From the Markey Molecular Medicine Center, Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195
Received for publication, November 13, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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Severe congenital neutropenia is a heritable
human disorder characterized by neutropenia and acute myelogenous
leukemia. We recently determined that the majority of cases result from
de novo or autosomal dominantly inherited heterozygous
mutations in ELA2, encoding neutrophil elastase. Neutrophil
elastase is a chymotryptic serine protease localized in granules of
neutrophils and monocytes and is the major target of inhibition of the
serpin Commencing at birth, the human disorder severe congenital
neutropenia (sometimes known as "Kostmann syndrome") is typified by
circulating neutrophil counts of less than 200 µl SCN was first described in consanguineous Swedish families, where it
results from autosomal recessive inheritance (1). More commonly,
however, the illness results from apparent autosomal dominant
inheritance or arises sporadically in the absence of a family history,
presumably as a consequence of new dominant mutations (3). SCN was once
thought to be caused by constitutional mutations of the gene encoding
the GCSF receptor (GCSFR) (4), but it is now known that
GCSFR mutations are not the cause of SCN, instead occurring
somatically in the bone marrow of some patients in association with
leukemia (5-9). We recently found that both cyclic neutropenia (10,
11), an autosomal dominant illness with a 21-day cycle of oscillating
neutropenia, and about 75% of cases of SCN (11) result from
heterozygous, autosomal dominant mutations in ELA2, encoding
neutrophil elastase.
Neutrophil elastase (EC 3.4.21.37) is a monomeric polypeptide of about
25 kDa synthesized in promyelocytes and promonocytes and predominantly
localized in azurophilic cytoplasmic granules (reviewed in Refs. 12 and
13). It possesses activity toward a wide variety of proteins, including
matrix components, clotting factors, immunoglobulins, and complement.
Released from neutrophils migrating to sites of inflammation, it is
thought responsible for tissue destruction in pulmonary emphysema,
rheumatoid arthritis, and the adult respiratory distress syndrome,
among other illnesses. Consequently, its activity is counteracted by a
variety of endogenous inhibitors, including the serpins
Neutrophil elastase was once purified as "medullasin" based on its
cytokine activity. It induces activated killer lymphocytes (14),
cleaves aminoacyl-tRNA synthetase to yield one polypeptide with
interleukin-8-like activity and another promoting leukocyte and
monocyte chemotaxis (15), and further modulates the expression of
components of the inflammatory response, including ICAM-1 (16).
Given the plurality of its activities and hence myriad potential
pathogenic mechanisms, we here undertake to biochemically characterize
the properties of the variety of mutant forms of neutrophil elastase
causing SCN. We have considered three genetic hypotheses compatible
with the autosomal dominant inheritance of SCN resulting from mutant
forms of neutrophil elastase. First, it is possible that the mutations
merely disable protease activity, and the disease is a result of
haploinsufficiency. We have therefore measured enzymatic activity of
each of the mutants. Second, the mutant enzymes could gain a novel
function. We examine two potential gain of function activities, toxic
gain of function and loss of inhibition by
cDNA, Mutagenesis, and Construction of Cell Expression
Vectors--
cDNAs of neutrophil elastase and dipeptidyl
aminopeptidase I (DPPI) were reverse transcribed from total RNA of
human HL-60 leukemia cells (10). By designing appropriate polymerase
chain reaction primers, the Kozak consensus leader sequence for maximum translational efficiency was introduced, and the flanking restriction enzyme sites EcoRI and XbaI were included for
subsequent subcloning into the PCS2+ expression vector (17, 18)
containing a simian cytomegalovirus promoter/enhancer. Individual
clones were isolated and sequenced to verify the integrity of reading
frame and confirm that polymerase chain reaction introduced no
mutations. The Cell Lines and Cultivation--
The rat basophilic/mast cell
leukemia line RBL-1 (20) was purchased from ATCC. Mouse 32D
myoblast-like cells (21) were the gift of Dr. Joel Greenberger
(University of Pittsburgh, Pittsburgh, PA), by way of Drs. Laurie
Milner (Fred Hutchinson Cancer Research Center, Seattle, WA) and Thalia
Papayannopoulou (University of Washington, Seattle, WA). Media for
RBL-1 cells consisted of minimal essential medium supplemented with
12.5% fetal bovine serum (FBS), 1 mM sodium pyruvate and
1% penicillin/streptomycin. 32D cells were grown in McCoy's 5A medium
with 15% FBS, with the addition of 1% essential amino acids, 0.5%
nonessential amino acids, 1% sodium pyruvate, 1% glutamine, 15 mg/ml
L-asparagine, 5 mg/ml L-serine, 0.4% minimal
essential medium vitamins, 1% penicillin/streptomycin, and 10% WEHI-3
conditioned medium. WEHI-3 conditioned medium was generated by growing
WEHI-3 cells, a gift of Dr. Papayannopoulou, in RPMI medium containing
2% FBS.
Transient Transfection--
106 or half that
quantity of cells were seeded onto 10-cm cell culture dishes for
enzymatic assays and immunoprecipitation or microscope coverslips in
6-cm cell culture dishes for immunofluorescent staining and apoptosis
assays, respectively, then transiently transfected by LipofectAMINE
Plus reagent (Life Technologies, Inc.) with 4 or 2 µg of DNA,
respectively (unless stated otherwise), following the manufacturer's
instructions. 1 µg of a Preparation of Cell Extract and Enzymatic Assays--
A
modification of a prior protocol (23) was used to prepare cell
extracts. 106 transfected cells were washed in
phosphate-buffered saline (PBS), and the cell pellet was resuspended in
150 µl of 250 mM Tris-Cl, pH 7.4. 30 µl was aliquoted
for Western Blotting Analysis--
Denaturing polyacrylamide gel
electrophoresis (SDS-PAGE) with transfer to nitrocellulose membranes
was used for separation of cell extracts. Primary antibodies were
1:1,000 rabbit polyclonal antibody to human neutrophil elastase
(Calbiochem) or 1:600 murine monoclonal antibody AHN-10 to
Coimmunoprecipitation--
Following transient cotransfection of
either wild type or mutants of neutrophil elastase with
Immunofluorescent Staining and Photomicroscopy--
Cells were
fixed and stained as described previously (22, 24) 24 h following
transfection using 1:500 of rabbit polyclonal anti-neutrophil elastase
(Calbiochem) as a primary antibody and 1:200 dilution of
rhodamine-conjugated secondary anti-rabbit serum (Jackson
Immunoresearch Laboratories). Nuclei were counterstained for 1 min in
0.5 µg/ml 4,6-diamidino-2-phenylindole. Fluorescence and phase
contrast photomicroscopy were performed on a Zeiss Axiophot microscope
equipped with fluorescein isothiocyanate and rhodamine filter sets with
15-30-s exposure on Kodak Elitechrome 400. Transparencies were scanned
onto a Kodak Photo CD and composited with Adobe Photoshop software (to
adjust size, brightness, and contrast). Confocal microscopy was
performed on a Bio-Rad Radiance system.
