Download Deletion of the Zinc-Binding Motif of CD13

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Deletion of the Zinc-Binding Motif of CD13/Aminopeptidase N Molecules
Results in Loss of Epitopes That Mediate Binding of Inhibitory Antibodies
By Richard A. Ashmun, Linda H. Shapiro, and A. Thomas Look
The myeloid cell-surface glycoprotein CD13/ aminopeptidase
N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its
extracellular domain that is characteristic of many zincdependent metalloproteinases. This region contains residues
important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an
internal fragment of 117 base pairs (bp) from the human
CD13/APN cDNA, resulting in an in-frame deletion that
included the sequences coding for this pentapeptide motif.
The mutant cDNA was subcloned into a retroviral expression
vector, and polypeptidesencoded by the altered cDNA were
expressed in transfected murine NIH-3T3 fibroblasts. The
mutant CD13/APN molecules lacked enzymatic activity, and
their intracellular processing to the cell surface was retarded
by comparison with normal CD13/APN polypeptides. The
mutant molecules also lacked epitopes required for binding
of four of 19 CD13-specific monoclonal antibodies (MoAbs)
tested in flow cytometricassays. Each of the four MoAbs also
inhibitedthe enzymatic activity of wild-type APN molecules,
suggesting that these antibodies may inhibit aminopeptidase activity by interferingwith the enzyme’s zinc-coordinating properties. Cells engineered to express mutant CD13/
APN polypeptides at the cell surface provide a tool for
defining the physiologic role of this enzyme on normal and
malignant myeloid cells and marrow stromal cells.
o 1992 by The American Society of Hematology.
T
metallopr~teinases.~~
We have now created a deletion
mutant of APN that lacks a 39-amino acid segment in the
extracellular domain, including the critical pentapeptide
motif. Presented here are findings that indicate the location
of epitopes that may be important in the zinc-coordinating
activities of CD13/APN.
HE CELL-SURFACE glycoprotein CD13 (also known
as gp150) was first identified on leukocytes of the
myeloid series and was later shown to be identical to
aminopeptidase N (APN; EC 3.4.11.2),1,2 a prominent
membrane-bound metallopeptidase of epithelial cells that
form the brush borders of the small intestine and renal
tubules. CD13/APN exemplifies a group of cell-surface
peptidases that have been found on discrete subsets of
hematopoietic cells. While CD13/APN expression is restricted to myeloid progenitors and mature monocytes,
macrophages, and granulocytes within the hematopoietic
system, other membrane-bound peptidases show activity on
granulocytes and lymphoid progenitors (CDlO/neutral endopeptidase; EC 3.4.24.11),3-5 activated T lymphocytes
(CD26/dipeptidyl peptidase IV, EC 3.4.14.5),6-s and early
B-lymphocyte progenitors (murine BP1/6C3, identical to
aminopeptidase A, EC 3.4.11.7).9-”
We previously demonstrated that the APN activity of
CD13 molecules on myeloid cells can be inhibited with two
of a panel of 11 monoclonal antibodies (MoAbs) that
specifically bind epitopes of human CD13.12 APN has an
absolute requirement for zinc for its enzymatic activity, and
its extracellular domain includes a pentapeptide sequence
(HExxH, at amino acid positions 388 to 392) that mediates
zinc binding and catalytic activity in a series of related
From the Departments of Hematology-Oncology and Tumor Cell
Biology, St Jude Children’s Research Hospital, Memphis, TN; and the
Division of Hematology-Oncology, Department of Pediatrics, The
University of Tennessee, Memphis, College of Medicine, Memphis, TN.
Submitted October 21, 1991; accepted February 13, 1992.
Supported in part by National Institutes of Health Grant No.
ROI -CA42804, Leukemia Program Project Grant No. POI-CA20180,
Cancer Center Research Grant No. P3O-CA21765, and the American
Lebanese Syrian Associated Charities (ALSAC).
Address reprint requests to Richard A . Ashmun, PhD, Department
of Hematology-Oncology, St Jude Children’s Research Hospital, PO
Box 318, Memphis, TN38IOI.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I992 by The American Society of Hematology.
