Proc. Nati. Acad. Sci. USA Vol. 86, pp. 297-301, January 1989 Immunology Common acute lymphoblastic leukemia antigen (CALLA) is active neutral 24.11 ("enkephalinase"): Direct evidence by cDNA transfection analysis (metalloprotease/zinc/lymphocytes) MARGARET A. SHIPP*t, JAYANTHI VIJAYARAGHAVAN*, EMMETT V. SCHMIDT§, EMMA L. MASTELLER*, LUCIANO D'ADAMIO*, Louis B. HERSHt, AND ELLIS L. REINHERZ*t *Laboratory of Immunobiology, Dana-Farber Cancer Institute, tDepartment of Medicine, and §Howard Hughes Medical Institute and Department of Genetics, Harvard Medical School, Boston, MA; and tDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX Communicated by Arthur B. Pardee, October 10, 1988 (receivedfor review September 10, 1988)

ABSTRACT The common acute lymphoblastic leukemia To elucidate the primary structure of CALLA. we purified antigen (CALLA) is a 749- type H integral mem- the to homogeneity, obtained NH2-terminal sequence brane protein expressed by most acute lymphoblastic leuke- from both the intact protein and derived tryptic and Staphy- mias, certain other lymphoid malignancies with an immature lococcus aureus V8 , and isolated CALLA phenotpe, and normal lymphoid progenitors. A computer cDNAs from a Nalm-6 cell line AgtlO library by using re- search against the most recent GenBank release (no. 56) indi- dundant oligonucleotide probes (12). The CALLA cDNA cates that human CALLA cDNA encodes a protein nearly sequence predicts a 750-amino acid integral membrane pro- identical to the rat and rabbit neutral endopeptidase 24.11 tein with a single, 24-amino acid hydrophobic segment that ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopep- could function as both a transmembrane region and a signal tidase, which has been shown to inactivate a variety of peptide (12). The COOH-terminal 700 amino acids, including hormones including enkephalin, chemotactic peptide, sub- six potential N-linked glycosylation sites, compose the extra- stance , oxytocin, , and angiotensins cellular protein segment, whereas the 25 NH2-terminal amino P, acids remaining after cleavage of the initiation methionine I and II, had not been identified in lymphoid cells. To determine form the cytoplasmic tail (12). CALLA' cells contain whether CALLA cDNA derived from human acute lympho- CALLA transcripts of 2.7-5.7 kilobases (kb) with the major blastic leukemia cells (Nalm-6 cell line) encodes functional 5.7- and 3.7-kb mRNAs being preferentially expressed in neutral endopeptidase activity, we generated CALLA' stable specific cell types (12). Molecular cloning of CALLA and its transfectants in the CALLA- murine myeloma cell line J558 identification as a type II transmembrane glycoprotein do not and analyzed them for enzymatic activity in a fluorometric allow inference of its role in lymphoid function or differen- assay based upon cleavage of the substrate glutaryl-Ala-Ala- tiation. in this class have diverse functions ranging Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total from receptors to membrane-bound enzymes and include the lysates as well as whole-cell suspensions of the Nalm-6 line and transferrin receptor, the asialoglycoprotein receptor, influ- of the CALLA' transfectants, but not of the CALLA- J558 enza viral neuraminidase, y-glutamyl transpeptidase, prosu- cells, possessed neutral endopeptidase activity. This enzymatic crase-isomaltase complex, and the invariant chain of HLA activity was associated with the cellular membrane fraction and proteins (13). was abrogated by the specific neutral endopeptidase inhibitor In the present study, a computer search against the most . The unequivocal identification of CALLA as recent GenBank release (no. 56) uncovered the surprising a functional neutral endopeptidase provides insight into its finding that human CALLA has 94% identity at the ahiino potential role in both normal and malignant