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The Role of the Propeptide for Processing and Sorting of Human
Myeloperoxidase*
(Received for publication, June 6, 1997, and in revised form, December 4, 1997)
Elinor Andersson‡, Lars Hellman§, Urban Gullberg‡, and Inge Olsson‡
¶
From the ‡Department of Hematology, Research Department 2, E-blocket, University Hospital, S-221 85 Lund
and the §Department of Medical Immunology and Microbiology, University of Uppsala Biomedical Center,
Box 582, S-751 23 Uppsala, Sweden
Myeloperoxidase (MPO), stored in azurophil granules
of neutrophils, is critical for an optimal oxygen-depend-
ent microbicidal activity of these cells. Pro-MPO goes
through a stepwise proteolytic trimming with elimina-
tion of an amino-terminal propeptide to yield one heavy
and one light polypeptide chain. The propeptide of MPO
may have a role in retention and folding of the nascent
protein into its tertiary structure or in targeting of pro-
MPO for processing and storage in granules. A propep-
tide-deleted pro-MPO mutant (MPODpro) was con-
structed to determine if deletion of the propeptide
interferes with processing and targeting after transfec-
tion to the myeloid 32D cell line. Transfection of full-
length cDNA for human MPO results in normal process-
ing and targeting of MPO to cytoplasmic dense
organelles. Although the efficiency of incorporation was
lower for MPODpro, both pro-MPO and MPODpro
showed heme incorporation indicating that the propep-
tide is not critical for this process. Deletion of the
propeptide results in synthesis of a protein that lacks
processing into mature two-chain forms but rather is
degraded intracellularly or secreted. The finding of con-
tinued degradation of MPODpro in the presence of lyso-
somotrophic agents or brefeldin A rules out that the
observed degradation takes place after transfer to gran-
ules. Intracellular pro-MPO has high mannose oligosac-
charide side chains, whereas stored mature MPO was
found to have both high mannose and complex oligosac-
charide side chains as judged by only partial sensitivity
to endoglycosidase H. The propeptide may normally in-
terfere with the generation of certain complex oligosac-
charide chain(s) supported by the finding of high man-
nose side chains in secreted pro-MPO and lack of them
in MPODpro that contained complex oligosaccharide
side chains only. In conclusion, elimination of the
propeptide of pro-MPO blocks the maturation process
and abolishes accumulation of the final product in gran-
ules suggesting a critical role of the propeptide for late
processing of pro-MPO and targeting for storage in
granules.
Neutrophil granulocytes are specialized for a role in host
defense. A regulated pathway targets enzymes and antibiotic
proteins to a storage compartment in these cells consisting of
cytoplasmic azurophil, specific, and gelatinase granules formed
sequentially, whereas a constitutive pathway exports proteins
to the cell surface (1). Azurophil granules are thought to be
specialized lysosomes, and their protein constituents are often
subject to posttranslational glycosylation and proteolytic trim-
ming similar to that of lysosomal enzymes (2). A retention
mechanism may be necessary to avoid constitutive secretion of
granule proteins, and a condensation mechanism is necessary
for efficient packaging. Signals for targeting storage in gran-
ules have been sought within the structure of neutrophil gran-
ule proteins. For instance, a pro-region segment is necessary
for targeting to granules of neutrophil defensins (3), but car-
boxyl-terminal prodomains or asparagine-linked carbohy-
drates of hematopoietic serine proteases are not required in
targeting storage in granules (4, 5). Myeloperoxidase (MPO)
1
of
azurophil granules plays a major role in the oxygen-dependent
killing of microorganisms after release into phagolysosomes by
amplifying the effects of oxygen derivatives formed during the
respiratory burst (6). Pro-MPO undergoes extensive process-
ing, including the removal of an amino-terminal propeptide not
found in mature MPO. Therefore, in this work we have inves-
tigated whether the propeptide of MPO has a role in intracel-
lular trafficking and targeting to granules.
The processing steps for MPO are shown in Fig. 1. Mature
MPO is a 150-kDa tetramer composed of two glycosylated 59–
64-kDa heavy subunits and two unglycosylated 14-kDa light
subunits as a pair of protomers linked together by a disulfide
bond (7). Each heavy subunit carries a covalently bound heme
prosthetic group (8), although the crystal structure of canine
MPO suggests that heme of the intact molecule associates with
both subunits (9). The primary translation product undergoes
cotranslational glycosylation with production of 89-kDa heme-
free apopro-MPO followed by incorporation of heme and con-
version into enzymatically active pro-MPO (7). Processing and
maturation of pro-MPO is a slow process (10) that can be
accomplished only after acquisition of heme (11–13). Calreticu-
lin, a calcium-binding protein that resides in the ER, has been
suggested to function as a molecular chaperone and facilitate
the critical folding of apopro-MPO to allow insertion of heme
followed by conversion to pro-MPO (14). The stepwise process-
ing of pro-MPO has been investigated in myeloid cells (10,
15–20), and the results obtained are consistent with those later
deduced from cDNA sequence data. Thus, during subsequent
processing of pro-MPO the amino-terminal propeptide, a small
peptide between the light and heavy chains, and a single serine
residue at the carboxyl-terminal are removed (21). Intermedi-
* This work was supported by the Swedish Cancer Foundation, the
Swedish Medical Research Council Project No. 11546, the Alfred O
¨
ster-
lund Foundation, and the Greta and Johan Kocks Foundation. The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
¶
To whom correspondence should be addressed: Research Dept. 2,
E-blocket, University Hospital, S-221 85 Lund, Sweden. Tel.: 46-46-
173533; Fax: 46-46-184493; E-mail: inge.olsson@hematologi.lu.se.