Apoptosis Assay--
RBL-1 cells were grown on
poly-L-lysine-coated glass slides and then transiently
transfected with wild type or mutant neutrophil elastase constructs.
The ApoAlert DNA fragmentation kit (CLONTECH) was
used with these modifications; cells were fixed in 4% paraformaldehyde in PBS at 4 °C for 25 min and permeabilized by incubation for 5 min
in ice-cold 0.2% Triton X-100 in PBS. After incorporating fluorescein-dUTP at the 3'-hydroxyl termini of the fragmented DNA from
apoptotic cells, staining was performed using 1:500 diluted rabbit
polyclonal antibody to human neutrophil elastase (Calbiochem) and 1:200
dilution of rhodamine-conjugated secondary antibody (Jackson
Immunoresearch Laboratory) in PBS. The ApoAlert Annexin V apoptosis kit
(CLONTECH) was used following manufacturer's directions.
Mutations in SCN--
The ELA2 mutations responsible
for SCN are depicted in Fig. 1. We list
here an additional person with the G185R mutation, whom we have not
previously reported. Among 22 unique patients we have identified 17 different mutations, predicted to result in 16 distinctive mutant
neutrophil elastase polypeptides, comprising eight single-base amino
acid missense substitutions (A28T, I31T, C42S, V72M, S97L, P110L,
G181V, and G185R), three single-base nonsense mutations (G192ter,
S196ter, and Y199ter), two single-base deletion mutations causing
frameshifts with premature termination near the carboxyl terminus
(P200fs and P205fs), one 24-base pair in-frame deletion of exon 4 ( Expression of Mutant Neutrophil Elastase Enzymes in RBL-1
Cells--
We have elected to express the mutant proteins in
vitro, rather than purifying the enzymes from patient neutrophils,
because the mutations occur heterozygously, in which case it would
prove difficult to purify the mutant away from the wild type protein, and because many of the patients with this rare disease are no longer
living or reside in geographically disperse locales. The rat
basophilic/mast cell line RBL-1 does not demonstrate endogenous neutrophil elastase activity and has previously been used successfully to express human neutrophil elastase (23, 28). We engineered expression
vectors for the wild type, 14 of the 16 different mutant polypeptides,
and a negative control (S173A), in which the catalytic serine residue
was substituted. (We did not analyze the second premature termination
mutation, G196ter, because this mutation is adjacent to the other two
premature termination mutants, G192ter and G199ter. We also did not
analyze the truncating mutation P200fs, because this mutation is
adjacent to Y199ter, which similarly prematurely terminates the
polypeptide.) We then transiently transfected each of this panel
expressing the 16 different versions of neutrophil elastase in RBL-1
cells (Fig. 2A). To ensure
that each of the mutants was appropriately expressed, we
immunofluorescently stained the cells with a polyclonal neutrophil
elastase antiserum. Each of the mutants, except
Neutrophil elastase is a highly processed enzyme that undergoes
extensive subcellular trafficking and compartmentalization. We
therefore also attempted to determine whether the mutant forms of the
enzyme were intracellularly mislocalized by examining the transfected
cells with confocal microscopy. Although it can be seen that the wild
type enzyme translocates to the granules of RBL-1 cells (Fig.
1B), as has been reported previously (23, 28), light
microscopic resolution proves inadequate to determine conclusively
whether the mutants were similarly correctly localized (data not
shown). We are addressing this problem in more extensive, pending
experiments employing biochemical methods to subfractionate the
cellular compartments.
Test of the First Hypothesis (Haploinsufficiency): Neutrophil
Elastase Mutations Do Not Uniformly Abrogate Proteolytic
Activity--
The first hypothesis is that the effect of the
neutrophil elastase mutations is to merely inactivate enzymatic
activity, thereby causing the SCN phenotype through haploinsufficiency.
To test this possibility, we transfected the panel of mutant neutrophil elastase constructs into RBL-1 cells and assayed their activity in a
post-nuclear supernatant on peptide substrates.
We first tested activity (Fig. 3A) on suc-Ala-Ala-Ala-pNA
(25), as it is perhaps the most commonly employed experimental substrate. As an internal control, we cotransfected a lacZ
expression vector, simultaneously assayed for
Three residues in the active site of neutrophil elastase,
His41-Asp88-Ser173, comprise a
"charge-relay" system in which His41 and
Asp88 transiently bind protons from Ser173 to
facilitate nucleophilic attack on peptide bonds undergoing hydrolysis
(12, 13). The first two substrates that we examined are tripeptides.
Extended substrates more efficiently form specific interactions with
the transition site and can more fully engage the catalytic machinery
of the charge-relay system (12). In order therefore to determine
whether the mutations have differing effects on the efficiency of
proteolysis of a substrate interfacing with the active site of the
enzyme in a somewhat different manner, we tested the activity of the
panel of mutants on MeOsuc-Ala-Ala-Pro-Val-pNA (Fig. 3C).
The results are largely similar to that seen with the prior two
substrates, suggesting that, collectively, the effect of the mutations
is not substrate-dependent.