lymphoid function. acid sequence level with neutral endopeptidase 24.11 ("en- kephalinase;" EC 3.4.24.11), a membrane-bound zinc met- The common acute lymphoblastic leukemia antigen (CALLA) alloendopeptidase cloned from rat brain (14) and rabbit is a 100-kDa cell surface glycoprotein originally identified oh kidney (15). Herein we prove, by using CALLA-transfected human acute lymphoblastic leukemia cells (1, 2). Subsequent murine cell lines and a sensitive enzymatic assay in conjunc- studies indicated that CALLA was also expressed by certain tion with a specific neutral metalloendopeptidase inhibitor, other lymphoid malignancies with an immature phenotype (3) that CALLA is a functional form of this membrane-bound and by early lymphoid progenitors from fetal liver, fetal and enzyme. Our results raise the interesting possibility that this adult bone marrow, and thymus (3-7). These CALLA' cells enzyme could play a role in lymphoid cell development have the phenotypic characteristics of cells that are either and/or function. uncommitted to B- or T-cell lineage or committed to only the earliest stages of B-cell differentiation. The temporally re- MATERIALS AND METHODS stricted expression ofCALLA during lymphoid development suggests that the antigen may play a role at an early stage of CALLA Open Reading Frame Construct. In order to gen- lymphoid differentiation. However, CALLA has also been erate a CALLA cDNA containing the entire open reading detected on the surface of nonlymphoid cell types including frame [base pairs (bp) 12-2261], two recombinant AgtlO phage renal cells, peripheral blood granulocytes, bone marrow clones containing CALLA cDNA fragments that spanned bp stromal cells, and cultured fibroblast lines (8-11), implying -132 to +583 (clone 1.1) and bp + 127 to +3723 (clone 2) were that its biological function is not restricted to lymphoid utilized (Fig. 1). The numbering system for the two AgtlO development. clones is derived from ref. 12, in which bp 1 is located 11 bp 5' to the initiation methionine codon. DNA from clone 1.1 The publication costs of this article were defrayed in part by page charge was digested with. EcoRI and Ava I, yielding a 0.435-kb payment. This article must therefore be hereby marked "advertisement" fragment (Fig. L4). Aliquots of DNA from clone 2 were in accordance with 18 U.S.C. §1734 solely to indicate this fact. digested with Ava I and Cla I, yielding a 0.499-kb fragment, 297 Downloaded by guest on September 26, 2021 298 Immunology: Shipp et al. Proc. Natl. Acad. Sci. USA 86 (1989)

A

4.? (,9 9b B a aI If tATG I U~ X1.1 il pBS Mouse Ig Human Ig SV40 Intron Human Ig -132 -11 +12 303 583 Enhancer Promotor and Poly-A Enhancer

0.435 kb -

f, X2 /\ CMV-Neo for 127 303 583 621 802 1503 2222 12348 3723 G418 Selection 2321 /\ 0.499 kb CALLA cDNA Open 1.546 kb Reading Frame

FIG. 1. (A) Restriction map of the CALLA cDNA clones utilized in the pIGTE/N CALLAS construct. Clone 1.1 (bp -132 to +583) was digested with EcoRI and Ava I, yielding a 0.435-kb fragment. Aliquots of clone 2 (bp 127-3723) were digested with Ava I and Cla I, yielding a 0.499-kb fragment, and with Cla I and Apa I, yielding a 1.546-kb fragment. After ligation, the reconstructed CALLA cDNA fragment contained an intact open reading frame (bp 12-2261). The CALLA open reading frame fragment was excised from pBluescript SK(+) (not shown) with Dra I and Apa I and modified at the 5' and 3' ends to generate Sal I sites. (B) Diagram of the pIGTE/N CALLAS construct. The CALLA open reading frame fragment was ligated into the Xho I site of pIGTE/N. The pIGTE/N plasmid contains a portion of pBluescript SK(+) (pBS), the human immunoglobulin promoter, human and murine immunoglobulin enhancers, a simian virus 40 (SV40) intron and polyadenylylation signal, and a neomycin-resistance gene governed by the cytomegalovirus (CMY) promoter.