0006-4971/92/7912-0019$3.00/0
3344
MATERIALS AND METHODS
Construction and expression of a mutant APN cDNA. The
full-length human CD13/APN cDNA subcloned in a pBluescript
plasmid (Stratagene, La Jolla, CA)’ was digested with the BstEII
restriction endonuclease, which excised an internal 117-base pair
(bp) restriction fragment from the cDNA, but did not cut elsewhere in the cDNA or in the vector. The deleted fragment included
the coding sequences for the extracellular zinc-binding motif
(HExxH, at amino acid positions 388 to 392; see Fig 1). The
digested cDNA molecules were separated from the small insert
fragment by electrophoresis and gel elution, religated, and used to
transform XL1 blue bacteria (Stratagene). Digested DNA lacking
the deleted sequences was religated correctly to maintain the
CD13/APN open-reading frame, as confirmed by sequence analysis using the dideoxynucleotide chain termination method14 as
applied to double-stranded DNA templates (Stratagene). Mutant
cDNA molecules were excised from the pBluescript plasmid by
digestion with Sal1 andXbaI, which cut within the polylinker of the
plasmid and in the 3’ noncoding sequences of the cDNA, respectively. The insert DNA was recovered by electrophoresis and gel
elution and blunt-end-ligated into the unique BamHI site of the
retroviral expression vector ~ Z I P ~ ~ O S V ( X provided
) - ~ , ’ ~ by Dr
Richard A. Mulligan (Whitehead Institute, Boston, MA). Colonies
containing recombinant plasmids were identified by hybridization,
and a retroviral construct with the mutant CD13/APN cDNA
cloned in the correct orientation was isolated and selected on the
basis of restriction endonuclease mapping. This construct was
transfected into NIH-3T3 cells by calcium phosphate precipitation
techniques as previously described.I6 The cells were then cultured
in complete medium containing 800 pg/mL G418 (Geneticin;
Sigma Chemical, St Louis, MO) to select for transfectants expressing the neomycin resistance gene contained in the vector. Stable
G418-resistant transformants were isolated after 2 to 3 weeks.
We had previously generated NIH-3T3 transfectants that expressed high levels of normal human APN,’JZ thus providing a
basis for comparison with the deletion mutant. The predicted
amino acid sequences of normal and deletion mutant APN
molecules are illustrated in Fig 1.
Flow cytomeh’c analysis of MoAb binding to transfectants expressing mutant APN polypeptides. NIH-3T3 transfectants expressing
Blood, Vol79, No 12 (June 15). 1992: pp 3344-3349
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CD13/AMINOPEPTIDASE N DELETION MUTANT
1
I
Cytoplasmic Transmembrane
Segment
Domain
I
Extracellular
Domain
3345
I
7
Normal
CD13
COOH
NHz
I
., ,,.
I ,
I ,,
928
Deletion
Mutant
CD13
NH,
i
i
i
COOH
Fig 1. Schematic of the predicted structure of normal and mutant
CD13/APN molecules. The structure of the protein encoded by the
CD13/APN deletion mutant cDNA (928 amino acids, bottom) was
predicted to lack 39 amino acids as compared with the normal
CD13/APN molecules (967 amino acids, top). The deleted residues
(shaded region) included those residues encoded by the portion of
CD13/APN cDNA between twoBstEll sites, and includedthe extracellular pentapeptide sequence HELAH, which is associated with catalytic activity and zinc coordination.
normal CD13/APN (NIHKD13wt) or the deletion mutant (NIH/
CD13del) were analyzed by flow cytometry as previously described.16Briefly, cells were incubated at 4°C for 30 minutes in a
titered excess of either a CD13-specific MoAb or an isotypematched control mouse myeloma protein. After being washed with
cold staining medium (Dulbecco's modified Eagle's medium,
supplemented with 1.0% fetal calf serum, L-glutamine, 10 mmol/L
HEPES, antibiotics, and 2 mmol/L sodium azide), cells were
incubated for 30 minutes with fluoresceinated affinity-purified goat
antiserum to mouse immunoglobulin (Coulter, Hialeah, FL). After
further washing in cold staining medium, the cells were resuspended in staining medium containing 0.25 mmol/L propidium
iodide and analyzed with a Coulter EPICS 753 flow cytometer.