Among proteases with elastase activity (i.e. neutrophil
elastase, pancreatic elastase, and macrophage elastase), there is a
variable relationship between enzymatic activity on model substrates and activity toward elastin. This is a result of the fact that elastin
is largely insoluble; neutrophil elastase inefficiently forms the
productive complexes required for surface proteolysis (30). In order
therefore to further determine whether the effect of the SCN mutations
is substrate-dependent, we tested activity on the substrate
DQ-elastin. Because DQ-elastin can also be digested by proteases other
than neutrophil elastase, a selective inhibitor of elastase,
MeOsuc-Ala-Ala-Pro-Val-chloromethyl ketone (31), was used to confirm
the identity of the enzyme responsible for hydrolysis of this
substrate. Once again, the panel of mutations tested similarly on this
substrate (Fig. 3D), and the activity was selectively
inactivated by MeOsuc-Ala-Ala-Pro-Val-chloromethyl ketone. The
mutations causing SCN do not appear to have differing effects on the
ability of neutrophil elastase to form productive catalytic complexes
on elastin.
To assure that there were neither significant differences in
transfection efficiency or differences in the stability of the mutant
enzymes, we used Western blots, rather than cotransfection with a
lacZ standard, as an independent internal control, for the
experiments in which the elastin substrate was tested. Western blots
(Fig. 3E) reveal no gross differences in the levels of
expression across the panel of neutrophil elastase mutations and are
themselves internally controlled for total protein concentration by
reprobing with anti-glyceraldehyde-3-phosphate dehydrogenase,
endogenously expressed by RBL-1 cells.
Neutrophil elastase expressed in RBL-1 cells is biochemically
indistinguishable from the enzyme purified from neutrophils (23, 28).
Nevertheless, to control for the possibility that the activity in the
panel of mutants arises artifactually as a result of unique properties
of RBL-1 cells, we assayed activity in promyelocytes. For this purpose
we used the murine myeloblast-like cell line 32D, which has previously
been employed for the in vitro expression of the homologous
neutrophil granule serine protease, proteinase 3 (31). Activity in 32D
cells toward the substrate suc-Ala-Ala-Ala-pNA (Fig. 3F) is
generally indistinguishable from that observed in RBL-1 cells, and we
conclude that the differences in proteolytic activity in the panel of
mutant enzymes is unlikely to result from cell-specific factors.
In summary, no consistent effect of the
mutations was observed when the collection of mutants was assayed on
four different substrates, using two different cell types for
expression, and when controlling for levels of expression by three
different standards. The SCN phenotype is therefore unlikely to result
from haploinsufficient reductions in proteolytic activity toward native substrates.
Test of Second Hypothesis (Gain of Function Activity): Mutant
Neutrophil Elastase Is Not Intracellularly Toxic and Retains
Sensitivity to Inhibition by and Interaction with
A second potential novel activity that the mutants could acquire is
invulnerability to inhibition. To determine the likelihood of this
hypothesis, we assayed the mutant enzymes for their ability to be
inhibited by the serpin
As another approach to determining whether there are disrupted
interactions between the mutant forms of neutrophil elastase and
Test of Third Hypothesis (Dominant Negative): Co-expression of
Mutant Neutrophil Elastase Inhibits Wild Type Activity--
A third
potential genetic explanation for the dominant inheritance of SCN is
that the mutant enzyme is capable of dominantly negatively inhibiting
the activity of the normal, wild type allele. To test this possibility,
we performed two experiments.
First, we mixed the mutant enzyme with the wild type enzyme (both
derived from RBL-1 cell extracts following transient transfection). The
combined extracts were coincubated at 37 °C for 30 min and then
assayed for activity on the substrate suc-Tyr-Leu-Val-pNA (Fig.
6A). The total activity
appears to represent the sum of the activities of the two enzymes in
isolation. For example, addition of extract from cells transfected with
the inactive negative control S173A was largely indistinguishable from
the addition of an extract obtained from cells transfected only with
the empty expression vector. In contrast, combining preparations of
mutant enzymes with near wild type levels of activity (G199ter or
P205fs) led to a near doubling of activity. We conclude that, when
mutant and wild type enzyme are added together, there is no inhibition of the wild type activity by the mutant.
Given that individuals with SCN resulting from mutations in the gene
encoding neutrophil elastase are heterozygous, then the mutant and wild
type enzyme will be coexpressed within the cell. There is thus a
possibility of the two forms of protein aberrantly interacting within
the context of the compartments in which subcellular trafficking and
post-translation modification takes place. A well known example of such
a situation involves liver toxicity and cirrhosis resulting from
polymerization in the endoplasmic reticulum of the Z form of mutant
Normal Processing of the Amino Terminus of Neutrophil Elastase Is
Required for Activity--
Neutrophil elastase undergoes extensive
post-translational processing in which it is synthesized as an inactive
zymogen and fully activated by removal of the amino and COOH-terminal
extensions (38). It is possible that the mutations could interrupt
trafficking, processing, or folding events governed by either terminus.
For example, the mutations could interfere with a protein-protein interaction mediated by either terminus and thereby prevent it from
undergoing subsequent post-translational modifications, intracellular routing, or appropriately folding. If this situation were to hold, then
it is possible that deletion of either terminus, to shortcut potentially aberrant processing events occurring in the mutants, could
intragenically complement the mutation. To test for this possibility,
we endeavored to genetically delete the amino and COOH-terminal extensions.
We first sought to delete the amino terminus from the wild type enzyme.
Normal processing of the amino terminus of neutrophil elastase involves
two steps (12, 38). First, the signal peptidase cleaves the signal
peptide portion. Second, DPPI (also known as cathepsin C) cleaves the
remaining two amino acid leader sequence, resulting in a mature protein
with an isoleucine residue at the amino terminus. We prepared a
construct in which the entire amino terminus was deleted, but note that
translation initiation in the resulting protein necessarily must begin
with a methionine and therefore differs from the mature protein
resulting from normal processing. Expression of this construct resulted
in no neutrophil elastase activity (data not shown), either because the
addition of the amino-terminal methionine inactivates proteolytic
function or because at least one of the two steps of amino-terminal
processing is necessary for appropriate post-translational
modification, folding, or subcellular localization of the enzyme. We
then tested a construct in which we allowed the two peptide target for
DPPI to remain in place (again with an amino-terminal methionine), but,
again, this construct was not active (data not shown). Next, we
inserted variable lengths of arbitrary protein-encoding DNA sequence
into the above construct to distance the NH2-terminal methionine from the DPPI recognition sequence, in case it was interfering with the second processing step, but this also did not
yield activity (data not shown). Finally, we coexpressed DPPI along
with these neutrophil elastase constructs, but again failed to
demonstrate activity (data not shown). We conclude that either the
necessary presence of an amino-terminal methionine in all engineered
forms of the enzyme lacking an amino terminus disables proteolytic
activity or disrupts the second processing step by DPPI or, else,
normal processing is required for correct post-translational modification, folding, or subcellular trafficking of neutrophil elastase. In any event, it is not possible to experimentally test for
intragenic complementation of the SCN mutations through deletion of the
amino terminus.