or with Cla I and Apa I, yielding a 1.546-kb fragment, respec- reading frame in the sense orientation (pIGTE/N CALLAs) tively (Fig. 1A). The plasmid vector pBluescript SK(+) was obtained. (Stratagene) was digested with EcoRI and Apa I. The Generation of CALLA' Cell Lines. The pIGTE/N CALLAS 0.435-kb EcoRI-Ava I, 0.499-kb Ava I-Cla I, and 1.546-kb construct was transfected into the CALLA- murine myeloma Cla I-Apa I CALLA cDNA fragments and the EcoRI/Apa cell line J558 by electroporation (17). In brief, 2 x 107 cells were I-digested plasmid vector were purified by gel electrophore- washed once in ice-cold phosphate-buffered saline (145 mM sis, ligated, and used to transform Escherichia coli DHSa+ NaCl/2.3 mM KH2PO4/7.7 mM K2HPO4) and resuspended in cells (Bethesda Research Laboratories). Recombinants were 500 ,ul of ice-cold phosphate-buffered saline containing 50 ,g of identified on the basis of blue/white color selection on LB pIGTE/N CALLAS. The cell/DNA mixture was transferred to plates containing ampicillin (100 ttg/ml), 5-bromo-4-chloro- an electroporation cuvette and, after 5 min on ice, electroporated 3-indolyl 8-D-galactoside (X-Gal, 80,4g/ml), and isopropyl at 2000 V. After a 10-min incubation at room temperature, cells fB-D-thiogalactoside (IPTG, 20 mM). Following a large-scale were diluted in 10 ml of RPMI-1640 medium supplemented with 10%o plasmid preparation, the reconstructed CALLA cDNA con- fetal bovine serum, 2 mM glutamine, penicillin (100 units/ ml), and streptomycin (100 ,ug/ml) and cultured for 48 hr at 37°C taining the intact open reading frame was excised from the in a 5% CO2 atmosphere. Thereafter, cells were maintained in plasmid vector by using Dra I and Apa I and blunted at its 3' RPMI-1640 supplemented as above but with the addition of end with phage T4 DNA polymerase. In order to generate a antibiotic G418 at 900 ,ug/ml for 14 days before assay for CALLA 5' Sal I site, the resulting 5' and 3' blunt-ended CALLA expression with the anti-CALLA monoclonal antibody J5 (2). cDNA fragment was ligated into EcoRV-digested pBluescript Subsequently, JS+ cells were selected by fluorescence-activated SK(+), which contains a Sal I site in the polylinker. After cell sorting (Epics V) using standard techniques (2) and transformation, recombinants were identified and analyzed cloned by limiting dilution at 0.5 cell per well in G418- for orientation by using a panel of diagnostic restriction containing medium. endonucleases. An appropriate clone was further analyzed Enzymatic Assay for Neutral Endopeptidase 24.11 Activity. by sequencing the 5' and 3' ends and the EcoRI-Ava I, Ava Neutral endopeptidase 24.11 activity was measured fluoro- I-Cla I, and Cla I-Apa I junctions of the CALLA insert by metrically in a coupled assay using glutaryl-Ala-Ala-Phe the dideoxy chain-termination method (16). 4-methoxy-2-naphthylamide (Enzyme Systems Products, In order to obtain the CALLA open reading frame with the Livermore, CA) as substrate (18). Cleavage of this substrate Sal I site from the polylinker, the fragment was excised with by neutral endopeptidase 24.11 yields Phe 4-methoxy-2- Sma I and Apa I, which cleaved at polylinker sites 3' of the naphthylamide, which, in the presence of aminopeptidase CALLA fragment and 5' of the CALLA fragment and Sal I activity, is converted to the fluorescent product 4-methoxy-2- site, respectively. The Apa I end was blunted with T4 DNA naphthylamine. Reaction mixtures contained 0.1 mM sub- polymerase and the modified CALLA open reading frame strate, 100 mM 2-(N-morpholino)ethanesulfonate (Mes) insert was ligated into EcoRV-cut pBluescript SK(+). After buffer at pH 6.5, 0.3 M NaCl, 0.5 milliunit ofpurified rat brain transformation, recombinants containing a resulting 3' Sal I aminopeptidase (19), and enzyme in a final volume of 100 ,u1. end from the polylinker were identified with diagnostic Sal I Reactions were initiated with enzyme and followed at 30°C at digestions. Following a large-scale plasmid preparation of a an excitation