Nineteen different CD13/APN-specific MoAbs were used in this
analysis. They were gifts from the following investigators: SJ-1D1,
Dr J. Mirro, St. Jude Children's Research Hospital, Memphis, TN;
MCS-2, Dr K. Sagawa, Kurume University, Fukuoka, Japan;
WM15 and WM47, Drs E. Favaloro and K. F. Bradstock, ICPMR,
Westmead Hospital, NSW, Australia; F23, Dr C.L. Finstad, Memorial Sloan-Kettering Cancer Center, New York, Ny,Mou28 and
Mou48, Dr R. Winchester, Hospital for Joint Diseases, New York,
Ny, CLB/Mon/Gran/2, Dr P.M. Lansdorp, British Columbia
Cancer Research Center, Vancouver, BC, and Dr A.E.G. van dem
Borne, Central Laboratory of the Netherlands, Amsterdam;
RMAG6, Drs P.J. O'Connell and A.J. d'Apice, Royal Melbourne
Hospital, Victoria, Australia; 22A5, Dr M.A. Horton, St. Bartholomew's Hospital, London, U K MY7, Dr J.D. Griffin, DanaFarber Cancer Institute, Boston, MA; 3D8, Dr A.E. Koch, Northwestern University Medical School, Chicago, IL; 46All and 43E6,
Dr H.-J. Buhring, Medizinische Klinik 11, Tubingen, Germany;
U71 and U81, Dr D. Bourel, Centre Regional de Tranfusion
Sanguine, Rennes, France; MY32, Dr C.I. Civin, Johns Hopkins
Oncology Center, Baltimore, MD; 72a, Dr R.F. Todd, University of
Michigan Medical Center, Ann Arbor, MI; and TUK1, Dr B.
Uchanska-Ziegler, Institut fur Experimentelle Immunologie der
Philipps Universitat Marburg, Marburg, Germany.
Analysis of peptidase activiy. Cell-surface APN activity was
measured with a sensitive spectrophotometric assay, as previously
described.12 Briefly, intact NIH/CD13wt cells were incubated at
37°C in isotonic buffer containing 6 mmol/L alanine-p-nitroanilide
(Sigma), a substrate for CD13/APN. Samples were periodically
removed and chilled to arrest enzymatic activity. After centrifugation at PC, cell-free supernatants were assayed for optical density
at 405 nm to detect the presence of freep-nitroaniline liberated by
cleavage of the substrate by CD13/APN. All measurements were
made in triplicate. This assay specifically detects APN activity, as
demonstrated by the ability of the aminopeptidase-specific inhibitors bestatin and actinonin to block all catalytic activity.I2To test
for inhibition of cell-surface APN activity by CD13/APN-specific
MoAbs, NIH/CD13wt cells were preincubated for 1 hour at 37°C
in the presence of saturating concentrations of individual CD13/
APN-specific MoAbs, followed by addition of substrate and measurement of cell-surface APN activity by the above method.
Immunoprecipitation of mutant CD13IAPN molecules. NIH/
CD13wt cells, NIH/CD13del cells, or parental NIH-3T3 cells were
metabolically labeled with [35S]methioninefor 1 hour, incubated an
additional hour in medium containing unlabeled methionine,
lysed, and immunoprecipitated with either CD13/APN-specific
MoAb (WM15 or MY7) or isotype-matched control mouse myeloma protein. Protein-A-Sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, NJ) coated with goat antibody to mouse immunoglobulin was used as the immunoabsorbent. Immunoprecipitates
were heat-denatured and analyzed on polyacrylamide gels containing sodium dodecyl sulfate (SDS), as previously described.16
For kinetic studies, parallel cultures of NIH/CD13wt or NIH/
CD13del cells were labeled for 15 minutes with [35S]methionine,
followed by incubation in medium containing an excess of cold
methionine. At various times of chase, cells were lysed and
immunoprecipitated with MoAb MY7, and the immunoprecipitates were analyzed as described above.
RESULTS
Inhibitory MoAbs do not recognize mutant CD13IAPN
molecules. NIH/CD13wt transfectants and NIH/CD13del
transfectants were stained with a panel of 19 MoAbs
specific for defined extracellular epitopes of CD13/APN,
and then were analyzed by flow cytometry. All of the
MoAbs recognized epitopes of normal CD13/APN molecules (Fig 2A and C), whereas four of the 19 (F23, WM15,
U71, and U81) did not react with the deletion mutant (Fig
2F), suggesting that the epitopes normally recognized by
these antibodies were either abolished or modified by the
deletion of 39 amino acids from the extracellular domain.