The Mutations Do Not Exert Their Effect through Processing of
Folding Mediated by the Carboxyl Terminus--
Proceeding similarly to
the above, we sought to determine whether deletion of the portion of
the gene encoding the COOH-terminal extension would render the wild
type enzyme active. These experiments proved straightforward, as
expression of the wild type cDNA deleted of the carboxyl terminus
demonstrated full proteolytic activity (see below) and correctly
translocated to the granules of RBL-1 cells (data not shown), in accord
with prior reports that this region of the molecule is not necessary
for enzymatic activity and granule localization (23).
We next tested whether deletion of the carboxyl terminus from the SCN
mutations would restore wild type characteristics in the previously
conducted assays of proteolytic activity, Some of the biochemical consequences of the mutations occurring in
SCN can be inferred from the distribution of the mutations with respect
to the known crystal structure (Fig. 7)
of neutrophil elastase and by sequence alignments with other serine
proteases. First, the mutations are unlikely to have too disruptive of
an effect upon protein structure; note that all five of the mutations causing premature protein truncation are clustered near the carboxyl terminus. This distribution suggests that the mutations cause the SCN
phenotype only when a substantial portion of the amino terminus of
neutrophil elastase, including the catalytic serine and the charge
relay triad, is intact. Presumably, truncating mutations occurring in
closer proximity to the amino terminus would not be capable of causing
SCN and are therefore not observed. Second, in contrast to the
mutations responsible for cyclic neutropenia (10, 11), the mutations
causing SCN are generally spatially distant from the active site in the
structure of the enzyme, and therefore might not be expected to have
significant effects upon catalytic activity. Third, based on protein
sequence homology (data not shown), the mutated amino acids tend to
represent conserved, signature residues defining the
chymotryptic-family of serine proteases. Collectively, these
observations suggest that the mutations causing SCN might, in common,
alter a core function of this family of serine proteases. We
consequently sought to define biochemical abnormalities common to all
forms of mutant neutrophil elastase. The first two predictions were
largely borne out, as we found no consistent effects upon catalysis or
vulnerability to proteolytic inhibition. The possibility of potentially
disruptive effects of the mutants upon post-translational processing
and subcellular trafficking is suggested by evidence of a dominant
negative effect.
1-antitrypsin. The mutations causing severe
congenital neutropenia consist of amino acid missense substitutions,
in-frame deletion, splice donor mutation producing a deletion, splice
acceptor mutation causing insertion of novel residues, and protein
truncating mutations of the carboxyl terminus resulting from nonsense
substitutions and deletions leading to frameshifts. We have expressed
14 mutant forms of neutrophil elastase in vitro and have
characterized their biochemical properties. The mutations have variable
effects on proteolytic activity, eliminating the possibility that the
disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by
1-antitrypsin, but demonstrate variable
avidity for interaction with this serpin. Somewhat surprisingly, the
mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal
subcellular trafficking or post-translational processing.
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INTRODUCTION
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1 (normal >1,800
µl
1) but generally normal quantities of
other blood cells. Since neutrophils are the primary phagocyte of the
blood responsible for defending against sepsis, affected individuals
develop chronic infections. The bone marrow in
SCN1 is characterized by a
promyelocytic maturation arrest (1). About 10% of individuals with SCN
develop acute myelogenous leukemia or myelodysplasia (2), often with
characteristic bone marrow cytogenetic abnormalities comprised of
monosomy 7 and trisomy 21.
1-antitrypsin and ELANH2 and the nonserpin elafin.
1-antitrypsin. Third, the mutant enzymes may have the
potential to be a dominant negative inhibitor of the wild type
protease; we address this hypothesis through coexpression of both wild
type and mutant enzymes.
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ABSTRACT
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1-antitrypsin cDNA (19) was a gift of
Dr. Andre Lieber (University of Washington, Seattle, WA).
Site-directed mutagenesis employing oligonucleotide cassettes was used
to construct each of the neutrophil elastase mutations and the M358R
1-antitrypsin negative control.
-galactosidase (encoded by the
lacZ gene in pCS2+C-
-gal (22)) expression plasmid was
included in each transfection as an internal control for enzymatic assays. 24 h after transfection, neutrophil elastase and
-galactosidase activities were measured, with normalization of
neutrophil elastase activity to
-galactosidase activity.
Transfections were performed in triplicate, repeated at least three
times, and the mean and standard error for a representative set of
triplicates were reported.
-galactosidase assay, performed as described previously (24).
The concentration was adjusted to 100 mM Tris-Cl, pH 7.4, 1 M MgCl2, 0.1% Triton X-100 in a 300-µl volume. Cells were lysed with one freeze-thaw cycle, followed by
homogenization for 20 s at position 3 using an ultrasonicator (Model W185F, Heat Systems-Ultrasonics). The lysate was then diluted to
a 500-µl volume with a concentration of 700 mM NaCl, 60 mM Tris-Cl, pH 8.5, 600 mM MgCl2,
0.1% Triton X-100, followed by centrifugation at 15,000 × g at 4 °C for 1 h (to remove DNA). The collected
supernatant was adjusted to a final volume of 600 µl at a
concentration of 100 mM Tris-Cl, pH 8.5, 1 M
NaCl, 500 mM MgCl2, 0.1% Triton X-100 and
mixed with 20 µl of substrate: 100 mM suc-Ala-Ala-Ala-pNA
(Elastin Products Co.) (25, 26), 10 mM suc-Tyr-Leu-Val-pNA
(Bachem) (27), or 1 mM MeOsuc-Ala-Ala-Pro-Val-pNA (Bachem)
(26), with all substrates dissolved in 1-methyl-2-pyrrolidinone. The
reaction mixture was incubated for 30 min at 40 °C and terminated with the addition of 300 µl of 200 µg/ml soybean trypsin inhibitor (Sigma), and spectrophotometrically measured absorbance at 405 nm.