wavelength of 340 nm and an emission wave- representative clone, the CALLA open reading frame was length of 425 nm by using an Aminco-Bowman spectrofluo- excised with Sal I and ligated into the Xho I site of pIGTE/N rometer equipped with a thermostatted cell holder and strip (Fig. 1B). The pIGTE/N plasmid (E.V.S. and P. Leder, chart recorder. Inhibition by phosphoramidon (2 ,uM) was unpublished results) contains the human immunoglobulin used to confirm the specificity of the assay (20). Protein was promoter, both human and murine immunoglobulin enhanc- determined by the bicinchoninic acid (BCA) method (21). ers, a simian virus 40 intron and polyadenylylation signal, and Cell extracts were prepared by resuspending washed cells the neomycin-resistance gene driven by a cytomegalovirus in 200-400 ,ul of 20 mM Mes (pH 6.5) containing 1% (wt/vol) promoter. After transformation, recombinants were ana- n-octyl ,-D-glucopyranoside (Sigma). After a 1-hr incubation lyzed for orientation with a panel of diagnostic restriction at room temperature, the samples were centrifuged for 30 min endonucleases and a plasmid containing the CALLA open in an Eppendorf centrifuge and the supernatants were taken Downloaded by guest on September 26, 2021 Immunology: Shipp et al. Proc. Nati. Acad. Sci. USA 86 (1989) 299

for enzyme assays. Whole-cell suspensions were washed cell line Nalm-6 under the control of an immunoglobulin once in RPMI-1640 and then utilized at a concentration of promoter and enhancer and transfected it into the murine 103-104 cells per 100-pl reaction mixture. For preparation of myeloma cell line J558, which lacks cell surface CALLA the membrane fraction, washed cells were homogenized in 1 expression (see Fig. 1 and Materials and Methods). Follow- ml of Tris-buffered saline 0.2 M NaCl/50 mM Tris, pH 7.8) ing G418 selection, CALLA' J558 cells were identified by with a Teflon/glass homogenizer and the membrane fraction phenotyping with the JS anti-CALLA monoclonal antibody, was isolated by centrifugation at 40,000 x g for 30 min. This sorted, and cloned by limiting dilution. Two J5' subclones, fraction was resuspended in the Mes/octyl glucoside buffer A2-3 and A2-2, which had high and low levels of CALLA and treated as described above. expression, respectively, were chosen for further analysis. Computer Search. The 5508-bp CALLA cDNA sequence Fig. 2 contains a comparison ofrelative CALLA fluorescence (12) was utilized in a computer search of the updated Gen- of these two CALLA' stable transfectants, the parental Bank data base (release 56). The DASHER program (D. V. CALLA- multiple myeloma line J558, and the CALLA' Faulkner, Molecular Biology Computer Resource Center, acute lymphoblastic leukemia line from which the CALLA Dana-Farber Cancer Institute) was used to compare CALLA cDNA was isolated, Nalm-6. As indicated, J558 lacks de- cDNA segments of 600 bp, each with 100-bp overlaps, to tectable cell surface CALLA expression (mean channel 600-bp segments with 100 bp overlaps ofeach sequence in the fluorescence 0), whereas A2-3 and A2-2 express cell surface GenBank data base. CALLA. Note that A2-3 expresses a substantially higher number of CALLA sites per cell than does A2-2 (mean RESULTS AND DISCUSSION channel fluorescence 116.8 and 7.6, respectively). Compar- ison of the mean channel fluorescence of A2-3 and A2-2 with Virtual Identity of Human CALLA and Neutral Endopep- the mean channel fluorescence of Nalm-6 (172.6) indicates tidase 24.11 (Enkephalinase) by Sequence Analysis. Compar- that A2-3 expresses CALLA at a level 67.7% that of Nalm-6, ison of the CALLA cDNA sequence against the GenBank whereas A2-2 expresses CALLA at only 4.4% the level of sequence data base (release 56; June 1988) revealed striking Nalm-6. similarities with the rat and rabbit neutral endopeptidase Analysis of Neutral Endopeptidase Activity. To assess the 24.11, commonly referred to as enkephalinase (14, 15). neutral endopeptidase activity associated with the A2-3, Related segments of the