & Sabatini, D. D. (1971) in Biomembranes, ed. 11. Gullberg, U., Widegren, B., Arivason, U., Egesten, A. & Manson, L. A. (Plenum, New York), Vol. 2, pp. 193-195. Olsson, I. (1986) Biochem. Biophys. Res. Commun. 139, 1239- 48. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67 835-851. 1242. 49. Milstein, C., Brownlee, G. G., Harrison, T. M. & Mathews, 12. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. M. B. (1972) Nature (London) New Biol. 239, 117-120. 13. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 50. Olsson, I., Persson, A. M. & Winqvist, I. (1986) Blood 67, Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold 498-503. Spring Harbor, NY). 51. Tai, P. C., Spry, C. J. F., Peterson, C., Venge, P. & Olsson, I. 14. Kozak, M. (1986) Cell 44, 283-292. (1984) Nature (London) 309, 182-184. 15. Wallace, R. B. & Miyada, C. G. (1987) Methods Enzymol. 152, 52. Barker, R. L., Gleich, G. J. & Pease, L. R. (1988) J. Exp. Med. 432-442. 168, 1493-1498. 16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. 53. McGrogan, M., Simonsen, C., Scott, R., Grittith, J., Ellis, N., Acad. Sci. USA 74, 5463-5467. Kennedy, J., Campanelli, D., Nathan, C. & Gabay, J. (1988) J. 17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, Exp. Med. 168, 2295-2308. W. J. (1979) Biochemistry 18, 5294-5299. 54. Fischkoff, S. A., Pollak, A., Gleich, G. J., Testa, J., Misawa, 18. Rosmarin, A. G., Weil, S., Rosner, G., Griffin, J. D., Arnaout, S. & Reber, T. (1984) J. Exp. Med. 160, 179-196. M. A. & Tenen, D. G. (1989) Blood 73, 13-21. 55. Collins, S. J. (1987) Blood 70, 1233-1244. 19. Tomonaga, M., Gasson, J. C., Quan, S. G. & Golde, D. W. 56. Rosenberg, H. F., Ackerman, S. J. & Tenen, 0. G. (1989) J. (1986) Blood 67, 1433-1441. Exp. Med., in press. Downloaded by guest on September 23, 2021