1
The abbreviations used are: MPO, myeloperoxidase; [4-
14
C]ALA,
d
-[4-
14
C]aminolevulinic acid hydrochloride; ER, endoplasmic reticulum;
Endo-H, endoglycosidase H; N-glycanase, N-glycosidase F; PCR, polym-
erase chain reaction; BFA, brefeldin A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 8, Issue of February 20, pp. 4747–4753, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 4747
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ate processing forms have been observed with molecular
masses of 81 and 74 kDa (10, 18, 19, 22) of which the smaller
can be converted directly into mature MPO after cleavage
between the heavy and the light subunit (19, 22). This finding
suggests that the amino-terminal propeptide, which does not
seem to be part of the 74-kDa form, is removed during an
intermediate step before final processing.
One can envision a role for the amino-terminal propeptide of
MPO in retention and folding of the nascent protein into its
tertiary structure or in targeting pro-MPO to pregranule struc-
tures for further processing and storage in granules. A propep-
tide-deleted pro-MPO mutant (MPODpro) was constructed to
determine if propeptide deletion interferes with processing and
targeting. In this work, we describe the consequences of these
manipulations for posttranslational processing, intracellular
sorting, and constitutive secretion after transfection of the
cDNA for MPO and MPODpro into the murine myeloid 32D
clone 3 cell line.
EXPERIMENTAL PROCEDURES
Materials—The eucaryotic expression vector pCDNA 3 was from
Invitrogen, British Biotechnology, Oxon, UK. The vector provides a
cytomegalovirus promoter-driven expression of introduced cDNA. The
plasmid also confers resistance to geneticin, allowing selection of re-
combinant cells. [
35
S]Methionine/[
35
S]cysteine (cell labeling grade) was
from Amersham International (Buckinghamshire, UK).
d
-[4-
14
C]Ami-
nolevulinic acid hydrochloride ([4-
14
C]ALA) was from DuPont, Belgium.
Prior to use, [4-
14
C]ALA was concentrated 10-fold in a vacuum centri-
fuge and pH adjusted with NaOH. Percoll and protein A-Sepharose
CL-4B were from Pharmacia (Uppsala, Sweden). Protein G-Sepharose
was from Sigma. Geneticin, N-glycosidase F (N-glycanase) and endogly-
cosidase H (Endo-H) were from Boehringer Mannheim (Mannheim,
Federal Republic of Germany). Brefeldin A (BFA), a gift from Sandoz
AB, was dissolved in methanol and stored at 220 °C.
cDNA, Mutagenesis, and Construction of Expression Vector—A par-
tial cDNA clone encoding approximately 80% of human prepro-MPO
was obtained from the American Type Culture Collection (clone
pMP503, ATCC 57694). This clone lacks the coding region for the
amino-terminal part of the protein. To obtain a full-length clone for
transfection studies, an approximate 500-base pair fragment, originat-
ing from the 59 end of the mRNA, was isolated by PCR amplification.
This fragment was isolated by using one primer directed against a
region starting approximately 80 base pairs upstream of the start codon
and a second primer directed against a region just downstream of a
single XbaI site present in the 59 end of the clone pMP503 (in the coding
region of the clone). The PCR fragment was cloned as an RsaI/XbaI
fragment, and the entire nucleotide sequence was determined for two
separate clones. One of the clones was found to have an identical
sequence to that for MPO. To obtain the full-length MPO clone, two
separate fragments were ligated into the vector pcDNAIneo, one XbaI/
EcoRI fragment originating from the pMP503 clone and one HindIII/
XbaI fragment from the PCR clone. After sequence analysis of 59 and 39
ends of the resulting clone and restriction mapping for a panel of
internal sites, this clone was used for the subsequent transfection
studies and as starting material for the construction of the MPODpro.