CALLA cDNA and rat and rabbit A2-2, J558, and Nalm-6 lines, we used a sensitive fluoromet- neutral 24.11 (bp 1-600, 501-1100, 1001- ric assay based upon the cleavage of the substrate glutaryl- 1600, 1501-2100, and 2001-2600) had homology scores of Ala-Ala-Phe 4-methoxy-2-naphthylamide. When whole-cell 121-236. In the DASHER program, homology scores greater suspensions of the individual cell populations were assayed than 12 are thought to be of potential significance. Thus, the (Table 1), Nalm-6, A2-3, and A2-2 cells exhibited neutral high scores noted herein are indicative of near identity. endopeptidase activity of4789, 1906, and 446 nmol ofproduct Subsequent comparison of amino acid sequences of human per hr per 106 cells. The value for J558 cells, of 0.2 nmol per CALLA and rat and rabbit neutral endopeptidase molecules hr per 106 cells, is within the experimental error ofno activity. showed 94% identity in each case (12, 14, 15). Analysis of the Addition of the specific inhibitor phosphoramidon (20) to the recently reported human homologue (22) shows virtual iden- Nalm-6, A2-3, and A2-2 cell suspensions reduced the neutral tity with CALLA; the latter two sequences differ by one endopeptidase activity by factors of 90, 37, and 25, respec- amino acid representing either a sequencing error or a genetic tively (Table 1). Cell lysates (Table 2) from Nalm-6, A2-3, polymorphism. A2-2, and J558 contained neutral endopeptidase specific Neutral endopeptidase 24.11 is a cell-membrane-associ- activities of 9.96, 2.35, 1.18 and 0.08 nmol per min per mg of ated enzyme that cleaves peptide bonds on the amino side of protein, respectively. When the assays were performed in the hydrophobic amino acids (23). The enzyme was identified in presence of phosphoramidon, the neutral endopeptidase brain as an enkephalinase because it cleaved the Gly3-Phe4 bond of enkephalins (24). However, the enzyme was subse- Nalm6 J558 quently found in many other tissues, including kidney, where it was present in high levels (25). In kidney, enkephalinase activity was shown to be identical to that of neutral endopep- tidase 24.11, which had been identified several years earlier by using the B chain of as substrate (26, 27). Neutral endopeptidase 24.11 has been shown to react with a variety of physiologically active peptides including chemotactic Zzz A2-3 A2-2 peptide (28), substance P and neurotensin (29, 30), oxytocin (31), bradykinin, angiotensins I and 11 (32), and a variety of opioid peptides (33). This enzyme has also been shown to hydrolyze the lymphokine interleukin 1 (34). Neutral en- dopeptidase 24.11 has been found in numerous tissues other than kidney and brain, including peripheral blood granulo- Log ChannelFluorescence cytes (28), fibroblasts (35), small intestine (36), and placenta (22). However, lymphoid cells have not been shown to FIG. 2. Comparison of relative cell surface CALLA expression possess this enzymatic activity (37). Given that enkephali- on the Nalm-6, J558, A2-3, and A2-2 cell lines. The human CALLA' nase is a zinc-binding metalloendopeptidase, it is of interest acute lymphoblastic leukemia line Nalm-6, the CALLA- murine that the chromosomal location of the CALLA gene is 3q21- multiple myeloma line J558, and the two CALLA' stable transfec- 27, a region rich in genes encoding metal-bihding proteins, tants, A2-3 and A2-2, were phenotyped with the anti-CALLA including transferrin, lactotransferrin, melanotransferrin, the monoclonal antibody J5 (thick traces). Background fluorescence was and determined by phenotyping the cell lines with an anti-CD4 mono- transferrin receptor, ceruloplasmin (43). clonal antibody (19Thy5D7, ref. 38) (thin traces). The CALLA mean Expression of Human CALLA in Murine Transfectants. To channel fluorescence for each cell line was determined by subtracting determine whether CALLA derived from a lymphoblastic the mean channel fluorescence generated by staining with the leukemia cell line has functional neutral endopeptidase 24.11 anti-CD4 antibody