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Proc. Nati. Acad. Sci. USAVol. 86, pp. 297-301, January
1989Immunology
Common acute lymphoblastic leukemia antigen (CALLA) is
activeneutral endopeptidase 24.11 ("enkephalinase"): Direct
evidenceby 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 (received for
review September 10, 1988)
ABSTRACT The common acute lymphoblastic leukemiaantigen (CALLA)
is a 749-amino acid type H integral mem-brane protein expressed by
most acute lymphoblastic leuke-mias, certain other lymphoid
malignancies with an immaturephenotpe, and normal lymphoid
progenitors. A computersearch against the most recent GenBank
release (no. 56) indi-cates that human CALLA cDNA encodes a protein
nearlyidentical to the rat and rabbit neutral endopeptidase
24.11("enkephalinase;" EC 3.4.24.11). This zinc
metalloendopep-tidase, which has been shown to inactivate a variety
of peptidehormones including enkephalin, chemotactic peptide,
sub-stance P, neurotensin, oxytocin, bradykinin, and angiotensinsI
and II, had not been identified in lymphoid cells. To
determinewhether CALLA cDNA derived from human acute lympho-blastic
leukemia cells (Nalm-6 cell line) encodes functionalneutral
endopeptidase activity, we generated CALLA' stabletransfectants in
the CALLA- murine myeloma cell line J558and analyzed them for
enzymatic activity in a fluorometricassay based upon cleavage of
the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the
Ala-Phe bond. Totallysates as well as whole-cell suspensions of the
Nalm-6 line andof the CALLA' transfectants, but not of the CALLA-
J558cells, possessed neutral endopeptidase activity. This
enzymaticactivity was associated with the cellular membrane
fraction andwas abrogated by the specific neutral endopeptidase
inhibitorphosphoramidon. The unequivocal identification ofCALLA asa
functional neutral endopeptidase provides insight into itspotential
role in both normal and malignant lymphoid function.
The common acute lymphoblastic leukemia antigen (CALLA)is a
100-kDa cell surface glycoprotein originally identified ohhuman
acute lymphoblastic leukemia cells (1, 2). Subsequentstudies
indicated that CALLA was also expressed by certainother lymphoid
malignancies with an immature phenotype (3)and by early lymphoid
progenitors from fetal liver, fetal andadult bone marrow, and
thymus (3-7). These CALLA' cellshave the phenotypic characteristics
of cells that are eitheruncommitted to B- or T-cell lineage or
committed to only theearliest stages of B-cell differentiation. The
temporally re-stricted expression ofCALLA during lymphoid
developmentsuggests that the antigen may play a role at an early
stage oflymphoid differentiation. However, CALLA has also
beendetected on the surface of nonlymphoid cell types
includingrenal cells, peripheral blood granulocytes, bone
marrowstromal cells, and cultured fibroblast lines (8-11),
implyingthat its biological function is not restricted to
lymphoiddevelopment.
To elucidate the primary structure ofCALLA. we purifiedthe
protein to homogeneity, obtained NH2-terminal sequencefrom both the
intact protein and derived tryptic and Staphy-lococcus aureus V8
protease peptides, and isolated CALLAcDNAs from a Nalm-6 cell line
AgtlO library by using re-dundant oligonucleotide probes (12). The
CALLA cDNAsequence predicts a 750-amino acid integral membrane
pro-tein with a single, 24-amino acid hydrophobic segment thatcould
function as both a transmembrane region and a signalpeptide (12).
