From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Characterization of Defensin Precursors in Mature Human Neutrophils
By Sylvia S.L. Harwig, Alane S.K. Park, and Robert I. Lehrer
Human defensins HNP-1 and -3 are broad spectrum antimicrobial peptides that are synthesized by human neutrophils as
94 amino acid (aa) precursors that require proteolytic removal of 64 amino-terminal residues to produce the mature
defensins. Recent studies have shown that the early proteolytic processing events include two sequential cleavages,
each removing 19 amino-terminal aa residues, that yield 75
aa and 56 aa prodefensins, respectively. The subsequent
processing steps that convert these 56 aa prodefensins to
mature (30 aa) HNP-1 and HNP-3 are not yet known. We
identified four new defensin precursors in mature normal
neutrophils. The most abundant of these were two 39 aa
forms that resulted from the monobasic endoproteolytic
cleavage of proHNP-I and proHNP-3. The presence of two
proline residues in the vicinity of this newly defined scission
site suggestedthat this cleavage might be "proline-directed."
Smaller amounts of the 34 aa and 32 aa prodefensin forms
were also found. It remains to be established if these 39,34,
and 32 aa prodefensins are obligate intermediates in the
prodefensin processing pathway, or arise from side reactions. In either event, because these prodefensin intermediates accounted for only 0.25% of the total defensin content,
proteolytic conversion of 56 aa prodefensins to mature
defensins appears to be a highly efficient process.
o 1992 by TheAmerican Society of Hematology.
D
clues that may be relevant to the final processing steps
involved in forming mature defensins from their precursors.
From the Department of Medicine, UCLA-Center for the Health
Sciences; School of Medicine, University of Southem Califomia; and
the Department of Medicine, Wadsworth Veterans Administration
Hospital, Los Angeles, CA.
Submitted August 19, 1991; accepted November 6, 1991.
Supported by Grant No. AI22839 from the National Institutes of
Health. Protein sequencing was performed at the UCLA Protein
Microsequencing Facility, which is supported by a BRS Shared
Instrumentation Grant (ISlORR05554) from the National Institutes of
Health.
Address reprint requests to Robert I. Lehreq MD, Department of
Medicine, UCLA-Centerfor the Health Sciences, 10833 LeConte Ave,
Los Angeles, CA 90024-1876.
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 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-49711921 7906-0015$3.00/0
Chemicals. High performance liquid chromatography (HPLC)grade water and acetonitrile, as well as analytical reagent grade
acetic acid, formic acid, and hydrogen peroxide were purchased
from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid (TFA),
heptafluorobutyric acid (HFBA), ethyldimethylaminopropylcarbodiimide (EDC), phenylisothiocyanate (PITC), and triethylamine
were from Pierce Chemical (Rockford, IL). Acrylamide and
molecular weight markers were from Bethesda Research Laboratories, Inc (Bethesda, MD). Lysine methyl ester, dithiothreitol,
iodoacetamide, nitro blue tetrazolium (NBT), and 5-bromo-4chloro-3-indolyl phosphate (BCIP) were obtained from Sigma (St
Louis, MO). Sephacryl S-200 for gel permeation chromatography
was from Pharmacia LKB Biotechnology (Piscataway, NJ). Alkaline phosphatase-conjugated goat antimouse or antirabbit IgG
were purchased from Biorad (Richmond, CA).
Procurement of human neutrophil granules. Human neutrophils
were obtained from seven normal donors by leukophoresis and
were processed within 2 hours of harvest as previously described,"
except that the cells were disrupted by nitrogen cavitation at 750
psi essentially as described by Klempner et al,I4 rather than by
homogenization. The granule-rich, 27,OOOg postnuclear sediments
were collected and pooled in two separate batches, derived from
four and three donors, respectively, and stored at -70°C until
used. The pooled granules were extracted overnight in 5% acetic
acid at 4"C, and after the extract was cleared at 27,OOOg for 20
minutes, the pellet was extracted two more times in the same way.