Although 15 of the MoAbs did recognize epitopes on the
CD13/APN mutant (Fig 2B and D), the extent of binding
was generally less than one tenth of that with the NIH/
CD13wt transfectants.
Sixteen of the 19 MoAbs, including the four that failed to
recognize the deletion mutant, were tested for their ability
to inhibit cell-surface A€" activity. A subset of seven
MoAbs significantly inhibited the activity of the peptidase,
whereas the other nine had little or no effect. The seven
inhibitory MoAbs were in two groups: (1) four MoAbs that
were highly inhibitory (WM15, F23, U71, and USl), and
were the same MoAbs that failed to recognize the mutant
CD13/AF"; and (2) three MoAbs with intermediate inhibitory effects (3D8, MY7, and CLB/Mon/Gran/2), which
did bind to the CD13/APN mutant (Fig 3). All nine of the
noninhibitory MoAbs recognized epitopes on the CD13/
AI" mutant by flow cytometric assays.
Mutant CDISIAPN molecules lack enzymatic activity.
APN activity was measured on the surface of intact parental
NIH-3T3 cells, NIH/CD13wt transfectants, and NIH/
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ASHMUN, SHAPIRO, AND LOOK
3346
NIH3T3 / Normal CD13
120 r
NIH3T3 I Mutant CD13
"I
B
2
s
100
..
B
BO
E
p
.c
2
I]
60
40
m
0"
50 1
W
n
500
3 :
F
E l ,
IA
m..
250-
0
E,
P 20
D
1
10
100
1
10
100
Log Fluorescence Intensity
Fig 2. Flow cytometric analysis of normal and deletion mutant
CD13/APN on NIH-3T3 transfectants. NIH/CD13wt or NIH/CD13del
transfectants were stained with a panel of CD13/APN-specific MoAbs; binding was compared with that of an isotype-matched control
mouse myeloma protein (dotted lines). All 19 MoAbs recognized the
normal CD13/APN on NIH/CD13wt cells, as shown for MoAbs Mou28
(A), MY7 (C), and WM15 (E). Fifteen of the 19 MoAbs also bound t o the
CDl3/APN deletion mutant on NIH/CD13del cells, as shown for
Mou28 (B) and MY7 (D). However, four MoAbs (WM15, F23. U71, and
U81) failed to bind t o the deletion mutant, as shown for WM15 (F).
CD13del transfectants (Fig 4). The mutant cells lacked
detectable APN activity, indicating that the CD13/APN
molecules expressed on their cell surface were enzymatically inactive. NIH/CD13wt cells, by contrast, displayed
high levels of APN activity, more than 15 times that of
parental NIH-3T3 cells, whose aminopeptidase activity is
characteristically 10w.l~
Immunoprecipitated mutant CD13IAPN molecules display
alteredprocessing. CD13/AI" molecules were immunoprecipitated from NIH/CD13wt cells and from NIH/CD13del
cells with either MY7 or WM15 MoAbs. The MY7 antibody
recognized both normal and mutant CD13/APN molecules
in flow cytometric assays (Fig 2), and immunoprecipitated
both normal and mutant molecules (Fig 5, lanes 3 and 6).
By contrast, WM15 recognized normal but not mutant
CD13/APN molecules in flow cytometric assays (Fig 2); it
also immunoprecipitated normal CD13/APN molecules
(Fig 5, lane 2), but not the mutant polypeptides (Fig 5,
lane 5).
The majority of the normal CD13/APN molecules were
present as 150-Kd species, previously shown to be the
mature cell-surface form of this enzyme; a minority were
present as 130-Kd species, previously shown to be the
transient intracellular precursor.lb The CD13/APN deletion mutant also appeared as two species, with electro-
MAb Binding\/
Normal CD13
Deletion Mutant CD13
+
-
+
+
+
+
Fig 3. Inhibition of cell-surface APN activity by CDl3/APN-specific
MoAbs. Cell-surface APN activity on intact NIH/CD13wt cells was
measured after the binding of each of 16 CDlB/APN-specific MoAbs,
as described in Materials and Methods. The measured activity is
expressedas a percentage of control activity measured in the absence
of any MoAb. The four MoAbs (WM15, F23, U71, and U81) that failed
t o recognize mutant CD13/APN molecules corresponded t o the most
inhibitory for cell-surface CD13/APN a c t i v i i . Three MoAbs displaying intermediate inhibition of APN (3D8, MY7, and CLB/Mon/Gran/2)
recognized both native and deletion mutant enzyme molecules, as did
the nine MoAbs that were noninhibitory.