The COOH-terminal 700 amino acids, includingsix potential N-linked
glycosylation sites, compose the extra-cellular protein segment,
whereas the 25 NH2-terminal aminoacids remaining after cleavage of
the initiation methionineform the cytoplasmic tail (12). CALLA'
cells containCALLA transcripts of 2.7-5.7 kilobases (kb) with the
major5.7- and 3.7-kb mRNAs being preferentially expressed
inspecific cell types (12). Molecular cloning of CALLA and
itsidentification as a type II transmembrane glycoprotein do
notallow inference of its role in lymphoid function or
differen-tiation. Proteins in this class have diverse functions
rangingfrom receptors to membrane-bound enzymes and include
thetransferrin receptor, the asialoglycoprotein receptor,
influ-enza viral neuraminidase, y-glutamyl transpeptidase,
prosu-crase-isomaltase complex, and the invariant chain of
HLAproteins (13).
In the present study, a computer search against the mostrecent
GenBank release (no. 56) uncovered the surprisingfinding that human
CALLA has 94% identity at the ahiinoacid sequence level with
neutral endopeptidase 24.11 ("en-kephalinase;" EC 3.4.24.11), a
membrane-bound zinc met-alloendopeptidase cloned from rat brain
(14) and rabbitkidney (15). Herein we prove, by using
CALLA-transfectedmurine cell lines and a sensitive enzymatic assay
in conjunc-tion with a specific neutral metalloendopeptidase
inhibitor,that CALLA is a functional form of this
membrane-boundenzyme. Our results raise the interesting possibility
that thisenzyme could play a role in lymphoid cell
developmentand/or function.
MATERIALS AND METHODS
CALLA Open Reading Frame Construct. In order to gen-erate a
CALLA cDNA containing the entire open readingframe [base pairs (bp)
12-2261], two recombinant AgtlO phageclones containing CALLA cDNA
fragments that spanned bp-132 to +583 (clone 1.1) and bp + 127 to
+3723 (clone 2) wereutilized (Fig. 1). The numbering system for the
two AgtlOclones is derived from ref. 12, in which bp 1 is located
11 bp5' to the initiation methionine codon. DNA from clone 1.1was
digested with. EcoRI and Ava I, yielding a 0.435-kbfragment (Fig.
L4). Aliquots of DNA from clone 2 weredigested with Ava I and Cla
I, yielding a 0.499-kb fragment,
297
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page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 86 (1989)
A
a aItATG I U~
X1.1 il-132 -11 +12 303 583
0.435 kb
4.? (,9 9bIf B
pBS Mouse Ig Human Ig SV40 Intron Human IgEnhancer Promotor and
Poly-A Enhancer
127 303 583 621 802 1503 2222 12348 37232321
0.499 kb
1.546 kb
f,
/\
/\
CALLA cDNA OpenReading 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)
wasdigested 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, yieldinga 0.499-kb fragment, and with Cla I and Apa I, yielding
a 1.546-kb fragment. After ligation, the reconstructed CALLA cDNA
fragment containedan intact open reading frame (bp 12-2261). The
CALLA open reading frame fragment was excised from pBluescript
SK(+) (not shown) withDra 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 openreading frame fragment was ligated into
the Xho I site of pIGTE/N. The pIGTE/N plasmid contains a portion
of pBluescript SK(+) (pBS), thehuman 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-tively (Fig. 1A). The plasmid vector pBluescript
SK(+)(Stratagene) was digested with EcoRI and Apa I. The0.435-kb
EcoRI-Ava I, 0.499-kb Ava I-Cla I, and 1.546-kbCla I-Apa I CALLA
cDNA fragments and the EcoRI/ApaI-digested plasmid vector were
purified by gel electrophore-sis, ligated, and used to transform
Escherichia coli DHSa+cells (Bethesda Research Laboratories).