The supernatants were pooled and concentrated to approximately
60 mL containing 8 to 10 x 108 cell-equivalentslml in a vacuum
centrifuge (Speed Vac; Savant Instruments, Hicksville, NY).
Purification of prodefensin peptides and defensins. The concentrated extract of human neutrophil granules was loaded on a 4.8 x
105 cm (V, = 1,900 mL) Sephacryl S-200 column that had been
equilibrated and was eluted with 5% acetic acid at a flow rate of
108 mL/h. The column effluent was monitored at 280 nm and
18-mL fractions were collected. Fractions containing peptides for
further purification were identified by their electrophoretic mobility on AU-PAGE. Appropriate fractions were pooled and lyophilized, and their contents were further purified by reversed-phase
HPLC (RP-HPLC) on a 4.6 x 250 mm Vydac C-18 column
(Separations Group, Hesperia, CA) using water-acetonitrile gradients that contained 0.1% TFA. Fractions that contained mature
defensins HNP-1, -2, and -3 were further purified by a combination
of RP-HPLC (Vydac C-18) and Hydrophilic Interaction HPLC
(Hydroxyethyl aspartamide column, PolyLC, Columbia, MD).
EFENSINS ARE SMALL, cysteine-rich antimicrobial
peptides that are abundant in human, rabbit, guinea
pig, and rat neutrophils.'a2 Although attention has centered
on their broad spectrum microbicidal and cytotoxic activity,
some defensins are selective monocyte chemoattractants?
and others cause nonspecific opsonization of bacteria:
interference with ACTH-receptor-mediated functions,536
release of histamine from mast cells,' or potent inhibition of
protein kinases.' Thus far, four human defensins have been
described in human neutrophils. Three of these (HNP-1,
HNP-2, and HNP-3) are highly homologous9and constitute
between 30% and 50% of the total protein in azurophil
granules of human neutrophils." The fourth, HNP-4, has a
distinctly different primary structure" and accounts for only
1% to 2% of the neutrophil's total defensins."
We began this project to see if any additional, quantitatively minor defensins existed in mature human neutrophils. Because HNP-4 and all of the currently known
nonhuman defensins are more cationic than HNP-1, -2,
or -3, we concentrated our search on the neutrophil's small
cationic peptides, using acid-urea polyacrylamide gel electrophoresis (AU-PAGE) as a screening tool. This strategy
led us to identify four "new" peptides, all of which proved
to be incompletely processed forms of either HNP-1 or
HNP-3. The newly characterized prodefensins provide
1532
MATERIALS AND METHODS
Blood, Vol79, No 6 (March 15), 1992: pp 1532-1537
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DEFENSIN PRECURSORS
1533
Purity of the final product was assessed by tricine sodium dodecyl
sulfate-PAGE (Tricine SDS-PAGE),by AU-PAGE, and by analytical RP-HPLC, as previously described."
Composition analysis and sequence determination. The purified
peptides were hydrolyzed in vacuo in 5.7 N HCI for 36 to 40 hours.
Amino acids were quantitated by RP-HPLC on a Novapak C-18
column (Millipore Waters, Milford, MA) after derivatizationwith
phenylisothiocyanate (Picotag technique; Millipore Waters). For
quantificationof half-cystineresidues, hydrolysis was performed on
performic acid-oxidized peptides as previously described? Prodefensins were reduced and carboxyamidomethylated, desalted by
RP-HPLC, and subjected to gas-phase Edman degradation on a
model 475 A sequencing system (Applied Biosystem, Inc, Foster
City, CA).
Because some of the purified fractions were available only in
minute amounts, the protein amounts reported in Table 2 were
determined by quantitative amino acid analysis. This calculation
was based on measuring their total content of phenylthiocarbamylated (FTC) amino acids and converting this data to total protein
content by applying appropriate weighting factors derived from the
sequence data.