Construction of cDNA of MPO Lacking the Propeptide (MPODpro)—For
site-directed mutagenesis cDNA of human MPO (pcDNA1neo/MPO) was
used as template in a two-step “spliced overhang extension” polymerase
chain reaction in the following way. In the first reaction two separate
amplifications with 100 ng of DNA template in a 20-cycle PCR produced two
fragments of myeloperoxidase positioned amino-terminally and carboxyl-
terminally of the propeptide (Pro
43
-Gly
164
), respectively (Fig. 2). By design of
the primers, the “Kozak” consensus leader sequence for maximum transla-
tional efficiency was introduced 59 to the ATG initiation codon, and the
flanking restriction enzyme sites HindIII and BamHI were included for
subsequent cloning into plasmid. The PCR primers in the two amplifications
were upstream 59-GACTTCAAGCTTGCCACCATGGGGGTTCCCTTCTT-
CTCT-39 (primer 1) plus downstream 59-CGGGCAAGTCACCCCCACGTC-
GGGCTGGGGCGTGGCCAGAAT-39 (primer 2), and upstream 59-GACGT-
GGGGGTGACTTGCCCG-39 (primer 3) plus downstream 59-CTTCAGGGA-
TCCCTAGGAGGCTTCCCTCCAGGA-39 (primer 4), respectively (start and
stop codons in boldface and restriction enzyme sites underlined). The PCR
products were isolated on agarose gel, mixed, and subjected to a second
20-cycle splicing PCR amplification with primers 1 and 4, thus creating
MPO lacking the propeptide (MPODpro). The resulting PCR product was
digested by HindIII and BamHI, followed by isolation on agarose gel and
cloning into plasmid (pcDNA3) to create the expression vector
pcDNA3/MPODpro.
All PCRs were performed in a Perkin-Elmer 480 Thermal Cycler
using Pfu polymerase (Stratagene, La Jolla, CA) according to the man-
ufacturer’s instructions.
Cell Culture—32D clone 3 cells (23, 24), kindly provided by G. Rovera
(Philadelphia, PA) were grown in complete medium consisting of
Iscove’s modified Dulbecco’s medium supplemented with 10% heat-
inactivated fetal bovine serum and 30% WEHI-conditioned medium as
a source of interleukin 3 (25). The cell cultures were kept in 5% CO
2
at
37 °C in a fully humidified atmosphere. Exponentially growing cells
were used in all experiments.
Transfection Procedure—32D cells were transfected with
pcDNA1neo/MPO and pcDNA3/MPODpro using the Bio-Rad Electropo-
ration Apparatus (Bio-Rad) with electrical settings of 960 microfarads
and 300 V as described previously (5). Forty-eight hours after electro-
FIG.1. The processing steps of MPO. The primary translation
product undergoes cotranslational cleavage of the signal peptide fol-
lowed by N-linked glycosylation to generate apopro-MPO. Initial proc-
essing also includes acquisition of heme, which yields enzymatically
active pro-MPO. The stepwise processing into mature dimeric MPO
includes removal of the propeptide.
FIG.2.Schematic view of MPO and of the MPO deletion mu-
tant (MPODpro). PCR primers used for construction as described
under “Experimental Procedures” are indicated with arrows. Amino
acids are indicated by three-letter symbols. SP, signal peptide; PRO
propeptide; LIGHT, light chain of mature MPO; HEAVY, heavy chain of
mature MPO.
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poration, geneticin (1 mg/ml) was added to select for recombinant clones
expressing the geneticin resistance of pcDNA 3. Individual clones grow-
ing in the presence of antibiotic were isolated, expanded into mass
cultures, and screened by biosynthetic radiolabeling for expression of
the protein encoded by the transfected cDNA. Clones with the most
pronounced expression were chosen for further experiments.
Biosynthetic Radiolabeling—Biosynthetic radiolabeling of newly
synthesized proteins was performed as described (26). Briefly, cells
were starved for 30 min in methionine/cysteine-free medium, followed
by radiolabeling with 15 or 30
m
Ci/ml [
35
S]methionine/[
35
S]cysteine for
30 or 60 min. In experiments with [4-
14
C]ALA labeling, cells were
incubated with 25
m
Ci/ml for 3 h for radiolabeling. In chase experi-
ments, following radiolabeling, cells were resuspended in complete me-
dium. At timed intervals, cells were withdrawn and lysed or homoge-
nized for subcellular fractionation.
Subcellular Fractionation—Subcellular fractionation was performed
as described (26). Briefly, the postnuclear cell homogenate was fraction-
ated in a Percoll density gradient, after which nine fractions were
collected with all cytosol in fraction 9. The distribution of lysosomes and
Golgi elements in the density gradient was determined by assaying
b
-hexosaminidase and
b
-galactosyltransferase as described elsewhere
(27, 28). Peak activities of
b
-hexosaminidase and galactosyltransferase
were localized in fractions 2 and 6, respectively (data not shown).
Immunoprecipitation—For immunoprecipitation, whole cells or Per-
coll-containing subcellular fractions were solubilized, and biosyntheti-
cally radiolabeled MPO or MPODpro was precipitated with polyclonal
anti-MPO (29) and subjected to electrophoretic analysis followed by
fluorography as described previously (26, 30).
Digestion with Endo-H and N-Glycanase—The susceptibility of MPO
and MPODpro to digestion with Endo-H and N-glycanase was deter-
mined as described (26, 30).