Recombinants wereidentified on the basis of blue/white color
selection on LBplates containing ampicillin (100 ttg/ml),
5-bromo-4-chloro-3-indolyl 8-D-galactoside (X-Gal, 80,4g/ml), and
isopropylfB-D-thiogalactoside (IPTG, 20 mM). Following a
large-scaleplasmid preparation, the reconstructed CALLA cDNA
con-taining the intact open reading frame was excised from
theplasmid vector by using Dra I and Apa I and blunted at its 3'end
with phage T4 DNA polymerase. In order to generate a5' Sal I site,
the resulting 5' and 3' blunt-ended CALLAcDNA fragment was ligated
into EcoRV-digested pBluescriptSK(+), which contains a Sal I site
in the polylinker. Aftertransformation, recombinants were
identified and analyzedfor orientation by using a panel of
diagnostic restrictionendonucleases. An appropriate clone was
further analyzedby sequencing the 5' and 3' ends and the EcoRI-Ava
I, AvaI-Cla I, and Cla I-Apa I junctions of the CALLA insert bythe
dideoxy chain-termination method (16).
In order to obtain the CALLA open reading frame with theSal I
site from the polylinker, the fragment was excised withSma I and
Apa I, which cleaved at polylinker sites 3' of theCALLA fragment
and 5' of the CALLA fragment and Sal Isite, respectively. The Apa I
end was blunted with T4 DNApolymerase and the modified CALLA open
reading frameinsert was ligated into EcoRV-cut pBluescript SK(+).
Aftertransformation, recombinants containing a resulting 3' Sal
Iend from the polylinker were identified with diagnostic Sal
Idigestions. Following a large-scale plasmid preparation of
arepresentative clone, the CALLA open reading frame wasexcised with
Sal I and ligated into the Xho I site of pIGTE/N(Fig. 1B). The
pIGTE/N plasmid (E.V.S. and P. Leder,unpublished results) contains
the human immunoglobulinpromoter, both human and murine
immunoglobulin enhanc-ers, a simian virus 40 intron and
polyadenylylation signal, andthe neomycin-resistance gene driven by
a cytomegaloviruspromoter. After transformation, recombinants were
ana-lyzed for orientation with a panel of diagnostic
restrictionendonucleases and a plasmid containing the CALLA
open
reading frame in the sense orientation (pIGTE/N CALLAs)was
obtained.