Preparation of a polyclonal antibody to HNP-1. A 20-fold molar
excess of EDC (40 mg/mL) in 6 mol/L guanidine-HC1was added
to 1mg of lyophilizedHNP-1 in 0.5 mL of 6 mol/L guanidine-HC1,
pH 4.75, and the pH was adjusted to between 5.0 and 6.0with 0.1 N
NaOH. After this solution was incubated and mixed overnight, 50
pL of 1 mol/L sodium acetate was added and the mixture was
dialysed against a 1,000-fold excess of 1% acetic acid for 3 hours,
with hourly exchanges. At this point, SDS-PAGE analysis showed
that the mixture contained both monomers and covalent dimers of
HNP-1, in an approximate ratio of 3:l. Lysine methyl ester (0.29
pmol) was added to the mixture and the EDC coupling reaction
was repeated, as above. After this step, we observed higher order
multimers of HNP-1 (n = 3,4,5, and 6) on the SDS-PAGE gels as
well as additional conversion of the HNP-1 monomers to dimers.
This material was combined with Ribi MPL/TDM/CWS adjuvant
(RIB1 ImmunoChem, Hamilton, MT) and used to immunize
rabbits by conventional procedure^.'^ The resulting antibody,which
showed the same specificity as a murine monoclonal antibody
(MoAb) to HNP-1,16recognized HNP-1, -2, and -3 but not HNP-4
on Western blots (data not shown).
Immunostaining of prodefensin peptides. After Tricine SDS-gel
electrophoresis, the peptides of interest were electrotransferred
onto polyvinylidenedifluoride(PVDF) membranes using the buffer
system recommended by the apparatus manufacturer (Hoefer
Scientific Instruments, San Francisco, CA). The membranes were
then reacted with monoclonal or polyclonal antibody against
HNP-1. Detection was accomplished by an alkaline phosphataseconjugated second antibody system, using NBT and BCIP for
visualization.
0.5
1
1
LYs
0.4
3
0.2
p
0.1
P
4
20
40
60
80 100 120 140 160 180 2 0 0 220
Fraction Number
Fig 1. Chromatographic profile of human neutrophil granule extract on Sephacryl S-200. The fractions containing lysozyme (lys) and
the mature human defensins HNP-1 through -4 are shown by arrows.
The open box encloses the fractions containing the defensin precursors that are characterized in this report.
fractions also contained mature HNP-1 through -3, which
was present in relatively small quantities, compared with
the amounts recovered from the late-emerging fractions
shown in Fig 1.
The indicated (boxed) fractions were concentrated and
subjected to RP-HPLC on a C-18 column. As shown in Fig
2, the prodefensins emerged slightly before mature HNP-1
through -3 in this system. AU-PAGE was performed to
identify fractions for further chromatography and to monitor their purification. Such fractions were rechromatographed on the C-18 column using very shallow gradients of
acetonitrile, with either 0.1% TFA or 0.13% HFBA serving
as the ion pairing agent.
Gel electrophoresis and immunostaining. Figure 3A shows
the appearance by AU-PAGE of our starting material, a
crude acetic acid extract of mature human neutrophil
granules, and of human defensins HNP-1, -2, -3, and -4. It
also shows that prodefensis A and B, the two most abundant prodefensins in circulating neutrophils, are more
cationic than any of the cell's mature defensins.
Figure 3B shows an immunostain of prodefensins A and
B after their transfer from a Tricine SDS-PAGE onto a
PVDF membrane. The primary antibody used in this study
-
.....
8-
2.0
nl
1.5
Peptide purification. A typical chromatographic profile
of the human neutrophil granule extract on Sephacryl S-200
is shown in Fig 1.Note that HNP-1, -2, and -3 emerged with
an elution volume considerably larger than the column's
bed volume, probably as a consequence of nonspecific
interactions between these peptides and the gel matrix. In
contrast, HNP-4 emerged within the calculated bed volume, shortly after the lysozyme peak had passed through.
The fractions containing the four prodefensins described in
this report are enclosed within a box in Fig 1. These
$
30 0
2.
r+
1.0
t
$
e
%
c
Q,
RESULTS
$9
40
c3
M
20
z.
