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
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ABSTRACT |
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Myeloperoxidase (MPO), stored in azurophil
granules of neutrophils, is critical for an optimal
oxygen-dependent microbicidal activity of these cells.
Pro-MPO goes through a stepwise proteolytic trimming with elimination
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
propeptide-deleted pro-MPO mutant (MPOpro) was constructed to
determine if deletion of the propeptide interferes with processing and
targeting after transfection to the myeloid 32D cell line. Transfection
of full-length cDNA for human MPO results in normal processing and
targeting of MPO to cytoplasmic dense organelles. Although the
efficiency of incorporation was lower for MPO
pro, both pro-MPO and
MPO
pro showed heme incorporation indicating that the propeptide 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 continued degradation of MPO
pro in the presence of
lysosomotrophic agents or brefeldin A rules out that the observed
degradation takes place after transfer to granules. Intracellular
pro-MPO has high mannose oligosaccharide side chains, whereas stored
mature MPO was found to have both high mannose and complex
oligosaccharide side chains as judged by only partial sensitivity to
endoglycosidase H. The propeptide may normally interfere with the
generation of certain complex oligosaccharide chain(s) supported by the
finding of high mannose side chains in secreted pro-MPO and lack of
them in MPO
pro 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 granules suggesting a critical role of the propeptide for late
processing of pro-MPO and targeting for storage in granules.
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INTRODUCTION |
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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 trimming 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 granules have been sought within the structure of neutrophil granule proteins. For instance, a pro-region segment is necessary for targeting to granules of neutrophil defensins (3), but carboxyl-terminal prodomains or asparagine-linked carbohydrates 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 processing, including the removal of an amino-terminal propeptide not found in mature MPO. Therefore, in this work we have investigated whether the propeptide of MPO has a role in intracellular 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 conversion 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). Calreticulin, 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 processing 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). Intermediate 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.
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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 structures for further
processing and storage in granules. A propeptide-deleted pro-MPO mutant
(MPOpro) 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 MPO
pro into the murine myeloid 32D clone 3 cell line.
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EXPERIMENTAL PROCEDURES |
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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
recombinant cells.
[35S]Methionine/[35S]cysteine (cell
labeling grade) was from Amersham International (Buckinghamshire, UK).
-[4-14C]Aminolevulinic acid hydrochloride
([4-14C]ALA) was from DuPont, Belgium. Prior to use,
[4-14C]ALA was concentrated 10-fold in a vacuum
centrifuge 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 endoglycosidase 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
20 °C.
cDNA, Mutagenesis, and Construction of Expression
Vector--
A partial 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, originating from the 5' 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 5' 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 5' and 3' 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 MPOpro.
Construction of cDNA of MPO Lacking the Propeptide
(MPOpro)--
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
(Pro43-Gly164), respectively (Fig.
2). By design of the primers, the
"Kozak" consensus leader sequence for maximum translational
efficiency was introduced 5' 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
5'-GACTTCAAGCTTGCCACCATGGGGGTTCCCTTCTTCTCT-3' (primer 1) plus downstream
5'-CGGGCAAGTCACCCCCACGTCGGGCTGGGGCGTGGCCAGAAT-3' (primer 2), and
upstream 5'-GACGTGGGGGTGACTTGCCCG-3' (primer 3) plus downstream
5'-CTTCAGGGATCCCTAGGAGGCTTCCCTCCAGGA-3' (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 (MPO
pro). 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/MPO
pro.
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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% CO2 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/MPOpro using the Bio-Rad
Electroporation Apparatus (Bio-Rad) with electrical settings of 960 microfarads and 300 V as described previously (5). Forty-eight hours
after electroporation, geneticin (1 mg/ml) was added to select for
recombinant clones expressing the geneticin resistance of pcDNA 3. Individual clones growing 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 µCi/ml [35S]methionine/[35S]cysteine for 30 or 60 min. In experiments with [4-14C]ALA labeling, cells were incubated with 25 µCi/ml for 3 h for radiolabeling. In chase experiments, following radiolabeling, cells were resuspended in complete medium. At timed intervals, cells were withdrawn and lysed or homogenized for subcellular fractionation.
Subcellular Fractionation--
Subcellular fractionation was
performed as described (26). Briefly, the postnuclear cell homogenate
was fractionated 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 -hexosaminidase and
-galactosyltransferase as described elsewhere (27, 28). Peak
activities of
-hexosaminidase and galactosyltransferase were
localized in fractions 2 and 6, respectively (data not shown).
Immunoprecipitation--
For immunoprecipitation, whole cells or
Percoll-containing subcellular fractions were solubilized, and
biosynthetically radiolabeled MPO or MPOpro 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 MPOpro to digestion with Endo-H and N-glycanase
was determined as described (26, 30).
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RESULTS |
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Construction of Full-length and Mutated MPO and Establishment 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 (MPOpro) was constructed by polymerase chain
reactions as described under "Experimental Procedures." The
sequence encoding 122 amino acids from Pro43 to
Gly164 (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,
MPO
pro-protein would, unlike intact pro-MPO, not be targeted to
granules. Likewise, if the propeptide plays a role for folding of
nascent protein, the mutant protein might be misfolded and retained in
the ER.
Human Wild Type MPO in 32D Cells Is Processed and Targeted 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 expressing MPO. The initially detectable protein is a proform of molecular 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, representing 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 peptides 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).