phoretic mobilities consistent with slightly smaller molecular masses ( 145 Kd and 125 Kd, respectively) as predicted
by the modified cDNA sequence. As opposed to the normal
CD13/APN molecules, most of the mutants were present as
smaller 125-Kd species. Pulse-chase analysis demonstrated
processing of normal CD13/APN molecules to mature,
150-Kd cell-surface molecules within 2 hours (Fig 6, left),
whereas the deletion mutant remained incompletely processed as intracellular precursor molecules even after 8
-
+0.0
0.5
Time (hr)
1.o
Fig 4. Measurement of the enzymatic activity of mutant CD13/
APN molecules. Cell-surface APN activity was measured on NIH/
CDl3wt cells (0).
NIH/CD13del cells (O),or parental NIH3T3 cells (A).
Enzymatic activity on NIH/CD13del cells was indistinguishable from
that on parental NIH3T3 cells, indicating that the deletion mutant
lacked enzymatic activity.
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CD13/AMINOPEPTIDASE N DELETION MUTANT
NIH3T3
+
Normal
CD13
-0,
c,
c
0
0
s
3
h
NIH3T3
+
Mutant
CD13
-
0,,
c
c
s
NIH3T3
0
b
h
3347
c,
C
0
0
E3 >-I
h
1 2 3 4 5 6 7 8 9
- 200
- 116
-
97
Fig 5. Characterizationof normaland mutant CD13/APN glycoproteins. Cells were metabolically labeled with ["Slmethionine for 1
hour, followed by incubation for an additional hour in complete
medium; detergent lysates were immunoprecipitated,and immune
complexes were analyzed in polyacrylamide gels containing SDS.
CD13/APN molecules were immunoprecipitatedfrom NIH/CD13wt
cells (lanes 1 to 3). NIHICD13del cells (lanes 4 to 6). or parental
NIH3T3 cells (lanes 7 to 9). using MoAbs MY7 (lanes 3.6.9). WM15
(lanes 2,5,8), or isotype-matched control myeloma protein (lanes
1.4.7). Normal CD13/APN molecules were precipitated with either
MY7 or WMlS, and were present predominantlyas the mature, 150 kD
cell surface form (left arrow, lanes 2 and 3). Mutant CD13/APN
molecules were precipitatedonly by MY7, and were present predominantly as the lower-molecular weight precursor form (right arrow,
lane 6).
hours (Fig 6, right), with only a small percentage of the
molecules reaching the cell surface.
DISCUSSION
We have produced a deletion mutant of CD13/APN that
lacks a part of the extracellular domain, including a putative
zinc-binding motif. Many extracellular epitopes were preserved in the altered CD13/APN molecule, as 15 of 19
CD13/APN-specific MoAbs bound to the mutant glycoproteins. However, the four highly inhibitory MoAbs that
failed to bind must recognize epitopes in the vicinity of the
critical pentapeptide sequence associated with zinc coordination and catalytic activity. These MoAbs may exert their
inhibitory action by interfering with zinc coordination of the
native enzyme, by blocking a residue important in catalytic
activity, or both. The antigenic determinants of these four
MoAbs may also include residues elsewhere on the molecule that would normally be placed in juxtaposition with the
deleted amino acids on the normal CD13/APN protein."
By contrast, the three MoAbs with intermediate inhibitory
effects (3D8, MY7, and CLB/Mon/Gran/2) may recognize
epitopes lying near but outside of the deleted segment.
Flow cytometric profiles for these three MoAbs were
similar to those for the nine noninhibitory MoAbs (Fig
2A-D), indicating that the epitopes recognized by these
MoAbs were fully preserved on the deletion mutant molecules. Steric effects of binding in close proximity to the
native enzyme's active site could explain moderate, but incomplete, inhibition of APN activity by this subset of MoAbs.