&-
0.5
d
lo
10
20
30
40
Gradient time (min)
Fig 2. RP-HPLC of prodefensins. Lyophilized fractions from the
boxed area shown in the Sephacryl S-200 column profile were
dissolved in 0.1% TFA and loaded onto a 4.6 x 250 mm C-18 column
and eluted with a 0% to 60% gradient of acetonitrile over 60 minutes,
at a flow rate of 1 mL/min. The prodefensins (precursors) eluted
shortly before the mature HNPs, as shown.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1534
HARWIG, PARK, AND LEHRER
Fig 3. PAGE of mature human defensins and prodefensins. (A) AU-PAGE
(12.5%): lane 1, human PMN granule
extract equivalent t o 2 x 10' cells; lane
2.2 p g each of HNP-1, HNP-2, and HNP-3;
lane 3, 2 pg of HNP-4; lane 4, 2 pg of
purified prodefensin A; lane 5, 2 p g of
purified prodefensin B. The gel was
stained with Coomassie Brilliant Blue.
(6) lmmunostaining of mature defensin
peptides and prodefensin peptides after
transfer onto PVDF membrane. Lane 1,
prodefensin B; lane 2, prodefensin A;
lane 3, 2 p g HNP 1; lane 4, prestained
molecular weight markers (faintly visible). The reagents included a polyclonal
primary antibody against polymeric
HNP1 and an alkaline phosphataseconjugated second antibody with the
BCIP-NBT substrate system. (C) Tricine
SDS-PAGE (16.5%): lane 1, molecular
weight markers; lane 2, 2 pg HNP-1;
lane 3,2 p g HNP-3; lane 4,2 p g prodefensin A; lane 5,2 p g of prodefensin 6. Each
sample was boiled for 5 minutes in sample buffer containing 40 mmol/L dithiothreitol. The gel was stained with
Coomassie Brilliant Blue.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1535
DEFENSIN PRECURSORS
detected HNP-1, -2, and -3 with equal efficacy, but did not
recognize HNP-4.
Figure 3C shows a Tricine SDS-PAGE gel, run under
reducing conditions, and indicates that prodefensins A and
B migrated with an apparent molecular weight of approximately 4.3 Kd. The migration of HNP-1 and HNP-3 was
consistent with their known molecular weights (Mr), as
previously determined from sequence analysis (Mr HNP1 = 3,442; Mr HNP-3 = 3,486).
Taken together, these studies indicate that prodefensins
A and B are slightly larger and more cationic than any of
the mature human neutrophil defensins, and are immunologically related to HNP-1 through -3, but not to HNP-4.
Amino acid composition of the peptides. Amino acid
analysis (Table 1) showed that the compositions of prodefensin peptides A and B were similar to each other and that
both resembled mature defensin peptides HNP-1 or HNP-3.
However, both prodefensins A and B also contained certain
residues (most notably lysine and methionine) that were
not present in the aforementioned mature defensins. Prodefensins C and D were also subjected to amino acid analysis
(Table 1).Both contained a methionine residue and prodefensin C also contained a lysine in addition to those
residues also present in mature HNP-1 and HNP-3.