Activities were blanked against sample extracts prepared from cells
transfected with the empty expression vector. Neutrophil elastase
enzymatic activity was also determined with the EnzChek elastase assay
kit (Molecular Probes). Cell extracts were mixed with DQ-elastin
labeled with BODIPY FL dye in the presence or absence of 30 µM of the selective inhibitor
MeOsuc-Ala-Ala-Pro-Val-chloromethyl ketone. Fluorescence intensity was
measured following incubation of the reaction in the dark for
1 h at room temperature at excitation/emission of 485/530 nm.
1-antitrypsin (Research Diagnostics) diluted with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20, plus 5% nonfat dry milk. Secondary detection used a 1:10,000 dilution of peroxidase-conjugated anti-rabbit antibody (Jackson Immunoresearch Laboratory) or 1:1,500 dilution of peroxidase-conjugated anti-mouse antibody (Amersham Pharmacia Biotech) using ECL (Amersham Pharmacia Biotech). The blot was stripped and reprobed with mouse monoclonal antibody 6C5 to glyceraldehyde-3-phosphate dehydrogenase (Biodesign International) to standardize for variations in protein loading.
1-antitrypsin expression constructs, cells were lysed in
400 µl of ice-cold lysis buffer containing 20 mM
Tris-HCl, pH 8.0, 135 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, 1% Triton X-100, 10%
glycerol followed by a 10-s sonication. Cell lysates were cleared by
centrifugation at 15,000 × g for 30 min at 4 °C,
normalized for protein concentration, and incubated using 2 µg of
rabbit polyclonal anti-neutrophil elastase antiserum (Calbiochem) at
4 °C overnight on a rotating platform, followed by addition of 10 µl of protein A-Sepharose slurry (Sigma) and an additional 1-h
incubation. Immunoprecipitates were recovered by brief centrifugation and then washed three times with 1 ml of ice-cold lysis buffer and
resolved on 12.5% SDS-PAGE. Western blot conditions described above
were then used for detection.
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V145-C152), two single-base substitutions leading to splicing
errors (11) (one splice acceptor mutation causing insertion of two
novel amino acids ahead of exon 4 (IVS3-8SA/Q93+PQ) and a splice
donor mutation causing deletion of the last 10 amino acids from
exon 4 (IVS4+1SD/
V161-F170)), and a 5-base insertion (ins5T16063)
that also causes (11) the exon 4 splice donor error. In general, there
is no overlap between the mutations responsible for SCN and five
additional mutations (10), not shown, that cause cyclic neutropenia
(11), although two mutations, P110L and IVS4 +1SD, are also observed in
cyclic neutropenia; three of four patients in whom the P110L mutation
has been detected have SCN, and the fourth has a phenotype of cyclic
neutropenia suggesting that this mutation causes SCN in most of the
individuals who harbor it. One patient with the intron 4 splice donor
mutation has SCN, although the majority of people (11/12) in whom
mutations in this region are observed (10, 11) have cyclic neutropenia. Four of the mutations occur in SCN patients who have developed acute
myelogenous leukemia (I31T, V72M, and IVS3-8SA/Q93+PQ) or myelodysplasia (
V145-C152).
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Fig. 1.
Mutations in neutrophil elastase causing
SCN. The mutations are shown with respect to the sequence of
ELA2, the gene encoding neutrophil elastase, numbering from
the first residue after the amino-terminal pre-signal peptide has been
cleaved (45). Each asterisk (missense mutation),
oval (splicing mutation), cross (nonsense
mutation), triangle (insertion), or inverted
triangle (deletion) represents the number of unrelated
families or proband individuals in whom this mutation has been
observed. The description of the effect of the mutation with respect to
nucleotide sequence change is enclosed by parentheses, where
the numbering from exons 1 to 3 corresponds to cosmid R33516
(GenBankTM accession no. AC004799) and from intron 3 to exon 5 to
contig LLNL-R_285B9 (GenBankTM accession no. AC010648); when present,
a description of splicing errors is enclosed in brackets
where SD refers to activation of a cryptic splice donor
sites, SA refers to activation of cryptic splice acceptor
sites, and the relative nucleotide position with respect to the
intron/exon boundary is indicated. fs refers to deletions
causing frameshifts (the phase noted in parentheses).
The location of the charge relay triad is also shown.
V145-C152 and G185R,
can be detected by immunofluorescent staining in about 12% of cells
following transfection. We did not observe immunofluorescent staining
of
V145-C152 and G185R; however, all of the mutants, including
V145-C152 and G185R, can be detected by Western blot following
denaturing PAGE (see Fig. 3E).
Presumably these two mutations, in concert with the fixation process,
disrupt the epitope recognized by the antiserum for immunofluorescent
staining but not Western blots. Because each of the mutants can be
expressed as efficiently as the wild type enzyme, as shown by either
immunofluorescent staining, Western blot, or both, we can conclude that
the mutations are unlikely to grossly alter protein stability and that
expression of the mutants is unlikely to prove toxic to cells.
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Fig. 2.
Expression of wild type and mutant human
neutrophil elastase in RBL-1 cells by immunofluorescent staining.
A, low power conventional fluorescence photomicrographs.
B, photomicrograph of higher power confocal microscope image
of wild type (WT) neutrophil elastase expression. Nuclei
were counterstained with 4,6-diamidino-2-phenylindole (and false-color
imaged in green); three cells are apparent, only one of
which expresses neutrophil elastase, in a granular distribution.
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Fig. 3.
Activity of neutrophil elastase mutations
causing SCN. Cell extracts from the indicated transiently
transfected cells were prepared and assayed against the indicated
peptide substrate. -Galactosidase was cotransfected as an internal
control of transfection efficiency, and neutrophil elastase activity
was normalized to
-galactosidase activity. The black
bars represent neutrophil elastase activity (left
vertical axis), and the diagonally
striped bars represent
-galactosidase activity
(right vertical axis). Each
panel represents the mean of three different experiments.