RESULTS
Construction of Full-length and Mutated MPO and Estab-
lishment of Stable Transfectants—To determine whether the
propeptide of pro-MPO carries a targeting signal for granules,
a mutant form of MPO lacking the propeptide (MPODpro) was
constructed by polymerase chain reactions as described under
“Experimental Procedures.” The sequence encoding 122 amino
acids from Pro
43
to Gly
164
(numbered from the methionine
constituting the translation initiation site) was deleted from
MPO cDNA leaving the first three residues of the propeptide
and the entire signal peptide intact (Fig. 2). If the propeptide
plays a role for sorting, MPODpro-protein would, unlike intact
pro-MPO, not be targeted to granules. Likewise, if the propep-
tide plays a role for folding of nascent protein, the mutant
protein might be misfolded and retained in the ER.
Wild type MPO and MPODpro were transfected to 32D cells,
and stable cell clones were established. Clones with synthesis
of protein from transfected cDNA were chosen for further ex-
periments in which the consequences of the MPO mutation
were investigated. The murine origin of the cell line facilitates
the detection of expression of transfected human proteins by
biosynthetic radiolabeling followed by immunoprecipitation.
No endogenous synthesis of MPO in 32D cells is detected with
the antiserum used (data not shown). 32D cells have cytoplas-
mic granule-like vacuoles that have been shown to be able to
accumulate human neutrophil granule constituents such as
defensins expressed from DNA transfected into these cells (3).
In addition, the human hematopoietic serine proteases cathep-
sin G and proteinase 3 have been successfully transfected to
32D cells and targeted to the granule-like vacuoles (5, 32).
These cells were also found to have the machinery for process-
ing of MPO (see below).
Human Wild Type MPO in 32D Cells Is Processed and Tar-
geted to Granules—Stable 32D cells transfected with wild type
cDNA of human MPO show a biosynthesis and processing
pattern of MPO similar to that of human myeloid cells express-
ing MPO. The initially detectable protein is a proform of mo-
lecular mass 89 kDa (pro-MPO) (Fig. 3). As observed earlier in
promyelocytic HL-60 (9, 22, 33) and in PLB 985 cells (11),
processing of pro-MPO into the mature form is slow. A slow
processing of pro-MPO is also observed in transfected 32D cells.
Thus, a 64-kDa heavy chain and a 15-kDa light chain, repre-
senting mature MPO, begin to occur between 6 and 24 h of
chase of the radiolabel (Fig. 3). Additional MPO species with
molecular masses of approximately 45 kDa, precipitated with
anti-MPO, increase with chase of the radiolabel. These pep-
tides are known to be the result of autolytic cleavage of the
heavy subunit (34). Similar to the behavior of endogenous MPO
in myeloid cells, constitutive secretion of pro-MPO to medium
proceeds continually from 32D cells during chase of the
radiolabel (Fig. 3).
MPO is normally targeted to azurophil granules of the neu-
trophil series for storage. To investigate targeting in MPO-
transfected 32D cells, pulse-chase radiolabeling experiments
followed by subcellular fractionation were performed (Fig. 4).
Radiolabeled MPO was found to be slowly translocated to dense
fractions containing the granule-like vacuoles, where it is
clearly visible after 22 h of chase. Translocated (granule-asso-
ciated) MPO is present almost exclusively in fully mature form
consisting of a 64-kDa heavy and 15-kDa light chain. These
results are consistent with earlier data from investigations on
the processing of endogenous MPO in myeloid cells (10, 15–20)
and demonstrate that 32D cells can process human pro-MPO
into mature MPO that is at least partially targeted to a dense
subcellular fraction containing granules.
Both Wild Type MPO and Propeptide-deleted MPO (MPODpro)
Incorporate Heme—Proteolytic processing of endogenous wild type
MPO precursor to the mature storage form in granules requires
incorporation of heme into pro-MPO (11–13). Thus, heme incorpo-
ration occurs prior to removal of the propeptide, and it is therefore
of interest to determine whether the propeptide is necessary for
incorporation of heme. Wild type MPO and propeptide-deleted
MPO (MPODpro) in transfected 32D cells were therefore compared
in this respect. Cells were radiolabeled with [4-
14
C]ALA, a precur-
sor of heme synthesis, followed by immunoprecipitation with an
anti-MPO antibody. As expected, the proform of wild type MPO
incorporates heme, indicated by labeling of the protein with
[4-
14
C]ALA (Fig. 5), confirming earlier results (9). Incorporation of
heme into the propeptide-deleted MPO (MPODpro) is also seen
FIG.3. Processing of MPO in 32D cells. Cells transfected with
cDNA for human MPO were incubated with [
35
S]methionine/[
35
S]cys-
teine for 30 min followed by chase of the label for up to 24 h. At depicted
time points, 20 3 10
6
cells were removed and, after lysis, subjected to
immunoprecipitation with anti-MPO. In addition, MPO was also pre-
cipitated from the incubation medium at each chase time point. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel electro-
phoresis in a 5–20% gradient gel followed by fluorography. The fluoro-
gram was exposed for 3 days. The positions of the pro-MPO (pro), the
heavy (
a
) and the light (
b
) subunits are indicated to the right with
arrows. Numbers to the left in this and subsequent figures are the
values of molecular mass standards.