Primaiy sequences of prodefensins. Prodefensin A was
composed of 39 amino acids (aa), and included the entire
Table 1. Amino Acid Compositions of Mature Defensins and
Prodefensins
Mature Defensins
Amino
acids
Ala
Arg
Asx
Cyst
Glx
GlY
His
Ile
Leu
LYs
Met
Phe
Pro
Ser
Thr
Trp
Tyr
HNP-1
HNP-3
30
3.9 (4)
4.1 (4)
0.0 (0)
6.0 (6)
1.9 (2)
3.0 (3)
0.0 (0)
2.9 (3)
1.3 (1)
0.0 (0)
0.0 (0)
1.0 (1)
1.0 (1)
0.0 (0)
1.1 (1)
ND (1)
3.0 (3)
30
3.0 (3)
4.1 (4)
0.9 (1)
5.5 (6)
1.9 (2)
3.0 (3)
0.0 (0)
2.9 (3)
1.2 (1)
0.0 (0)
0.0 (0)
1.0 (1)
1.0 (1)
0.0 (0)
1.1 (1)
ND (1)
3.0 (3)
Prodefensins
A
B
39
2.9 (3)
5.2 (5)
2.3 (2)
4.7 (6)
2.2 (2)
3.9 (4)
0.8 (1)
2.8 (3)
1.3 (1)
2.0 (2)
1.0 (1)
0.9 (1)
1.9 (2)
1.2 (1)
1.0 (1)
ND (1)
2.6 (3)
39
4.0 (3 or 4)'
5.0 (5)
1.6 (1 or 2)*
4.8 (6)
2.2 (2)
4.2 (4)
1.0 (1)
3.1 (3)
1.3 (1)
2.1 (2)
0.7 (1)
1.1 (1)
2.3 (2)
1.2 (1)
1.2 (1)
ND (1)
2.9 (3)
C
34
3.1 (3)
4.8 (5)
2.1 (2)
5.0 (6)
2.2 (2)
3.4 (3)
0.0 (0)
2.8 (3)
1.1 (1)
1.0 (1)
0.6 (1)
0.9 (1)
1.3 (1)
0.4 (0)
1.2 (1)
ND (1)
3.2 (3)
D
32
3.1 (3)
3.8 (4)
2.2 (2)
4.6 (6)
2.1 (2)
3.0 (3)
0.0 (0)
2.8 (3)
1.1 (1)
0.0 (0)
0.7 (1)
0.9 (1)
1.2 (1)
0.4 (0)
1.1 (1)
ND (1)
2.7 (3)
Values determined from analysis of 40-hour hydrolysates. Numbers
within parentheses indicate residues as determined by sequence
analysis.
Abbreviations: Asx, aspartic acid or asparagine; Glx, glutamic acid or
glutamine; ND, not determined.
"This peptide contained a mixture of asp and ala in a ratio of 57:43
(D:A) on the 10th cycle of its sequence analysis.
tCysteine was determined as cysteic acid
IO
I
MRTLA I LAA I LLVALQAQA
30
20
I
I
Signal SEqUeilCe
YO
+
I
SO
I
60
i
EPLQARADEVAAAPEQ I A A D I PEVVVSLAWDESLAP KHPGS RK NM
!
70
EO
90
A
I
I
I
DCYCR I PAC IAGERRYGTC IYQGRLWAFCC
proplece
0 00
mature
...t+
HNRI
HNW
Fig 4. Proteolytic cleavage sites of HNP-1 through -3 precursor
peptides. The amino acid sequences of the HNP-1 and HNP-3 preprodefensins are shown in standard single letter code. The cleavage sites
that generate the 75 aa and 56 aa precursors" are shown by closed
arrows, those which generate the 39 aa, 34 aa, and 32 aa proHNPs
described in this report are indicatedby open arrows. The sites whose
cleavage would generate mature HNP-1 and -3 and HNP-2 and the
intramolecular cysteine disulfide pairs of the mature defensin molecule are also shown.
mature portion of HNP-3 plus 9 additional amino-terminal
residues. The amino acid sequence of prodefensin B was
determined independently for the materials purified from
batch 1 and batch 2, which had been derived from different
donor pools. Although the initial nine residues of both
batches of prodefensin B were identical to those of prodefensin A, the tenth residue contained a mixture of aspartic
acid and alanine. In contrast, we found only aspartic acid in
residue 10 of prodefensin A. The aspartic acid/alanine
ratio in residue 10of prodefensin B was the same (D:A = 57:
43) in the materials purified from batch 1 and batch 2. The
remaining amino acid residues of prodefensins A and B
were identical. Because an ala to asp change in this location
is the only difference between the HNP-1 and HNP-3
precursors, we concluded that prodefensin B represented a
mixture of the 39 aa precursors of HNP-1 and HNP-3 in
nearly equimolar amounts. Very small amounts of two
additional defensin precursors were also purified. When
sequenced, they proved to be 34 aa and 32 aa precursors of
HNP-3, with short amino-terminal extensions of 4 or 2
amino acid residues, respectively. Figure 4 shows the
prepropeptide sequences of HNP-1 and HNP-3, and indicates the proteolytic cleavage sites identified in this report
(open arrows) and those demonstrated in the report of
Valore and Ganz17(solid arrows).