from that generated by staining with the anti- activity, we engineered a construct (pIGTE/N CALLAS) CALLA antibody. Mean channel fluorescence values for Nalm-6, containing the CALLA open reading frame from the leukemic J558, A2-3, and A2-2 were 172.6, 0, 116.8, and 7.6, respectively. Downloaded by guest on September 26, 2021 300 Immunology: Shipp et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Neutral endopeptidase activity in whole-cell suspensions out the possibility that the CALLA substrate for lymphoid Specific activity, nmol per hr per 106 cells precursors is a previously defined peptide. Although neutral endopeptidase 24.11 has not previously Cell line - Phosphoramidon + Phosphoramidon been identified on lymphoid cells, the enzyme has been Nalm-6 4789 53 detected in nodal tissue (37). In porcine lymph nodes, the A2-3 1906 52 enzyme is found on a subpopulation ofadherent cells with the A2-2 446 18 morphological characteristics of fibroblasts (37). These cells J558 0.2 0.2 are most prevalent in medullary areas and are also found in the center of follicles and encircling them. Of interest, these activity associated with the Nalm-6, A2-3, and A2-2 cell neutral endopeptidase 24.11+ cells are observed to have lysates was reduced markedly (Table 2). The observation that clusters of lymphoid cells firmly attached to their cell surface the apparent activity in J558 cells is insensitive to phosphor- (37). In tonsil, spleen, thymus, and Peyer's patches, the this level activity is not neutral endopeptidase 24.11+ cells are present in a reticular amidon inhibition suggests that low of pattern similar to that seen in lymph nodes, where the due to neutral endopeptidase 24.11 and within the experi- enzyme is much more abundant (39). Recent studies mental error of no activity. Subcellular fractionation of the prompted by the identification of neutral endopeptidase Nalm-6 and A2-3 cells demonstrated that 99% of the Nalm-6 24.11+ reticular cells in lymphoid tissues indicate that the and 90% of the A2-3 neutral endopeptidase activity was enzyme inactivates interleukin 1 in vitro and inhibits thymo- associated with the membrane fraction (Table 2). Compari- cyte proliferation in a dose-dependent and specific fashion son of the levels of neutral endopeptidase activity (Tables 1 (34). and 2) and levels of CALLA expression of the four cell lines As noted above, CALLA/neutral endopeptidase 24.11 can (Fig. 1) indicated that there was a correlation between neutral cleave the chemotactic peptide fMet-Leu-Phe (28). The endopeptidase activity and cell surface CALLA expression. presence of CALLA/neutral endopeptidase 24.11 on the Implications. The observations (i) that the CALLA protein surface of mature neutrophils suggests that the enzyme may is found both on early normal lymphoid progenitors and on play an important role in the process of down-regulating their malignant counterparts and (ii) that CALLA cDNA chemotaxis, perhaps by reducing the local concentration of from an acute lymphoblastoid leukemia encodes neutral chemotactic peptides. Chronic treatment with morphine endopeptidase 24.11 (enkephalinase) activity indicate that induces a selective and specific increase in brain enkephalin- the enzyme functions at a critical stage in lymphoid differ- ase activity, likewise indicating that the enzyme may regulate entiation. This is of particular interest, given previous studies the local concentration of opioid neurotransmitters and that demonstrating that the cell-surface-bound enzyme has the the concentration of such neurotransmitters may also affect potential to mediate a wide range of biological activities in a enzyme levels (24). of tissues. For example, neutral endopeptidase 24.11 Earlier studies indicated that specific antibody treatment of variety CALLA' lymphoid cells resulted in rapid cell surface redis- has been shown to inactivate endogenous opioid pentapep- tribution, internalization, and degradation of the CALLA- tides on neurons in brain (23, 24), chemotactic