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(Fig. 5), and this form has a molecular mass of 76 kDa. Thus, the
presence of the propeptide is not necessary for incorporation of
heme into the proform of MPO. On the other hand, the relative
efficiency of insertion of heme seemed to be lower for MPODpro
compared with the normal proform. This comparison was possible
to make as control radiolabeling with [
35
S]methionine/[
35
S]cysteine
showed immunoprecipitates with similar density for both MPO and
MPODpro also when visualized through shorter exposure time of
the fluorogram than in Fig. 5 (not shown).
Lack of Processing and Targeting to Granules of Propeptide-
deleted MPO—32D cells expressing MPODpro show an abnor-
mal biosynthesis and processing pattern of MPO (Fig. 6). As
expected, a 76-kDa polypeptide is synthesized that may corre-
spond to the size of a pro-MPO that lacks propeptide. However,
deletion of the propeptide results in synthesis of a protein that
lacks processing into mature two-chain forms and is secreted
upon prolonged chase of the radiolabeled product or degraded
intracellularly (Fig. 6). Unlike secreted full-length pro-MPO
(Fig. 3), secreted MPODpro disappears with time (Fig. 6), sug-
gesting extracellular degradation. To investigate if MPODpro
could be transferred to granules, pulse-chase radiolabeling ex-
periments were carried out followed by subcellular fraction-
ation. Only trace amounts of radiolabeled MPODpro and deg-
radation products are visible in dense fractions containing the
granule-like vacuoles (Fig. 7).
Since only trace amounts of partially degraded MPODpro are
detected in dense fractions, the results suggest that degrada-
tion of non-secreted MPODpro preferentially takes place in a
pre-granule compartment. However, it is possible that substan-
tial degradation of MPODpro would still take place in granule-
like vacuoles but too rapidly to be detectable. Therefore, to rule
out considerable transfer to granules with degradation, exper-
iments were performed with chloroquine and NH
4
Cl, agents
that block lysosomal proteolysis. In particular, chloroquine
blocks late proteolytic processing of MPO (16). Biosynthetic
radiolabeling of MPODpro in transfected 32D cells and chase of
the radiolabel in the presence of NH
4
Cl or chloroquine does not
diminish the degradation rate of MPODpro, indicating that
lysosomes are not involved to any great extent in the degrada-
tion observed (Fig. 8). The secretion of MPODpro also occurs
FIG.4.Targeting of MPO to granules in 32D cells. Cells trans-
fected with cDNA for human MPO were incubated with [
35
S]methi-
onine/[
35
S]cysteine for 30 min followed by chase of the label for 5 and
22 h. At these time points, 100 3 10
6
cells were homogenized after
which subcellular fractionation of the postnuclear supernatant was
performed by centrifugation in Percoll followed by collection of nine
subcellular fractions, fraction 9 containing all the cytosol. The fractions
were lysed and subjected to immunoprecipitation with anti-MPO. Im-
munoprecipitates were analyzed as described in the legend to Fig. 3.
The fluorogram was exposed for 11 days. The positions of the pro-MPO
(pro), the heavy (
a
), and the light (
b
) subunits are indicated to the right
with arrows. Peak activities of
b
-hexosaminidase, fraction 2, and galac-
tosyltransferase, fraction 6, indicate the position of lysosomes and Golgi
elements, respectively.
FIG.5.Incorporation of heme into pro-MPO and propeptide-
deleted MPO (MPODpro). 32D cells (20 3 10
6
) transfected with
cDNA for MPO or MPODpro were incubated for 3 h with [4-
14
C]ALA
and for 2 h with [
35
S]methionine (
35
S-meth)/[
35
S]cysteine followed by
lysis and immunoprecipitation with anti-MPO. Immunoprecipitates
were analyzed as described in the legend to Fig. 3. The fluorogram was
exposed for 12 weeks. The positions of the proform of MPO (pro) and the
MPODpro (Dpro), respectively, are indicated with arrows.
FIG.6. Lack of processing of propeptide-deleted MPO
(MPODpro) in 32D cells. Cells transfected with cDNA for human
MPODpro were incubated with [
35
S]methionine/[
35
S]cysteine for 30 min
followed by chase of the label for up to 24 h. At depicted time points,
20 3 10
6
cells were removed and, after lysis, subjected to immunopre-
cipitation with anti-MPO. In addition, MPO was also precipitated from
the incubation medium at each chase time point. Immunoprecipitates
were analyzed as described in the legend to Fig. 3. The position of the
MPODpro (Dpro) is indicated with an arrow. The fluorogram was ex-
posed for 5 days.
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during incubation with NH
4
Cl and chloroquine, although it is
reduced by NH
4
Cl. Secreted MPODpro is of slightly higher
molecular weight than the protein retained intracellularly both
in the control and in the presence of NH
4
Cl or chloroquine,
indicating additional glycosylation during the secretory proc-
ess. To characterize the localization of the degradation further,
cell radiolabeling experiments were performed in the presence
of brefeldin A (BFA), which induces the disassembly of the
Golgi complex, thus blocking ER-Golgi transport (35, 36). As
expected, BFA blocks the secretion of MPODpro completely, but
total degradation is observed with time (Fig. 8), indicating that
degradation of MPODpro can occur in a pre-Golgi compart-
ment. A higher molecular weight form of MPODpro is observed
with time in the presence of BFA (Fig. 8). This form, which is
also degraded, is probably the result of aberrant glycosylation
in the presence of BFA.