Generation of CALLA' Cell Lines. The pIGTE/N CALLASconstruct was
transfected into the CALLA- murine myelomacell line J558 by
electroporation (17). In brief, 2 x 107 cells werewashed once in
ice-cold phosphate-buffered saline (145 mMNaCl/2.3 mM KH2PO4/7.7 mM
K2HPO4) and resuspended in500 ,ul of ice-cold phosphate-buffered
saline containing 50 ,g ofpIGTE/N CALLAS. The cell/DNA mixture was
transferred toan electroporation cuvette and, after 5 min on ice,
electroporatedat 2000 V. After a 10-min incubation at room
temperature, cellswere diluted in 10 ml of RPMI-1640 medium
supplemented with10%o fetal bovine serum, 2 mM glutamine,
penicillin (100 units/ml), and streptomycin (100 ,ug/ml) and
cultured for 48 hr at 37°Cin a 5% CO2 atmosphere. Thereafter, cells
were maintained inRPMI-1640 supplemented as above but with the
addition ofantibiotic G418 at 900 ,ug/ml for 14 days before assay
for CALLAexpression with the anti-CALLA monoclonal antibody J5
(2).Subsequently, JS+ cells were selected by
fluorescence-activatedcell sorting (Epics V) using standard
techniques (2) andcloned by limiting dilution at 0.5 cell per well
in G418-containing medium.Enzymatic Assay for Neutral Endopeptidase
24.11 Activity.
Neutral endopeptidase 24.11 activity was measured
fluoro-metrically in a coupled assay using
glutaryl-Ala-Ala-Phe4-methoxy-2-naphthylamide (Enzyme Systems
Products,Livermore, CA) as substrate (18). Cleavage of this
substrateby neutral endopeptidase 24.11 yields Phe
4-methoxy-2-naphthylamide, which, in the presence of
aminopeptidaseactivity, is converted to the fluorescent product
4-methoxy-2-naphthylamine. Reaction mixtures contained 0.1 mM
sub-strate, 100 mM 2-(N-morpholino)ethanesulfonate (Mes)buffer at
pH 6.5, 0.3 M NaCl, 0.5 milliunit of purified rat
brainaminopeptidase (19), and enzyme in a final volume of 100
,u1.Reactions were initiated with enzyme and followed at 30°C atan
excitation wavelength of 340 nm and an emission wave-length of 425
nm by using an Aminco-Bowman spectrofluo-rometer equipped with a
thermostatted cell holder and stripchart recorder. Inhibition by
phosphoramidon (2 ,uM) wasused to confirm the specificity of the
assay (20). Protein wasdetermined by the bicinchoninic acid (BCA)
method (21).
Cell extracts were prepared by resuspending washed cellsin
200-400 ,ul of 20 mM Mes (pH 6.5) containing 1% (wt/vol)n-octyl
,-D-glucopyranoside (Sigma). After a 1-hr incubationat room
temperature, the samples were centrifuged for 30 minin an Eppendorf
centrifuge and the supernatants were taken
X2 CMV-Neo forG418 Selection
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Proc. Nati. Acad. Sci. USA 86 (1989) 299
for enzyme assays. Whole-cell suspensions were washedonce in
RPMI-1640 and then utilized at a concentration of103-104 cells per
100-pl reaction mixture. For preparation ofthe membrane fraction,
washed cells were homogenized in 1ml of Tris-buffered saline 0.2 M
NaCl/50 mM Tris, pH 7.8)with a Teflon/glass homogenizer and the
membrane fractionwas isolated by centrifugation at 40,000 x g for
30 min. Thisfraction was resuspended in the Mes/octyl glucoside
bufferand treated as described above.Computer Search. The 5508-bp
CALLA cDNA sequence
(12) was utilized in a computer search of the updated Gen-Bank
data base (release 56). The DASHER program (D. V.Faulkner,
Molecular Biology Computer Resource Center,Dana-Farber Cancer
Institute) was used to compare CALLAcDNA segments of 600 bp, each
with 100-bp overlaps, to600-bp segments with 100 bp overlaps of
each sequence in theGenBank data base.
RESULTS AND DISCUSSIONVirtual Identity of Human CALLA and
Neutral Endopep-
tidase 24.11 (Enkephalinase) by Sequence Analysis. Compar-ison
of the CALLA cDNA sequence against the GenBanksequence data base
(release 56; June 1988) revealed strikingsimilarities with the rat
and rabbit neutral endopeptidase24.11, commonly referred to as
enkephalinase (14, 15).Related segments of the CALLA cDNA and rat
and rabbitneutral endopeptidases 24.11 (bp 1-600, 501-1100,
1001-1600, 1501-2100, and 2001-2600) had homology scores of121-236.
In the DASHER program, homology scores greaterthan 12 are thought
to be of potential significance. Thus, thehigh scores noted herein
are indicative of near identity.Subsequent comparison of amino acid
sequences of humanCALLA and rat and rabbit neutral endopeptidase
moleculesshowed 94% identity in each case (12, 14, 15). Analysis of
therecently reported human homologue (22) shows virtual iden-tity
with CALLA; the latter two sequences differ by oneamino acid
representing either a sequencing error or a
geneticpolymorphism.
Neutral endopeptidase 24.11 is a cell-membrane-associ-ated
enzyme that cleaves peptide bonds on the amino side ofhydrophobic
amino acids (23). The enzyme was identified inbrain as an
enkephalinase because it cleaved the Gly3-Phe4bond of enkephalins
(24). However, the enzyme was subse-quently found in many other
tissues, including kidney, whereit was present in high levels (25).
In kidney, enkephalinaseactivity was shown to be identical to that
of neutral endopep-tidase 24.11, which had been identified several
years earlierby using the B chain of insulin as substrate (26, 27).