Error bars indicate the standard error of the
mean. WT, wild type construct; S173A, engineered
negative control. A, neutrophil elastase activity assay
using substrate suc-Ala-Ala-Ala-pNA in RBL-1 cells. B,
neutrophil elastase activity assay using substrate suc-Tyr-Leu-Val-pNA
in RBL-1 cells. C, neutrophil elastase activity assay using
substrate MeOsuc-Ala-Ala-Pro-Val-pNA in RBL-1 cells. D,
neutrophil elastase activity assay using fluorescence-conjugated
substrate DQ-elastin in RBL-1 cells (black bars)
and in presence of inhibitor MeOsuc-Ala-Ala-Pro-Val-chloromethyl ketone
(spotted bars). E, Western blot
detection of transiently transfected neutrophil elastase in RBL-1 cells
using a polyclonal antibody against neutrophil elastase. The blots were
reprobed with monoclonal antibody to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) to ensure equal loading of sample. F,
neutrophil elastase activity assay using substrate suc-Ala-Ala-Ala-pNA
in 32D cells.
-galactosidase
activity, and normalized neutrophil elastase activity to
-galactosidase activity. (Given relatively uniform
-galactosidase
activity, normalization did not significantly change the specific
activity of the mutants, however.) Most of the mutations result in
reduced levels of proteolytic activity, but two mutations (G199ter and
P205fs) retain near wild type levels of activity, while others (S97L
and V72M) demonstrate about a third as much activity, and still others
(C42S) have levels of activity no different from the S173A negative
control. To confirm this result, we repeated the transfection and cell
extract preparation, but tested the activity on a second,
more-sensitive neutrophil elastase substrate, suc-Tyr-Leu-Val-pNA (29),
that differs at all three amino acid positions in the peptide. The
profile of activities (Fig. 3B) across the panel of
mutations was similar to that observed on the first substrate, with the
G199ter mutation demonstrating activity equivalent to that of the wild
type enzyme.
1-Antitrypsin--
An alternate genetic explanation for
the dominant inheritance of SCN is that the mutant neutrophil elastase
enzymes acquire a new activity. One possibility is a so-called "toxic
gain of function," in which the enzymes prove toxic to cells
expressing them. In particular, it has been reported that chymotrypsin
and other proteases can induce apoptosis (32) and that "accelerated apoptosis" in the bone marrow might be one mechanism for SCN (33). Cytotoxicity is somewhat unlikely because, by transient transfection (Fig. 2) or stable transfection (data not shown), the mutant enzymes were expressed as efficiently as the wild type enzyme and a
-galactosidase control. Nevertheless to determine whether the mutant
enzymes induced apoptosis, we subjected transfected RBL-1 cells to the terminal dUTP nick-end labeling assay (34) for apoptosis, in which
fragmented DNA is labeled at the 3'-hydroxyl termini by deoxynucleotidyl transferase mediated fluorescein-conjugated dUTP incorporation. As shown in Fig. 4,
transfected cells were detected by indirect immunofluorescent
staining of neutrophil elastase with rhodamine (red) and
apoptotic cells identified by fluorescein (green) stain.
There is a low background level of apoptosis, but the merged images
(yellow) indicate that neither the wild type nor the
indicated mutant enzymes induce significant apoptosis; these data are
representative of that obtained with the other mutants (data not
shown). Similar results (data not shown) were observed by using another
marker of apoptosis, annexin V (35). At least under the simple cell
culture conditions employed in this assay, then, there is no evidence
for a toxic gain of function activity in the mutants, which would lead
to an increased rate of cell death.
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Fig. 4.
Apoptosis assay. RBL cells transfected
with wild type or mutant neutrophil elastase constructs were subjected
to the terminal dUTP nick-end labeling assay, fixed and incubated with
fluorescein-dUTP, and then immunostained with a polyclonal antibody
against human neutrophil elastase (secondarily detected with
rhodamine-conjugated antibody). The resulting fluorescein-labeled
nuclei amid cells expressing neutrophil elastase were visualized by
fluorescence microscopy using fluorescein isothiocyanate and rhodamine
filters, respectively. The rightmost column
represents a merger of images (in false color yellow)
staining positively for both the fluorescein-dUTP and rhodamine
neutrophil elastase signals. Note that such overlap is rare.
WT, wild type.
1-antitrypsin. Under normal
conditions,
1-antitrypsin affords protection to tissues,
particularly the lung in pulmonary emphysema, by inhibiting the
neutrophil elastase discharged from neutrophils participating in the
inflammatory response. Among the mutant enzymes with residual
proteolytic activity, the ability to be inhibited by
1-antitrypsin in a concentration-dependent manner remains similar to that observed for the wild type (Fig. 5A). As expected, the inactive
enzymes remain inactive following the addition of
1-antitrypsin.
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Fig. 5.
Inhibition and coimmunoprecipitation of
neutrophil elastase with
1-antitrypsin. A, cell
extract prepared from cells that were transfected with constructs
encoding the wild type or indicated mutant neutrophil elastase then
assayed in the absence of (filled bars) or presence of 5 ng/µl (dotted bars) or 10 ng/µl
(empty bars) purified
1-antitrypsin on the substrate suc-Tyr-Leu-Val-pNA.
B, detection of the interaction between
1-antitrypsin and the wild type or mutant neutrophil
elastase by coimmunoprecipitation with anti-neutrophil elastase
antiserum followed by immunoblotting with
anti-
1-antitrypsin.