Relative abundance of defensins andprodefensinsin normal
human neutrophilgranules. Table 2 compares the amounts
of prodefensins and mature defensins we recovered from
normal neutrophils. Together, HNP-1, -2, and -3 constituted 98.2% of the total defensins. HNP-4 was substantially
less abundant, accounting for 1.65% of the total defensin
content. Prodefensins A and B, the 39 aa precursors of
HNP-3 and HNP-1, were approximately 14% as abundant
as HNP-4 and more than 27-fold more abundant than the
34 aa and 32 aa precursors of HNP-3. Overall, these 4
prodefensins were present in approximately 1/500 the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HARWIG, PARK, AND LEHRER
1536
Table 2. Relative Recovery of Prodefensin Peptides From Normal Human Neutrophils
Batch
No. of
Leukophoresis
Units
1
2
Combined
4
3
7
Mature Defensins
Prodefensins
Total Cells
HNP-1 Through -3
HNP-4
A
B
C
D
5.9 x 10'0
4.7 f 10'0
10.6 x 10"
% of total
72.5'
52.8
125.3
98.2
1.1
1.o
2.1
1.65
0.043
0.031
0.074
0.058
0.061
0.157
0.157
0.171
NA
0.0023
0.0023
0.002
NA
0.0045
0.0045
0.004
Abbreviation: NA, not analyzed.
'Amount purified (mg), determined by amino acid analysis.
abundance of the mature defensins HNP-1 through -4,
suggesting that the final steps in proteolytic processing of
defensins by neutrophils were highly efficient.
DISCUSSION
The human neutrophil's principal defensins (HNP-1, -2,
and -3) are synthesized as 94 aa prepropeptides.'* Consequently, to form the mature defensins, which are either 29
(HNP-2) or 30 (HNP-1 and -3) aa long, these precursors
must undergo proteolytic processing. Valore and Ganz
recently showed that the early processing steps entail
sequential formation of two intermediate prodefensin forms,
75 and 56 aa long, re~pectively.'~
This report describes an
additional 39 aa prodefensin precursor for both HNP-1 and
HNP-3, as well as the recovery of much smaller amounts of
32 and 34 aa precursors of HNP-3. Together, these prodefensins constituted approximately 0.23% of the total defensins recovered from circulating normal neutrophils.
Because the sequences of the preprodefensins are known
from their respective cDNAs,'* it is apparent that the 39 aa
precursors of both HNP-1 and HNP-3 resulted from endoproteolytic cleavage between pross and lyss6 in their
corresponding precursor peptides (numbered, in this case,
from the amino-terminal methionine residue of the preprodefensin). Although such an event could result from the
action of a trypsin-like enzyme, the arginine-rich and
potentially trypsin-susceptible nature of defensins suggests
that a more specific processing enzyme is likely to be
involved in generating the 39 aa forms. Given the existence
of vicinal proline residues, both amino- and carboxyterminal to the indicated scission site of proHNP-l and
proHNP-3, a proline-directed monobasic cleavage model,
deserves consideration. Although this type of monobasic
proteolytic processing has been noted for a wide variety of
peptides, including PGLa and xenopsin from frog skin and
yeast killer toxin,19it more often occurs at arginine than
lysine residues, as indicated in the formation of the 39 aa
prodefensins A and B.