Secreted MPODpro Is Resistant to Endo-H Indicating Com-
plex Oligosaccharide Side Chains—Both intracellular pro and
mature MPO are normally sensitive to digestion with Endo-H
indicating the presence of high mannose oligosaccharide side
chains (16, 17, 20). Consistent with published data, the intra-
cellular forms of pro and mature MPO in 32D cells transfected
with wild type MPO both show sensitivity to digestion with
Endo-H indicating the presence of high mannose groups (Fig.
9A). However, an additional reduction in size is observed for
the large subunit of mature MPO upon digestion with N-gly-
canase as compared with digestion with Endo-H. To ensure
that complete digestion had taken place with Endo-H and
N-glycanase, the concentrations of glycosidase were varied
(Fig. 9A). The results show that the molecular mass is reduced
by 4.5 kDa upon complete digestion with Endo-H and by 12.5
kDa upon complete digestion with N-glycanase (mean values
from two separate experiments). Thus, the large subunit con-
tains Endo-H-resistant oligosaccharides indicating the pres-
ence of complex oligosaccharides. The presence not only of high
mannose but also of complex oligosaccharides in the large
subunit of MPO has for technical reasons been overlooked in
previous studies. We observed both high mannose and complex
oligosaccharides also in the large MPO subunit of HL-60 cells
that normally produce MPO (data not shown). Also the secreted
pro-MPO shows partial Endo-H resistance both in the 32D cells
transfected with MPO (Fig. 9A) and in HL-60 cells (data not
shown). This indicates that the secreted pro-MPO achieves
some complex mannose groups during passage through the
Golgi compartment during constitutive secretion. The secreted
pro-MPO shows heterogeneity, and removal of all oligosaccha-
rides with N-glycanase reveals at least two distinct protein
forms that differ in molecular mass (Fig. 9A). The smaller
protein form is resistant and the larger is sensitive to Endo-H.
Two distinct protein forms are also observed for secreted pro-
MPO in the HL-60 cell line (data not shown).
The glycosylation pattern for MPODpro is shown in Fig. 9B.
Intracellular MPODpro is highly sensitive to digestion with
Endo-H. In contrast, the secreted MPODpro is homogeneous
and Endo-H-resistant corresponding to the presence of complex
oligosaccharide side chains. As judged by results from digestion
with N-glycanase, the secreted form contains slightly more
carbohydrate than the intracellular form consistent with proc-
essing into complex forms.
DISCUSSION
Neutrophils carry at least the following three types of gran-
ules: azurophil, specific, and gelatinase granules (37). Unique
constituents of azurophil granules such as MPO and serine
proteases are stored in enzymatically active forms, whereas
proteases of specific and gelatinase granules are stored in
FIG.7. Lack of targeting of propeptide-deleted MPO
(MPODpro) in 32D cells. Cells transfected with cDNA for human
MPODpro were incubated with [
35
S]methionine/[
35
S]cysteine for 1 h
followed by chase of the label for 3 and 12 h. At these time points, 100 3
10
6
cells were homogenized after which subcellular fractionation of the
postnuclear supernatant was performed by centrifugation in Percoll
followed by collection of nine subcellular fractions, fraction 9 containing
all the cytosol. The fractions were lysed and subjected to immunopre-
cipitation with anti-MPO. Immunoprecipitates were analyzed as de-
scribed in the legend to Fig. 3. The position of MPODpro (Dpro)is
indicated with an arrow. The fluorogram was exposed for 5 days. Peak
activities of
b
-hexosaminidase, fraction 2, and galactosyltransferase,
fraction 6, indicate the position of lysosomes and Golgi elements,
respectively.
FIG.8.The effect of NH
4
Cl, chloroquine, and brefeldin A on the
processing of propeptide-deleted MPO (MPODpro) in 32D cells.
Cells transfected with cDNA for human MPODpro were incubated with
[
35
S]methionine/[
35
S]cysteine for 30 min followed by chase of the label
for up to 24 h. Separate experiments were carried out with 10 mmol/
liter NH
4
Cl, 1 mmol/liter chloroquine, or 5 mg/ml brefeldin A; cells were
preincubated with these agents for 30 min, and the agents were also
present during pulse labeling and during chase of the label. At depicted
time points, 20 3 10
6
cells were removed and, after lysis, subjected to
immunoprecipitation with anti-MPO. In addition, MPO was also pre-
cipitated from the incubation medium at each chase time point. Immu-
noprecipitates were analyzed as described in the legend to Fig. 3. The
fluorograms were exposed for 5 days. The position of MPODpro (Dpro)is
indicated with an arrow.
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inactive forms to become activated first after exocytosis (1).