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Both Wild Type MPO and Propeptide-deleted MPO (MPOpro)
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 incorporation
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
(MPO
pro) in transfected 32D cells were therefore compared in this
respect. Cells were radiolabeled with [4-14C]ALA, a
precursor 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 [4-14C]ALA (Fig. 5),
confirming earlier results (9). Incorporation of heme into the
propeptide-deleted MPO (MPO
pro) is also seen (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 MPO
pro compared with the normal proform. This comparison
was possible to make as control radiolabeling with
[35S]methionine/[35S]cysteine showed
immunoprecipitates with similar density for both MPO and MPO
pro also
when visualized through shorter exposure time of the fluorogram than in
Fig. 5 (not shown).
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Lack of Processing and Targeting to Granules of Propeptide-deleted
MPO--
32D cells expressing MPOpro show an abnormal biosynthesis
and processing pattern of MPO (Fig. 6).
As expected, a 76-kDa polypeptide is synthesized that may correspond 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 MPO
pro disappears
with time (Fig. 6), suggesting extracellular degradation. To
investigate if MPO
pro could be transferred to granules, pulse-chase
radiolabeling experiments were carried out followed by subcellular
fractionation. Only trace amounts of radiolabeled MPO
pro and
degradation products are visible in dense fractions containing the
granule-like vacuoles (Fig. 7).
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Secreted MPOpro Is Resistant to Endo-H Indicating Complex
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 intracellular 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-glycanase 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 contains Endo-H-resistant oligosaccharides indicating the presence 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
oligosaccharides 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).
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DISCUSSION |
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Neutrophils carry at least the following three types of granules: 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 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 intracellular trafficking. Likewise, the propiece of prodefensin is removed before storage in azurophil granules of mature antibiotic defensin, which is non-catalytic. In this case the propiece is essential for subcellular trafficking and sorting, e.g. by interaction with a complementary hydrophobic part of mature defensin peptide or with a chaperone protein that facilitates transit and protects against adverse effects of the mature peptide (3). MPO, on the other hand, is enzymatically active prior to proteolytic removal of its propiece (1). Therefore, the propiece 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 granule-like vacuoles of 32D cells. The same cell line has previously been utilized for the investigation of the posttranslational processing 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 baculovirus-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 biosynthesis 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 (MPOpro) was found to lack processing into mature light and heavy chain MPO and primarily became secreted to
the exterior or degraded in a pregranule compartment. Therefore, the propiece is necessary for subcellular trafficking. The finding of
continued degradation of MPO
pro 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 formation 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,
-lactalbumin, did not redirect it
for lysosomes indicating that the propeptide might not be necessary for
the sorting process as such (45). If a sorting machinery were to
recognize 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 consistent with the
finding that a large part of MPO
pro is secreted, whereas almost none
is transported to granules. However, a part of MPO
pro is retained,
most likely in the ER, and degraded. Proteasomes may have a role,
although unproven, in proteolysis of MPO
pro. One theoretical
explanation is misfolding; 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 MPO
pro do not indicate misfolding, because MPO
pro 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 MPO
pro
were degraded within this period processing forms would not be visible.
Thus, it is suggested that undegraded MPO
pro 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 MPOpro differs during secretion. First,
the molecular mass of secreted MPO
pro, but not of secreted pro-MPO,
is slightly higher than that of the corresponding intracellular forms.
Second, in contrast to MPO
pro, intracellular pro-MPO in HL-60 cells
(16) or transfected pro-MPO of 32D cells of the present work does not
acquire detectable complex oligosaccharide side chains. Therefore, the
propeptide of pro-MPO may promote resistance to mannosidases and/or
glycosyltransferases whose action is required for production of complex
mannose groups in a late Golgi compartment. However, results from baby
hamster kidney 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 MPO
pro, in contrast, contains complex mannose
groups exclusively suggests that the propiece can, when present,
prevent the generation of certain complex mannose side chains.
MPOpro lacks targeting to granules and is instead conveyed to the
secretory path with concomitant synthesis of complex mannose groups
during passage of trans-Golgi. The finding 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. Similar extracellular pro-MPO forms were observed in
supernatants 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 lacking complex mannose groups, whereas
the lower molecular mass form might have come through another path. We
speculate 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
involved 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
MPO
pro, can re-enter the cell through receptor-mediated uptake into
the endocytic pathway with transport to late endosomes 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 calreticulin, present in the ER of many
cells, was shown to interact specifically with fully glycosylated
apopro-MPO during a relatively 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 processing and
targeting. Our results show that pro-MPO and MPOpro both have
incorporated heme although the relative efficiency of incorporation is
lower for MPO
pro. 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 biosynthesis, processing, and targeting. Elimination of the propeptide from pro-MPO blocks the maturation process, allows secretion, but abolishes accumulation of the final product for storage, suggesting a critical role of the propeptide for late processing of pro-MPO.
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ACKNOWLEDGEMENTS |
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We greatly appreciate the skilled technical assistance of Eva Nilsson and Ann-Maj Persson.
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FOOTNOTES |
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* This work was supported by the Swedish Cancer Foundation, the Swedish Medical Research Council Project No. 11546, the Alfred Österlund 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. 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: Research Dept. 2, E-blocket, University Hospital, S-221 85 Lund, Sweden. Tel.: 46-46-173533; Fax: 46-46-184493; E-mail: inge.olsson{at}hematologi.lu.se.
1
The abbreviations used are: MPO,
myeloperoxidase; [4-14C]ALA,
-[4-14C]aminolevulinic acid hydrochloride; ER,
endoplasmic reticulum; Endo-H, endoglycosidase H;
N-glycanase, N-glycosidase F; PCR, polymerase
chain reaction; BFA, brefeldin A.
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REFERENCES |
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