The next most abundant small defensin precursor that we
found was the 34 aa form of HNP-3, which would have
arisen from hydrolysis of a bond between serm and the
dibasic, arg61-1ys62doublet in the precursor (again numbered from the amino-terminal methionine of preprodefensin). Although dibasic residues are common sites of proteolytic processing in prohormones, bond scission usually
occurs between or carboy-terminal to the basic residues
rather than amino-terminal to them?' However, at least two
processing enzymes that cleave amino-terminal to a dibasic
arg-lys pair, as required to generate the 34 aa prodefensin
form of HNP-3, have been reported in other systems. One
of these cleaves a bond between glulz and lys,,-arg,, in
somatostatin*' and the other cleaves the bond between leu,
and ark-lys, of neoendorphin as well as the bond between
met, and ark-arg, of BAMP-12.22
Although the final trimming of the 34 aa precursors to
the 30 aa mature defensins could be mediated by sequential
aminopeptidase cleavages, our detection of small amounts
of the 32 aa precursor of HNP-3 without discerning the 33
aa and 31 aa forms raises the possibility that this final
trimming may involve two sequential cleavages mediated by
a dipeptidyl aminopeptidase, analogous to the process
recently described for two insect antimicrobial peptides,
cecropins A and B.23
Recently, Bateman et a1 isolated two prodefensin peptides from human HL-60 promyelocytic leukemia cells."
One was a 56 aa long precursor of HNP-1, which they
named "HP 1-56." Although they found this 56 aa form to
be prominent in HL-60 cells and immature leukocytes, they
reported difficulty in detecting it in mature neutrophils. We
have also recovered substantial amounts of this 56 aa form
from the immature leukocytes of patients with chronic
myelogenous leukemia, but not from mature neutrophils
(data not shown), consistent with the formation of this
prodefensin in immature myeloid cells that are not represented in normal circulating blood. Bateman et a1" did not
identify the additional putative HNP precursor. However,
speculating that it might result from cleavage at a dibasic
sequence, they inferred that it might be the 32 aa prodefensin form. Although we identified the hypothecated 32
defensin precursor in the present study, it was present in
minute concentrations, compared with the 39 aa precursor
forms of HNP-1 and HNP-3. Consequently, we suggest that
the unidentified small defensin precursor observed by
Bateman et a1" was more likely to have been the same 39 aa
prodefensin reported here.
It remains to be determined if the minor prodefensins
identified in this report are side products of defensin
biosynthesis or obligatory intermediates in this process. If it
is assumed that these minor prodefensins are produced by
the mainstream defensin processing mechanism, a model
for the final steps in the proteolytic processing of defensins
by human neutrophils can be formulated (Fig 4). Formal
proof that the prodefensins characterized in this report
accurately mark the mainstream pathway of defensin pro-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DEFENSIN PRECURSORS
1537
cessing must await further study with defensins whose
primary structures have been selectively altered in the
putative processing sites.
Although this model deals specifically with the production of mature (30 aa) HNP-1 and HNP-3, it should be
noted that either of these defensins could be converted to
HNP-2 simply by removing one additional amino-terminal
residue. Thus, the abundant messenger RNA for HNP-1'8,25
and HNP-318326
that has been found in immature myeloid
cells could also suffice for the production of HNP-2.
ACKNOWLEDGMENT
We thank Dr Tomas Ganz for helpful discussions and suggestions.
REFERENCES
1. Ganz T, Selsted ME, Lehrer RI: Defensins. Eur J Haematol
44:1,1990
2. Lehrer RI, Gam T: Antimicrobial polypeptides of human
neutrophils. Blood 76:2169,1990
3. Territo MC, Ganz T, Se1ste.d ME, Lehrer RI: Monocytechemotactic activity of defensins from human neutrophils. J Clin
Invest 84:2017,1989
4. Fleischmann J, Selsted ME, h h r e r RI: opsonic activity of
MCP-1 and MCP-2, cationic peptides from rabbit alveolar macrophages. Diagn Microbiol Infect Dis 3:233,1985
5. Zhu Q, B~~~~~~ A, singh A, solomon
S: ~
~and
biological activity of corticostatic peptides ( a n t i - A m ) . Endocrinol Res 15:129,1989
6. Tominaga T, Fukata J, Nakai Y, Funakoshi S, Fujii N, Imura
H: Effects of c0rticostatin-I on rat adrenal cells in vitro. J
Endocrinol125:287,1990
7. Yamashita T, Saito K Purification, primary structure and
biological activity of guinea pig neutrophil peptides. Infect Immun
57:2405,1989
"
Rice WG3
RL7
Khkade JM
Jr, Ganz T, Selsted ME, Lehrer RI, Kuo JF Inhibition of protein
kinase C by defensins, antibiotic peptides from human neutrophils.