Thus, azurophil granule enzymes are activated prior to storage,
e.g. by removal of an activation peptide from proforms of the
serine proteases (38). The activation peptide keeps enzyme
activity latent and is removed as a late step during intracellu-
lar trafficking. Likewise, the propiece of prodefensin is re-
moved before storage in azurophil granules of mature antibi-
otic defensin, which is non-catalytic. In this case the propiece is
essential for subcellular trafficking and sorting, e.g. by inter-
action with a complementary hydrophobic part of mature de-
fensin peptide or with a chaperone protein that facilitates
transit and protects against adverse effects of the mature pep-
tide (3). MPO, on the other hand, is enzymatically active prior
to proteolytic removal of its propiece (1). Therefore, the pro-
piece of pro-MPO probably does not have a role in protection
against peroxidation during intracellular travelling unless it
interacts with other molecules for this purpose. Rather, the
propiece may play a role in conformational stability, retention,
and/or sorting. The results of the present work are viewed in
this context.
The murine myeloid 32D cell line was successfully employed
for investigation of MPO synthesis. Thus 32D cells stably
transfected with the cDNA for human MPO demonstrate the
normal characteristics of MPO synthesis. Heme is incorporated
into apopro-MPO resulting in production of pro-MPO that is
processed into mature heterodimeric protein targeted to gran-
ule-like vacuoles of 32D cells. The same cell line has previously
been utilized for the investigation of the posttranslational proc-
essing of human neutrophil defensin (3) and neutrophil serine
proteases (5, 32). Previous attempts to use Chinese hamster
ovary cells (39–41), baby hamster kidney cells (42), or baculo-
virus-infected Sf9 cells (43) for expression of MPO cDNA have
not resulted in processing of the protein product. But, recently
the human erythroleukemia K562 cell line transfected with
MPO cDNA showed the typical processing seen during biosyn-
thesis of MPO in myeloid cells (44).
What do our results reveal about the role of the propiece for
post-translational processing, targeting, and secretion of MPO?
Propeptide-deleted pro-MPO (MPODpro) was found to lack
processing into mature light and heavy chain MPO and pri-
marily became secreted to the exterior or degraded in a
pregranule compartment. Therefore, the propiece is necessary
for subcellular trafficking. The finding of continued degrada-
tion of MPODpro in the presence of lysosomotrophic agents and
BFA rules out that the observed degradation should, after all,
take place upon transfer to granules but too rapidly to be
detectable. The results obtained for MPO processing can be
compared with those for the lysosomal hydrolase cathepsin D,
in which the precursor domains are indispensible for the for-
mation of a stable proenzyme (45). Thus, in the latter case the
propeptide appears to be necessary for the correct folding of the
proenzyme that is required for trafficking. However, it was not
possible to prove a direct role for the propeptide of cathepsin D
in sorting, because the propeptide when attached to a secretory
protein,
a
-lactalbumin, did not redirect it for lysosomes indi-
cating that the propeptide might not be necessary for the sort-
ing process as such (45). If a sorting machinery were to recog-
nize precursors rather than mature peptides, propiece-deleted
pro-MPO when available for sorting would be secreted instead
of being sorted for storage in granules. This seems to be con-
sistent with the finding that a large part of MPODpro is se-
creted, whereas almost none is transported to granules. How-
ever, a part of MPODpro is retained, most likely in the ER, and
degraded. Proteasomes may have a role, although unproven, in
proteolysis of MPODpro. One theoretical explanation is mis-
folding; if the propiece were required for folding, misfolding of
a propiece-deleted pro-MPO might lead to retention in the ER
because of lack of native conformation. On the other hand, the
characteristics of MPODpro do not indicate misfolding, because
MPODpro can incorporate heme, can be secreted, and can
achieve complex oligosaccharide side chains (see below) when
transferred to trans-Golgi. It is important to consider that
processing of pro-MPO is normally extremely slow, and it can
take as long as 6–15 h to chase radiolabel from the precursor
into mature MPO (10, 11). If MPODpro were degraded within
this period processing forms would not be visible. Thus, it is
suggested that undegraded MPODpro is transferred to Golgi
and secreted because the lack of propeptide prohibits targeting
to granules. In conclusion, removal of the propiece leads to a
block in the normal trafficking and maturation process of MPO.
The behavior of pro-MPO and MPODpro differs during se-
cretion. First, the molecular mass of secreted MPODpro, but
not of secreted pro-MPO, is slightly higher than that of the
corresponding intracellular forms. Second, in contrast to
MPODpro, intracellular pro-MPO in HL-60 cells (16) or trans-
fected pro-MPO of 32D cells of the present work does not
acquire detectable complex oligosaccharide side chains. There-
FIG.9. Oligosaccharide side chains of processing forms of
MPO and MPODpro. 32D cells transfected with MPO (A)orMPODpro
(B) were incubated with 30
m
Ci/ml [
35
S]methionine/[
35
S]cysteine for 30
min (pulse). Chase experiments of the radiolabel were performed for
24h(A)and6h(B), respectively. At depicted time points, 100 3 10
6
cells were removed and, after lysis, subjected to immunoprecipitation
with anti-MPO. In addition, MPO or MPODpro, respectively, was also
precipitated from the incubation medium at the end of chase. A, ali-
quoted immunoprecipitates were incubated with either Endo-H (Endo-
H1) (units 3 10
22
/ml) or N-glycanase (N-Glyc1) (units 3 10
21
/ml), or
served as controls (2). Immunoprecipitates of cells from 24-h chase
experiments were incubated with several different concentrations of
Endo-H or N-glycanase to ensure that complete digestion had taken
place, but results are given only for two concentrations of glycosidases.