Neutralendopeptidase 24.11 has been shown to react with a varietyof
physiologically active peptides including chemotacticpeptide (28),
substance P and neurotensin (29, 30), oxytocin(31), bradykinin,
angiotensins I and 11 (32), and a variety ofopioid peptides (33).
This enzyme has also been shown tohydrolyze the lymphokine
interleukin 1 (34). Neutral en-dopeptidase 24.11 has been found in
numerous tissues otherthan kidney and brain, including peripheral
blood granulo-cytes (28), fibroblasts (35), small intestine (36),
and placenta(22). However, lymphoid cells have not been shown
topossess this enzymatic activity (37). Given that enkephali-nase
is a zinc-binding metalloendopeptidase, it is of interestthat the
chromosomal location of the CALLA gene is 3q21-27, a region rich in
genes encoding metal-bihding proteins,including transferrin,
lactotransferrin, melanotransferrin, thetransferrin receptor, and
ceruloplasmin (43).
Expression of Human CALLA in Murine Transfectants. Todetermine
whether CALLA derived from a lymphoblasticleukemia cell line has
functional neutral endopeptidase 24.11activity, we engineered a
construct (pIGTE/N CALLAS)containing the CALLA open reading frame
from the leukemic
cell line Nalm-6 under the control of an immunoglobulinpromoter
and enhancer and transfected it into the murinemyeloma cell line
J558, which lacks cell surface CALLAexpression (see Fig. 1 and
Materials and Methods). Follow-ing G418 selection, CALLA' J558
cells were identified byphenotyping with the JS anti-CALLA
monoclonal antibody,sorted, and cloned by limiting dilution. Two
J5' subclones,A2-3 and A2-2, which had high and low levels of
CALLAexpression, respectively, were chosen for further
analysis.Fig. 2 contains a comparison ofrelative CALLA
fluorescenceof these two CALLA' stable transfectants, the
parentalCALLA- multiple myeloma line J558, and the CALLA'acute
lymphoblastic leukemia line from which the CALLAcDNA was isolated,
Nalm-6. As indicated, J558 lacks de-tectable cell surface CALLA
expression (mean channelfluorescence 0), whereas A2-3 and A2-2
express cell surfaceCALLA. Note that A2-3 expresses a substantially
highernumber of CALLA sites per cell than does A2-2 (meanchannel
fluorescence 116.8 and 7.6, respectively). Compar-ison of the mean
channel fluorescence of A2-3 and A2-2 withthe mean channel
fluorescence of Nalm-6 (172.6) indicatesthat A2-3 expresses CALLA
at a level 67.7% that of Nalm-6,whereas A2-2 expresses CALLA at
only 4.4% the level ofNalm-6.
Analysis of Neutral Endopeptidase Activity. To assess theneutral
endopeptidase activity associated with the A2-3,A2-2, J558, and
Nalm-6 lines, we used a sensitive fluoromet-ric assay based upon
the cleavage of the substrate glutaryl-Ala-Ala-Phe
4-methoxy-2-naphthylamide. When whole-cellsuspensions of the
individual cell populations were assayed(Table 1), Nalm-6, A2-3,
and A2-2 cells exhibited neutralendopeptidase activity of4789,
1906, and 446 nmol ofproductper hr per 106 cells. The value for
J558 cells, of 0.2 nmol perhr per 106 cells, is within the
experimental error ofno activity.Addition of the specific inhibitor
phosphoramidon (20) to theNalm-6, A2-3, and A2-2 cell suspensions
reduced the neutralendopeptidase activity by factors of 90, 37, and
25, respec-tively (Table 1). Cell lysates (Table 2) from Nalm-6,
A2-3,A2-2, and J558 contained neutral endopeptidase
specificactivities of 9.96, 2.35, 1.18 and 0.08 nmol per min per mg
ofprotein, respectively. When the assays were performed in
thepresence of phosphoramidon, the neutral endopeptidase
Nalm6 J558
Zzz A2-3 A2-2
Log Channel Fluorescence
FIG. 2. Comparison of relative cell surface CALLA expressionon
the Nalm-6, J558, A2-3, and A2-2 cell lines. The human CALLA'acute
lymphoblastic leukemia line Nalm-6, the CALLA- murinemultiple
myeloma line J558, and the two CALLA' stable transfec-tants, A2-3
and A2-2, were phenotyped with the anti-CALLAmonoclonal antibody J5
(thick traces). Background fluorescence wasdetermined by
phenotyping the cell lines with an anti-CD4 mono-clonal antibody
(19Thy5D7, ref. 38) (thin traces). The CALLA meanchannel
fluorescence for each cell line was determined by subtractingthe
mean channel fluorescence generated by staining with theanti-CD4
antibody from that generated by staining with the anti-CALLA
antibody. Mean channel fluorescence values for Nalm-6,J558, A2-3,
and A2-2 were 172.6, 0, 116.8, and 7.6, respectively.