1-antitrypsin, we performed immunoprecipitation
experiments in which both molecules were coexpressed in RBL-1 cells. As
shown in Fig. 5B, coimmunoprecipitation with anti-neutrophil
elastase antiserum brings down a complex detectable upon immunoblotting with anti-
1-antitrypsin antibody. Interestingly, the
strongest interaction is evident for the wild type enzyme and the
COOH-terminal mutants G199ter and P205fs, and the strength of the
interaction generally parallels the residual activity that is observed
for each of the mutant forms of neutrophil elastase. Confirmatory results were obtained both in 32D cells (data not shown) and through the use of a reverse set of antibodies (immunoprecipitation with anti-
1-antitrypsin and Western blot with anti-neutrophil
elastase; data not shown). As a negative control, we engineered
1-antitrypsin "Pittsburgh" (M358R) (36), a naturally
occurring
1-antitrypsin allele causing thrombosis, in
which a methionine residue critical for correct interaction with
neutrophil elastase is substituted; nevertheless, some interaction with
wild type
1-antitrypsin is still apparent. (Purified
1-antitrypsin Pittsburgh, however, did not inhibit the
mutants of neutrophil elastase causing SCN (data not shown).) A
potential explanation for this observation is that the mutations induce
a conformational change in neutrophil elastase that affects both
catalytic activity and the ability to interact with serpins.
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Fig. 6.
Test of dominant negative inhibition by
mutant neutrophil elastase. Each panel shows a
representative mean of an experiment repeated in triplicate.
Error bars indicate the standard error of the
mean. A, cell extracts prepared from cells transfected with
either the wild type (WT) or the indicated mutant neutrophil
elastase construct were mixed and incubated at 37 °C for 30 min,
then activity toward the substrate suc-Ala-Ala-Ala-pNA assayed.
B, the construct encoding the full-length wild type
neutrophil elastase was cotransfected with each of the constructs
encoding the mutant neutrophil elastase, then the cell extracts were
assayed on suc-Ala-Ala-Ala-pNA. The bars represent
transfection of the full-length wild type construct (1.8 µg) with
cotransfection with 0.9 µg (diagonally striped
bars) or 1.8 µg (black bars) each of
the mutant constructs. C, the experiments were similar to
those in panel B except constructs (1.8 µg)
encoding the COOH-terminal prodomain-deleted wild type neutrophil
elastase were used instead of the full-length construct.
1-antitrypsin in the human disorder involving deficiency
of this protein (37). To determine whether the mutant enzyme could
intracellularly inhibit the wild type activity, we cotransfected the
wild type expression vector along with each of the mutants, such that
each transfected cell would have an opportunity to simultaneously
express both mutant and wild type protein. We again assayed activity on
the substrate suc-Tyr-Leu-Val-pNA (Fig. 6B). In contrast to
the results observed in Fig. 6A, coexpression of the mutant
enzyme inhibits the activity of the wild type enzyme in a
concentration-dependent manner, when compared with
cotransfection with an empty expression vector (or a frameshifted wild
type construct; data not shown). The degree of inhibition roughly
parallels the proteolytic activity of the mutant. Generally, the
mutants with the least activity are the most inhibitory in this assay,
whereas the mutants with the greatest activity are the least
inhibitory. The total activity most closely matches that of expression
of the mutant enzyme alone. When both mutant and wild type are
coexpressed within the same cell, there is thus evidence for dominant
negative inhibition.
1-antitrypsin inhibition and coimmunoprecipitation, and dominant negative inhibition. Deletion of the COOH-terminal extension had no effect on any of the
biochemical properties of each of the tested mutant enzymes; the
COOH-terminal deleted constructs (with "
C" appended to their names) have nearly equal levels of proteolytic activity in comparison to the respective intact sequences (Fig. 3, A-C). The
COOH-terminal deletion constructs could be similarly inhibited by
1-antitrypsin (Fig. 5A) and demonstrated
equal avidity for
1-antitrypsin by immunoprecipitation
(Fig. 5B). Deletion forms of the COOH-terminal extension
behaved similarly to constructs containing this region (Fig.
6C). It appears that the mutations responsible for SCN do not influence functions mediated by the COOH-terminal extension of
neutrophil elastase, such as subcellular localization,
post-translational modification, or protein folding. Indeed, given the
lack of necessity of this tail for appropriate in vitro
expression and the fact that it is poorly conserved both across species
and between other members of the chymotryptic protease family (39), its
function remains obscure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
The distribution of the SCN mutations in the
tertiary structure of neutrophil elastase. Crystallography data
(46, 47) are taken from the Molecular Modeling Data Base and
displayed with WebLab ViewerPro software (Molecular Simulations).
Residues replaced by missense substitutions are shown in
yellow; the two deletions, one resulting from a splicing
error, are represented in blue; the location of nonsense
mutations are indicated with red arrowheads; the
positions of frameshift mutations are in black; a splicing
error causing insertion of two novel residues is localized in
purple. Secondary structure of the molecule is shown in
cyan ( -pleated sheet) and red (
-helix). The
charge relay triad, in green, is included for
reference.
Haploinsufficient reductions of proteolytic activity largely can be excluded, based on the fact that not all of the mutations causing SCN reduce neutrophil elastase activity. A potential objection to the interpretation of the data is that, because of the broad range of proteolytic activity of neutrophil elastase, the appropriate substrate was not examined. However, the panel of 16 mutant and control forms of neutrophil elastase individually demonstrated consistently similar relative activities on a variety of representative substrates, including those in which nucleophilic attack through the charge relay system proceeds via a single proton (suc-Ala-Ala-Ala-pNA and suc-Tyr-Leu-Val-pNA), two protons (MeOsuc-Ala-Ala-Prof-Val-pNA), or in which surface proteolysis occurs (elastin). Another potential objection to the interpretation of these experiments is that because activities were assayed in crude cell extracts, potential inhibitory effects of associating proteins cannot be excluded. In preliminary, unpublished experiments, however, we have detected the activities of the mutant enzymes by zymography conducted under SDS-PAGE. The mutant enzymes migrate according to their expected molecular weight while demonstrating reduced rates of catalysis toward elastin and casein; in isolation of other potential proteins, such as in a complex with a serpin, the mutant enzymes still seem to have diminished proteolytic activity. Exclusion of haploinsufficiency is compatible with reports that heterozygous or homozygous deficiency of neutrophil elastase in gene-targeted mice do not display neutropenia, but rather have a vulnerability to sepsis following exposure to fungi and enteroinvase bacteria (40-42).