B, aliquoted immunoprecipitates were incubated with either 0.1
units/ml of Endo-H (Endo-H1) or 7 units/ml of N-glycanase (N-glyc 1)
for 24 h, or served as controls (2). In both A and B SDS-polyacrylamide
gel electrophoresis and fluorography were performed, and the fluoro-
grams were exposed for 18 days.
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fore, the propeptide of pro-MPO may promote resistance to
mannosidases and/or glycosyltransferases whose action is re-
quired for production of complex mannose groups in a late
Golgi compartment. However, results from baby hamster kid-
ney cells transfected with MPO have shown that secreted pro-
MPO contains at least one Endo-H-resistant oligosaccharide
indicating the presence of complex mannose groups (42). Thus
the presence of the propeptide does not prevent generation of
complex oligosaccharides totally. Likewise, the present results
show that secreted pro-MPO contains complex mannose groups
which must have been added at a rapidly transient step as they
are not detectable in cellular pro-MPO. That MPODpro, in
contrast, contains complex mannose groups exclusively sug-
gests that the propiece can, when present, prevent the gener-
ation of certain complex mannose side chains.
MPODpro lacks targeting to granules and is instead con-
veyed to the secretory path with concomitant synthesis of com-
plex mannose groups during passage of trans-Golgi. The find-
ing of some complex mannose groups in secreted pro-MPO also
indicates that at least part of it has travelled the secretory
pathway through trans-Golgi. The secreted pro-MPO consists
of at least two protein forms with different molecular masses
easily seen after removal of carbohydrate with N-glycanase and
only the smaller one contains complex mannose groups. Simi-
lar extracellular pro-MPO forms were observed in superna-
tants from HL-60 cells (data not shown) indicating that their
occurrence may be a general phenomenon. It is possible that
the two forms have arrived at the cell surface through separate
routes. The higher molecular mass component might have
come through a secretory path excluding trans-Golgi and lack-
ing complex mannose groups, whereas the lower molecular
mass form might have come through another path. We specu-
late that the latter path is that for processing and storage of
MPO but that it is linked to the secretory pathway at a distal
point. Thus, the secreted lower molecular mass species could
represent an intermediate MPO processing form that is in part
released to the secretory pathway from an acidic pregranule
compartment in which intermediate processing forms have
been suggested to be produced (19, 22). Final processing occurs
later (in granules) when escape to the secretory pathway is
blocked. Intermediate MPO processing might take place in late
acidic endosomes after receiving contents, including pro-MPO,
from Golgi-derived vesicles. Because late endosomes are in-
volved in transport in and out of the cell, it is possible that some
material delivered to late endosomes escapes to the outside. An
additional unproven possibility is that secreted pro-MPO, but
not MPODpro, can re-enter the cell through receptor-mediated
uptake into the endocytic pathway with transport to late en-
dosomes and granules for processing and storage.
Acquisition of heme by heme-free apopro-MPO seems to be a
rate-limiting step in subsequent processing into mature MPO
of hematopoietic cells (11–13). The calcium-binding calreticu-
lin, present in the ER of many cells, was shown to interact
specifically with fully glycosylated apopro-MPO during a rela-
tively short period early in MPO synthesis and not with heme-
containing pro-MPO or mature MPO (14). These data suggest a
role of calreticulin as a molecular chaperone facilitating heme
insertion after which the calreticulin-MPO precursor complex
dissociates and pro-MPO can leave the ER for further process-
ing and targeting. Our results show that pro-MPO and
MPODpro both have incorporated heme although the relative
efficiency of incorporation is lower for MPODpro. In any case,
the lack of propeptide may not block the interaction between
apopro-MPO and calreticulin that is proposed to be necessary
for heme incorporation (14). The propeptide seems not to play a
major role for the initial processing of the translational product
but rather plays a role later in processing and trafficking.
Finally, our results provide novel information on MPO bio-
synthesis, processing, and targeting. Elimination of the
propeptide from pro-MPO blocks the maturation process, al-
lows secretion, but abolishes accumulation of the final product
for storage, suggesting a critical role of the propeptide for late
processing of pro-MPO.
Acknowledgments—We greatly appreciate the skilled technical as-
sistance of Eva Nilsson and Ann-Maj Persson.
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Gullberg and Inge Olsson
Elinor Andersson, Lars Hellman, Urban
and Sorting of Human Myeloperoxidase
The Role of the Propeptide for Processing
CELL BIOLOGY AND METABOLISM:
doi: 10.1074/jbc.273.8.4747
1998, 273:4747-4753.J. Biol. Chem.
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