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Table 1. Neutral endopeptidase activity in whole-cell
suspensions
Specific activity, nmol per hr per 106 cells
Cell line - Phosphoramidon + Phosphoramidon
Nalm-6 4789 53A2-3 1906 52A2-2 446 18J558 0.2 0.2
activity associated with the Nalm-6, A2-3, and A2-2 celllysates
was reduced markedly (Table 2). The observation thatthe apparent
activity in J558 cells is insensitive to phosphor-amidon inhibition
suggests that this low level of activity is notdue to neutral
endopeptidase 24.11 and within the experi-mental error of no
activity. Subcellular fractionation of theNalm-6 and A2-3 cells
demonstrated that 99% of the Nalm-6and 90% of the A2-3 neutral
endopeptidase activity wasassociated with the membrane fraction
(Table 2). Compari-son of the levels of neutral endopeptidase
activity (Tables 1and 2) and levels of CALLA expression of the four
cell lines(Fig. 1) indicated that there was a correlation between
neutralendopeptidase activity and cell surface CALLA
expression.
Implications. The observations (i) that the CALLA proteinis
found both on early normal lymphoid progenitors and ontheir
malignant counterparts and (ii) that CALLA cDNAfrom an acute
lymphoblastoid leukemia encodes neutralendopeptidase 24.11
(enkephalinase) activity indicate thatthe enzyme functions at a
critical stage in lymphoid differ-entiation. This is of particular
interest, given previous studiesdemonstrating that the
cell-surface-bound enzyme has thepotential to mediate a wide range
of biological activities in avariety of tissues. For example,
neutral endopeptidase 24.11has been shown to inactivate endogenous
opioid pentapep-tides on neurons in brain (23, 24), chemotactic
peptide(fMet-Leu-Phe) on polymorphonuclear granulocytes (28),and a
variety of regulatory peptides on the surface ofproximal tubule
epithelial cells ofthe kidney (32). Amino acidsequences of the
enzyme derived from three tissue sources(brain, kidney, and
placenta) in three species as well as froma human lymphoblastic
leukemia cell line are virtually iden-tical (i2, 14, 15, 22); these
results imply conservation ofcritical functional domains for zinc
binding, substrate bind-ing, and catalysis. Furthermore, given the
structural identityof the enzyme in various tissues, the biological
activity ofCALLA/neutral endopeptidase 24.11 is likely to be
dictatedby the availability of specific substrates in individual
organsrather than by the presence of different functional forms
ofthe enzyme. The substrate for the cell surface CALLA/neutral
endopeptidase 24.11 of early lymphoid progenitors isnot yet known.