Biochem Pharmacol37951,1988
9. Selsted ME, Harwig SSL, Ganz T, Schilling m,Lehrer RI:
Primary structures of three human neutrophil defensins. J Clin
Invest 76:1436,1985
10. Rice WG, Ganz T, Kinkade JM Jr, Selsted ME, Lehrer RI,
Parmley R T Defensin-rich granules of human neutrophils. Blood
70757,1987
11. Singh A, Bateman A, Zhu Q, Shimasaki S, Esch F, Solomon
s: Sh-~ctureof a novel granulocyte Peptide with anti-ACTH
activity. Biochem Biophys Res Commun 155524,1988
12. Gabay JE, Scott RW, Campanelli D, Grfith J, Wilde C,
Marra MN, Seeger M, Nathan C F Antibiotic proteins of human
polymorphonuclear leukocytes. Proc Natl Acad Sci USA 865610,
1989
13. Ganz T, Selsted ME, Szklarek D, Harwig SSL, Daher K,
Bainton DF, Lehrer RI: Defensins: Natural peptide antibiotics of
human neutrophils. J Clin Invest 76:1427,1985
14. Klempner MS, Mikkelsen RB, Corfman DH, AndreSchwartzA Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen
cavitation and differentialcentrifugation. J Cell Biol86:21,1980
15. Harlow E, Lane D: Antibodies, A Laboratory Manual. Cold
Spring Harbor, m,
Cold Spring Harbor Laboratory, 1988, P 92
16. Panyutich AN, Voitenok N, Lehrer RI, Ganz T An enzyme
immunoassay for human defensins. J h"mnol Methods 141:149,
1991
17. Valore EV, Ganz T: Posttranslational processing of defen~ sins in immature
l
~ human~myeloid icells. Blood
~ 79:1538,1992
~
18. D a h
Lehrer RI, Ganz T, Kronenberg M: Isolation
and characterization of human defensin cDNA clones. Proc Natl
USA 85:73177 1988
19.
Tw:The processing Of peptide precursors' FEBS
Lett 2001,1986
20. Gluschankof P, Cohen P: Proteolytic enzymes in the posttranslational processing of polypeptide hormone precursors. Biochem Res 12951,1987
21. Gluschankof P, Morel A, Benoit R, Cohen P: The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28(l-12). Biochem Biophys Res Commun
128:1051, 1987
22. Camargo ACM, Ribeiro MJVF, Schwartz W N Conversion
and inactivation of opioid peptides by rabbit brain endooligopeptidase A. Biochem Biophys Res Commun 130:932, 1985
23. Boman HG, Boman IA, Andreu D, Li Z-q, Merrifield RB,
Schlenstedt G, Zimmeman R Chemical synthesis and enzymic
processing of precursor forms of cecropins A and B. J ~ iChem
~ l
2645862,1989
24. Bateman A, Singh A, Shustik C, Mars WM, Solomon S: The
isolation and identification of multiple forms of the neutrophil
granule peptides from human leukemic cells. J Biol Chem 286:
7524,1991
25. Mars WM, vanTuinen P, Drabkin HA, White JW, Saunders
G F A myeloid-related sequence that localizes to human chromosome 8q21.1-22. Blood 71:1713,1988
26. Wiedemann LM, Francis GE, Lamb RF, Burns JH, Winnie
JN, MacKenzie ED, Birnie GD: Differentiation stage-specific
expression of a gene during granulopoiesis.Leukemia 3:227,1989
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1992 79: 1532-1537
Characterization of defensin precursors in mature human neutrophils
SS Harwig, AS Park and RI Lehrer
Updated information and services can be found at:
http://www.bloodjournal.org/content/79/6/1532.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.