We similarly did not find much support for a gain of function activity
arising from the SCN mutations. We specifically tested two potential
novel functions. First, it has been proposed that the promyelocytic
arrest characteristic of SCN might be attributable to an enhanced rate
of cell death (33). Although our simple cell culture model does not
fully reproduce events in the bone marrow, expression of wild type or
mutant forms of neutrophil elastase appeared to have no toxic effects
on the cells and specifically did not induce DNA fragmentation
characteristic of apoptotic induction nor externalization of
phosphatidyl serine to the plasma membrane as revealed by
immunoreactivity for annexin V (data not shown). As alternative
explanations, it may be that the mechanism for SCN involves either
deficient rates of production of mature neutrophils from myeloid
precursors or possibly defects in the mobilization of myeloid cells
from the bone marrow. Second, we considered the possibility that the
mutations result in an aberrant interaction with
1-antitrypsin or a related serpin.
1-Antitrypsin is still capable of inhibiting the mutant
forms of neutrophil elastase, and stable complexes between the mutant
forms of neutrophil elastase and
1-antitrypsin were
immunprecipitated from cells coexpressing both proteins. Nevertheless,
there is reduced formation of stable complexes with the mutants to a
degree that relates directly to loss of proteolytic activity. The most
likely explanation, given that serpin inhibition is an irreversible
process thought to require cleavage of the serpin by the protease, is
that the impairment of stable complex formation merely parallels the
inconsistent effects of the mutations upon catalytic activity. One
alternative possibility, given the number of serpin family members, is
that we have not yet tested interactions between the mutants and the appropriate serpin. Indeed, in preliminary experiments, we have found
that in a variety of myeloid cells neutrophil elastase interacts both
with
1-antitrypsin and another protein whose properties are most compatible for those previously reported for the serpin ELANH2
(43). A third potential novel gain of function activity not addressed
in this report is that of a change of proteolytic specificity. Given
the distribution of the SCN mutations away from the catalytic site and
with a tendency toward involving conserved residues of the chymotrypsin
family, we think this to be a less likely possibility. Unfortunately,
we have not been able to adequately address this hypothesis using the
in vitro expression system described in this report, as
there are other, nonelastase serine protease activities in RBL-1 and
32D cells that copurify in the cell extracts employed here. We are
presently attempting to express and more cleanly purify the mutant
enzymes in using the yeast species Pichia (44).
Somewhat to our surprise, there is the greatest support for the third
of the hypotheses, that of dominant negative inhibition of the wild
type enzyme by the mutant. Interestingly, the mutant enzyme appeared to
have no effect when simply added to wild type preparations; the total
activity was the sum of the two components. However, when the two were
cotransfected such that both the mutant and the wild type are expressed
within the same cell, then the total activity appears inhibited by the
mutant. It is possible that the mutant and the wild type enzyme form
misprocessed protein aggregates, in analogy to retention in the
endoplasmic reticulum of Z forms of 1-antitrypsin
causing cirrhosis (37) or that the two proteins compete for limiting
concentrations of associating cofactors. In support of the latter
possibility, it may be of relevance that SCN is a genetically
heterogeneous disease, i.e. not all cases of this disorder
can be attributed to mutations in the gene encoding neutrophil elastase
(11). It is possible that the individuals whose disease is unaccounted
for by ELA2 mutations could have defects in proteins
interacting aberrantly with the mutant forms of neutrophil elastase.
To discriminate among potential pathogenic mechanisms leading to
dominant negative inhibition, we sought to determine whether the
mutations causing SCN could interrupt the activation of the zymogen to
the mature form of neutrophil elastase in which both amino and
COOH-terminal extensions have been removed. Specifically, we attempted
to find out whether the mutant enzymes would demonstrate normal
function if activation to the zymogen state were artificially abridged
by deleting either terminal extension from the cDNA in the
expression vector. A variety of constructs were engineered in which
either the complete amino terminus or just the portion containing the
signal peptide was removed from the wild type sequence, but in all
cases expression resulted in a failure of proteolytic activity.
Coexpression of the amino-terminal processing enzyme DPPI had no
effect. Presumably the mandatory presence of an amino-terminal methionine residue in all such constructs interferes with proteolytic activity, interferes with DPPI-mediated cleavage, or else stepwise processing of the amino-terminal extension is required for appropriate intracellular post-translational modification, subcellular trafficking or folding. In any event, genetic deletion of the amino terminus is not
an experimentally tractable approach. Deletion of the COOH-terminal extension produced a more clear-cut result. The wild type enzyme demonstrates full proteolytic activity and correct subcellular localization when expressed from a cDNA in which this domain has been deleted. Removal of this domain from each of the mutants failed to
complement the observed defect in proteolysis, inhibition by or avidity
of association with 1-antitrypsin, or dominant negative
interference of wild type activity. The mutants are thus unlikely to
have effects upon functions of the protein that are mediated by the
COOH-terminal domain.
Any attempt to biochemically characterize the consequences of mutations
in neutrophil elastase must ultimately be reconciled with the
organismal biology of the phenotype. Key questions remain unanswered.
Why are most patients responsive to treatment with GCSF? What
distinguishing feature of the different ELA2 mutations accounts for hematopoietic oscillations in the allelic disorder cyclic
neutropenia? How does bone marrow failure in this disorder ultimately
lead to leukemic transformation? Continued molecular genetic and
biochemical dissection of the mutant enzymes, in concert with the
development of transgenic and gene-targeted mouse models, under way in
our laboratory, are likely to provide the best opportunity of answering
these questions and defining normal hemopoietic mechanisms governing
steady state levels of production of granulocytes.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK55820 and DK58161, Doris Duke Charitable Foundation Grant T98006, Leukemia and Lymphoma Society of America Grant 6443-00, and American Cancer Society Grant RPG-99-319-01-LBC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Markey
Molecular Medicine Center, Div. of Medical Genetics, Dept. of Medicine, University of Washington School of Medicine, 1705 N.E. Pacific St.,
HSB-K236B, Box 357720, Seattle, WA 98195. Tel.: 206-616-4566; Fax:
206-616-7288; E-mail: horwitz@u.washington.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M010279200
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ABBREVIATIONS |
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The abbreviations used are: SCN, severe congenital neutropenia; GCSF, granulocyte colony stimulating factor; GCSFR, gene encoding granulocyte colony stimulating factor receptor; DPPI, dipeptidyl aminopeptidase I; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; PBS, phosphate-buffered saline; pNA, p-nitroanilide.
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