However, previous studies of neutral en-dopeptidase 24.11 indicate
that optimal substrates for theenzyme are small peptides rather
than large proteins. Con-sequently, it is likely that CALLA/neutral
endopeptidase24.11 may also react with a small regulatory peptide
at the cellsurface of lymphoid precursors. Such an action could
lead tothe inactivation of a physiologically active peptide or
convertan inactive form of a peptide to an active one. We cannot
rule
Table 2. Neutral endopeptidase activity in total cell
lysatesSpecific activity, nmol per min per mg % activity
Cell of protein in membraneline - Phosphoramidon +
Phosphoramidon fraction
Nalm-6 9.96 0.2 99A2-3 2.35 0.08 90A2-2 1.18 0.29 NDJ558 0.08
0.05 NDND, not determined.
out the possibility that the CALLA substrate for
lymphoidprecursors is a previously defined peptide.Although neutral
endopeptidase 24.11 has not previously
been identified on lymphoid cells, the enzyme has beendetected
in nodal tissue (37). In porcine lymph nodes, theenzyme is found on
a subpopulation of adherent cells with themorphological
characteristics of fibroblasts (37). These cellsare most prevalent
in medullary areas and are also found inthe center of follicles and
encircling them. Of interest, theseneutral endopeptidase 24.11+
cells are observed to haveclusters of lymphoid cells firmly
attached to their cell surface(37). In tonsil, spleen, thymus, and
Peyer's patches, theneutral endopeptidase 24.11+ cells are present
in a reticularpattern similar to that seen in lymph nodes, where
theenzyme is much more abundant (39). Recent studiesprompted by the
identification of neutral endopeptidase24.11+ reticular cells in
lymphoid tissues indicate that theenzyme inactivates interleukin 1
in vitro and inhibits thymo-cyte proliferation in a dose-dependent
and specific fashion(34).As noted above, CALLA/neutral
endopeptidase 24.11 can
cleave the chemotactic peptide fMet-Leu-Phe (28). Thepresence of
CALLA/neutral endopeptidase 24.11 on thesurface of mature
neutrophils suggests that the enzyme mayplay an important role in
the process of down-regulatingchemotaxis, perhaps by reducing the
local concentration ofchemotactic peptides. Chronic treatment with
morphineinduces a selective and specific increase in brain
enkephalin-ase activity, likewise indicating that the enzyme may
regulatethe local concentration of opioid neurotransmitters and
thatthe concentration of such neurotransmitters may also
affectenzyme levels (24).
Earlier studies indicated that specific antibody treatment
ofCALLA' lymphoid cells resulted in rapid cell surface
redis-tribution, internalization, and degradation of the
CALLA-antibody complex (10, 40). The antibody-induced modulationof
CALLA was noted to resemble the specific down-regulation or loss of
cell surface receptors induced by peptidehormones (41, 42). Given
the fact that CALLA cDNAencodes functional lymphoid neutral
endopeptidase 24.11, itis possible that its peptide ligand will
also modulate cellsurface CALLA. This may result in efficient
internalizationof ligand. Whether such a putative peptide affects
migration,growth, or other functional aspects of immature normal
ormalignant B cells remains to be determined. However,
theunequivocal identification of CALLA as functional
neutralendopeptidase 24.11 (enkephalinase) and the availability
ofspecific endopeptidase inhibitors should make it possible
toassess the role of CALLA in lymphoid development.
Note Added in Proof. The partial deduced amino acid sequence
ofCALLA recently reported by LeTarte et al. (44) confirms
ourpreviously reported sequence (12) and independently identifies
theamino acid homology between CALLA and neutral endopeptidase.
We thank Dr. Frank Howard for assistance with the
computersearch. This work was supported in part by National
Institutes ofHealth Grant RO1 CA49232 to E.L.R. and M.A.S. and by
NationalInstitute on Drug Abuse Grant DA02243 and Welch Foundation
Grant1391 to L.B.H. M.A.S. was a recipient of a Clinical
InvestigatorAward (KO8 CA01057) from the National Institutes of
Health duringa portion ofthese studies. E.L.R. is a recipient ofan
American CancerSociety Faculty Award. E.V.S. was supported by the
Howard HughesMedical Institute and E. I. Dupont de Nemours and
Co.
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