peptide antibody complex (10, 40). The antibody-induced modulation (fMet-Leu-Phe) on polymorphonuclear granulocytes (28), of CALLA was noted to resemble the specific down- and a variety of regulatory peptides on the surface of regulation or loss ofcell surface receptors induced by peptide proximal tubule epithelial cells ofthe kidney (32). Amino acid hormones (41, 42). Given the fact that CALLA cDNA sequences of the enzyme derived from three tissue sources encodes functional lymphoid neutral endopeptidase 24.11, it (brain, kidney, and placenta) in three species as well as from is possible that its peptide ligand will also modulate cell a human lymphoblastic leukemia cell line are virtually iden- surface CALLA. This may result in efficient internalization tical (i2, 14, 15, 22); these results imply conservation of of ligand. Whether such a putative peptide affects migration, critical functional domains for zinc binding, substrate bind- growth, or other functional aspects of immature normal or ing, and catalysis. Furthermore, given the structural identity malignant B cells remains to be determined. However, the of the enzyme in various tissues, the biological activity of neutral CALLA/neutral endopeptidase 24.11 is likely to be dictated unequivocal identification of CALLA as functional by the availability of specific substrates in individual organs endopeptidase 24.11 (enkephalinase) and the availability of rather than by the presence of different functional forms of specific endopeptidase inhibitors should make it possible to the enzyme. The substrate for the cell surface CALLA/ assess the role of CALLA in lymphoid development. neutral endopeptidase 24.11 of early lymphoid progenitors is en- Note Added in Proof. The partial deduced amino acid sequence of not yet known. However, previous studies of neutral CALLA recently reported by LeTarte et al. (44) confirms our dopeptidase 24.11 indicate that optimal substrates for the previously reported sequence (12) and independently identifies the enzyme are small peptides rather than large proteins. Con- amino acid homology between CALLA and neutral endopeptidase. sequently, it is likely that CALLA/neutral endopeptidase 24.11 may also react with a small regulatory peptide at the cell We thank Dr. Frank Howard for assistance with the computer surface of lymphoid precursors. Such an action could lead to search. This work was supported in part by National Institutes of the inactivation of a physiologically active peptide or convert Health Grant RO1 CA49232 to E.L.R. and M.A.S. and by National an inactive form of a peptide to an active one. We cannot rule Institute on Drug Abuse Grant DA02243 and Welch Foundation Grant 1391 to L.B.H. M.A.S. was a recipient of a Clinical Investigator Table 2. Neutral endopeptidase activity in total cell lysates Award (KO8 CA01057) from the National Institutes of Health during Specific activity, nmol per min per mg % activity a portion ofthese studies. E.L.R. is a recipient ofan American Cancer Society Faculty Award. E.V.S. was supported by the Howard Hughes Cell of protein in membrane Medical Institute and E. I. Dupont de Nemours and Co. line - Phosphoramidon + Phosphoramidon fraction Nalm-6 9.96 0.2 99 1. Greaves, M. F., Brown, G., Rapson, N. T. & Lister, T. H. A2-3 2.35 0.08 90 (1975) Clin. Immunol. Immunopathol. 4, 67-84. A2-2 1.18 0.29 ND 2. Ritz, J., Pesando, J. M., Notis-McConarty, J., Lazarus, H. & Schlossman, S. F. (1980) Nature (London) 283, 583-585. J558 0.08 0.05 ND 3. Greaves, M. F., Hairi, G., Newman, R. A., Sutherland, D. R., ND, not determined. Ritter, M. A. & Ritz, J. (1983) Blood 61, 628-639. Downloaded by guest on September 26, 2021 Immunology: Shipp et al. Proc. Nati. Acad. Sci. USA 86 (1989) 301