Comparison of the deleted CD13/APN sequences with
sequences of other metalloproteases suggests that they are
pivotal in forming a tridentate pocket for zinc in the APN
extracellular domain. The pentapeptide sequence containing histidines 388 and 392 (HExxH) is characteristic of
zinc-binding metalloproteases.'3.'XStudies of CDlO/neutral
endopeptidase have also demonstrated the importance of
this extracellular region in enzymatic a~tivity.'~.~"
Point
mutations of either of the two analogous histidine residues
within its similar pentapeptide sequence (HExxH, at amino
acid positions 583 to 587) resulted in loss of zinc binding
and catalytic activity, whereas a point mutation of a
glutamic acid residue contained within the pentapeptide
sequence eliminated catalytic activity, apparently without
affecting zinc binding.I9 These findings suggest that while
zinc coordination is closely associated with the histidine
residues present within this critical region, catalytic activity
also depends on the presence of other important residues in
the enzyme's active site.
Dimerization of APN molecules, which occurs intracellularly during biosynthesis, while the molecules are still in the
transient precursor form, may be necessary for the transport of CD13/APN molecules through the Golgi, and is
sensitive to even small alterations in molecular structure, as
is ultimate processing of the protein to the cell surface.2'.22
In our study, the deletion of a 39-amino acid segment
resulted in markedly diminished processing and intracellular retention of mutant CD13/APN molecules, suggesting
that the critical dimerization and final processing of these
molecules were significantly affected by this deletion. This
raises the possibility that levels of normal CD13/APN could
be modulated on the surface of a cell by engineering
coexpression of mutant CD13/APN, leading to intracellular retention of heterodimers of normal and mutant molecules. Experiments to test this idea are under way.
APN is a widely distributed ectoenzyme found on a
spectrum of tissues, including the brush border epithelial
cells of small intestine, renal proximal tubules, and placenta. In the small intestine, APN is found on the apical
surface of epithelial cells, where it cleaves N-terminal
amino acids from small oligopeptides as part of protein
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ASHMUN, SHAPIRO, AND LOOK
3348
NIH3T3 + Mutant CD13
NIH3T3 + Normal CD13
I
Chase
0 0.51 2
Time (hr)
I
4 6 800.51
2 4
6
8’
- 200
F
-
116
- 97
Fig 6. Biochemical analysis of normal and mutant CD13/APN molecules. Parallel cultures of either NIH/CD13wt cells or NIH/CD13/del cells
were labeled with PsSJmethioninefor 15 minutes, and then incubated in medium containing unlabeled methionine for the indicated chase times.
Detergent lysates were immunoprecipitated with t h e MoAb MY7, and immune complexes were analyzed in polyacrylamide gels containing SDS.
Normal CD13/APN molecules were present in t h e transient precursor 130-Kd form, and were chased to the mature, 150-Kd cell-surfaceform by 2
hours (left). Deletion mutant CD13/APN molecules were detected as 125-Kd precursor forms predominantly, with only a small percentage
appearing as t h e processed mature cell surface form of approximately 145 Kd (right).
dige~tion.~
This
. ~ ~enzyme has also been implicated as
having a regulatory role in the inactivation of biologically
active oligopeptides, best exemplified by its participation in
the degradation of opioid dipeptides on synaptic membranes of the central nervous ~ y s t e m . ~ ~The
- ~ ’ precise
function of APN on the surface of myeloid cells is still
unknown, although some evidence suggests a regulatory
r0le,2~-~*
in which the enzyme either removes key residues
from active peptides or converts inactive peptides to active
forms. The lineage-specific pattern of expression of APN
within the hematopoietic system suggests that the enzyme’s
role may be central to myeloid cell function. Mutational
analysis to delineate structural motifs of CD13/APN polypeptides essential for enzymatic activity and binding of
inhibitory MoAbs, as described in this report, should prove
useful in delineating the role of APN on myeloid cells.
ACKNOWLEDGMENT
We thank Kevin Coleman, Elizabeth Mann, Edward Wingfield,
and Sam Lucas for excellent technical assistance, and John Gilbert
for editorial review. We thank the investigators listed in the
Methods for their gifts of MoAbs.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1992 79: 3344-3349
Deletion of the zinc-binding motif of CD13/aminopeptidase N
molecules results in loss of epitopes that mediate binding of
inhibitory antibodies
RA Ashmun, LH Shapiro and AT Look
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