4. Hokland, P., Rosenthal, P., Griffin, J. D., Nadler, L. M., 22. Malfroy, B., Kuang, W. J., Seeburg, P. H., Mason, A. J. & Daley, J., Hokland, M., Schlossman, S. F. & Ritz, J. (1983) J. Schofield, P. R. (1988) FEBS Lett. 229, 206-210. Exp. Med. 157, 114-129. 23. Hersh, L. B. (1982) Mol. Cell. Biochem. 47, 35-43. 5. Hokland, P., Nadler, L. M., Griffin, J. D., Schlossman, S. F. 24. Malfroy, B., Swerta, J. B., Guyon, A., Roques, B. P. & & Ritz, J. (1984) Blood 64, 662-666. Schwartz, J. C. (1978) Nature (London) 276, 523-526. 25. Llorens, C. & Schwartz, J. C. (1981) Eur. J. Pharmacol. 69, 6. Hoffman-Fezer, G., Knapp, W. & Thierfelder, S. (1982) Leuk. 113-116. Res. 6, 761-767. 26. Kerr, M. A. & Kenny, A. J. (1974) Biochem. J. 137, 477-488. 7. Neudorf,J. S. M., LeBien, T. W. & Kersey, J. H. (1984)Leuk. 27. Kerr, M. A. & Kenny, A. J. (1974) Biochem. J. 137, 489-495. Res. 8, 173-179. 28. Connelly, J. C., Skidgel, R. A., Schulz, W. W., Johnson, 8. Braun, M. P., Martin, P. J., Ledbetter, J. A. & Hanson, J. A. A. R. & Erdos, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, (1983) Blood 61, 718-725. 8737-8741. 9. Keating, A., Whalen, C. K. & Singer, J. W. (1983) Br. J. 29. Almenoff, J., Wilk, S. & Orlowski, M. (1981) Biochem. Bio- Haematol. 55, 623-628. phys. Res. Commun. 102, 206-214. 10. Pesando, J. M., Tomaselli, K. J., Lazarus, H. & Schlossman, 30. Mumford, R. A., Pierzchala, P. A., Strauss, A. W. & Zimmer- man, M. (1981) Proc. Natl. Acad. Sci. USA 78, 6623-6627. S. F. (1983) J. Immunol. 131, 2038-2045. 31. Johnson, A. R., Skidgel, R. A., Gafford, J. T. & Erdos, E. G. 11. Metzgar, R. S., Borowitz, M. J., Jones, N. H. & Dowell, B. L. (1984) Peptides 5, 789-796. (1981) J. Exp. Med. 154, 1249-1254. 32. Gafford, J. T., Skidgel, R. A., Erdos, E. G. & Hersh, L. B. 12. Shipp, M. A., Richardson, N. E., Sayre, P. H., Brown, N. R., (1983) Biochemistry 22, 3265-3271. Masteller, E. L., Clayton, L. K., Ritz, J. & Reinherz, E. L. 33. Hersh, L. B. (1984) J. Neurochem. 43, 487-493. (1988) Proc. Natl. Acad. Sci. USA 85, 4819-4823. 34. Pierart, M. E., Najdovski, T., Appelboom, T. E. & Dschodt- 13. Wickner, W. T. & Lodish, H. F. (1985) Science 230, 400-407. Lanckman, M. M. (1988) J. Immunol. 140, 3808-3811. 14. Malfroy, B., Schofield, P., Kuang, W. J., Seeburg, P. H., 35. Borkowski, G., Zigderhand-Bleekemolen, J. E., Erdos, E. G., Mason, A. J. & Henzel, W. J. (1987) Biochem. Biophys. Res. von Figura, K. & Hasilik, A. (1987) Biochem. J. 248, 345-350. 36. Gee, N. S., Matsas, R. & Kenny, A. J. (1983) Biochem. J. 214, Commun. 144, 59-66. 377-386. 15. Devault, A., Lazure, C., Nault, C., LeMoual, H., Seidah, 37. Bowes, M. A. & Kenny, A. J. (1986) Biochem. J. 236, 801-810. N. G., Chretian, M., Kahn, P., Powell, J., Mallet, J., Beau- 38. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, mont, A., Rogues, B. P., Crine, P. & Boileau, G. (1987) EMBO N. R., Chang, H. C., Siliciano, R. F., Dorfman, T., Walker, J. 6, 1317-1322. B., Sodroski, J. & Reinherz, E. L. (1988) Nature (London) 331, 16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. 78-81. Acad. Sci. USA 74, 5463-5467. 39. Bowes, M. A. & Kenny, A. J. (1987) Immunology 60, 247-253. 17. Patten, H., Weir, L. & Leder, P. (1984) Proc. Natl. Acad. Sci. 40. Ritz, J., Pesando, J., Notis-McConarty, J. & Schlossman, S. F. USA 81, 7161-7165. (1980) J. Immunol. 125, 1506-1514. 18. Orlowski, M. & Wilk, S. (1981) Biochemistry 20, 4942-4950. 41. Goldstein, J. L., Anderson, R. G. W. & Brown, M. S. (1979) 19. McLellan, S., Dyer, S. H., Rodriguez, G. & Hersh, L. B. Nature (London) 279, 679-685. (1988) J. Neurochem. 51, 1552-1559. 42. Catt, K. J., Harwood, J. P., Aguilera, G. & Dufan, M. C. 20. Hudgin, R. L., Charleson, S. E., Zimmerman, M., Mumford, (1979) Nature (London) 280, 109-116. R. & Wood, P. L. (1981) Life Sci. 29, 2593-2601. 43. Barker, P. E., Shipp, M. A., D'Adamio, L., Masteller, E. L. & 21. Smith, P. K., Krohn, R. I., Hermanson, C. T., Mallia, A. H., Reinherz, E. L. (1988) J. Immunol., in press. Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, 44. LeTarte, M., Vera, S., Tran, R., Addis, J. B., Onizuka, R. J., N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, Quackenbush, E. J., Jongeneel, C. V. & McInnis, R. R. (1988) 76-85. J. Exp. Med. 168, 1247-1253. Downloaded by guest on September 26, 2021