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(Journal of Leukocyte Biology. 2002;71:279-288.)
© 2002 by Society for Leukocyte Biology

Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor

E. Bülow*, W. M. Nauseef{dagger}, M. Goedken{dagger}, S. McCormick{dagger}, J. Calafat{ddagger}, U. Gullberg* and I. Olsson*

* Department of Hematology, Lund University, Sweden;
{dagger} Inflammation Program and Department of Medicine, Veterans Administration Medical Center and University of Iowa, Iowa City; and
{ddagger} The Netherlands Cancer Institute, Amsterdam

Correspondence: Elinor Bülow, M.D., Dept. of Hematology, C14, BMC, S-221 84 Lund, Sweden. E-mail: elinor.bulow{at}hematologi.lu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein {alpha}1-microglobulin ({alpha}1-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to {alpha}1-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of {alpha}1-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

Key Words: neutrophil • granule • secretory pathway • {alpha}1-microglobulin • secretion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human polymorphonuclear neutrophil (PMN) precursors in the bone marrow synthesize an array of biologically active granule proteins, among which is myeloperoxidase (MPO), and mature PMNs contain several different types of granules, each distinguished by their unique contents [1 ]. Targeting the granule proteins to their final intracellular storage compartments requires retrieval of newly synthesized protein from the constitutive secretory pathway as well as sorting into the distinct subtypes of storage granules for retention. The sorting of proteins into distinct granule subtypes reflects coordinated expression of the genes encoding these proteins [2 ]. Thus, the composition of a given granule reflects the array of granule protein genes expressed during that specific point of granulopoietic differentiation (reviewed in [1 ]). Whereas sorting into distinct subtypes of granules thus reflects the timing of transcriptional regulation, the mechanism that retrieves myeloid granule proteins from the secretory pathway for storage is not known (reviewed in [3 ]). In contrast to regulated delivery of lysosomal proteins for storage by the mannose-6-phosphate receptor system (reviewed in [4 ]), no specific targeting system for myeloid granule proteins has been identified yet. Moreover, heterologously expressed nonmyeloid proteins may be sorted for storage in myeloid cells. Thus, the normally secretory lipopolysaccharide-binding protein (LBP), which shares the same structure as the myeloid bactericidal/permeability-increasing protein (BPI), was sorted for storage when expressed in myeloid cell lines [5 ].

Optimal microbicidal activity of human PMNs requires the MPO-hydrogen peroxide-halide system. Upon stimulation, PMNs assemble and activate the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase, thus generating hydrogen peroxide from molecular oxygen, and degranulate, thereby releasing the azurophilic granule protein MPO into the phagolysosome. MPO catalyzes the formation of HOCl, which together with other highly reactive species generated within the phagosome constitutes a potent cytocidal system. Synthesized during the promyelocytic stage of myeloid development, the MPO precursor apoproMPO interacts transiently and reversibly with molecular chaperones in the endoplasmic reticulum (ER), including calreticulin (CRT) and calnexin (CLN), and subsequent incorporation of heme generates the enzymatically active 90-kDa proMPO (reviewed in [3 , 6 7 8 ]). Heme acquisition is required for proteolytic maturation and export from the ER, indicating that heme incorporation induces conformational changes necessary for successful maturation and intracellular targeting. Although a minor portion of newly synthesized proMPO is secreted constitutively from the cell, most of the precursor undergoes dimerization, modification of oligosaccharide sidechains, and proteolytic processing with final compartmentalization in the azurophilic granule. Among these proteolytic events is the cleavage of the amino terminal 125 amino acid propeptide [9 , 10 ], a modification necessary for the normal maturation of the precursor to the native form of MPO.

To investigate further the specificity of sorting for storage in myeloid cells, we stably expressed the genes for several nonmyeloid proteins in several myeloid cell lines. In addition, we assessed a possible role of the amino-terminal MPO propeptide in targeting by examining the impact of propeptide deletion and the intracellular trafficking of chimeric constructs containing the MPO propeptide (MPOpro) coupled with the amino terminus of nonmyeloid proteins. Our results indicate that normally secreted, nonmyeloid proteins can be targeted for storage granules of myeloid cells. We also observed that the chimeric proteins were retained in the ER to a greater degree than were native proteins. Although the ER retention observed may also result from slow or abnormal folding of the chimeric proteins, the propeptide may play an important role in the long retention time normally observed in MPO precursors in the ER. Prolonged retention may be required for the additional time needed for heme acquisition, a modification necessary for exit from the ER and functional maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Constructs were created for eukaryotic expression using vector pcDNA3 (Invitrogen, Groningen, The Netherlands) for transfection of 32D cells, pREP10 (Invitrogen, San Diego, CA) for stable transfection of K562 cells [11 ], and pEF-PGKpac for stable transfection of PLB-985 cells [12 ]. [35S]Methionine/[35S]cysteine (cell-labeling grade) was from ICN Pharmaceuticals (Costa Mesa, CA). Lymphoprep was from Nycomed (Oslo, Norway). X-vivo 15 and heat-inactivated fetal bovine serum (FBS) were from BioWhittaker (Verviers, Belgium). L-glutamine was from Gibco BRL (Life Technologies, Grand Island, NY). Human granulocyte colony-stimulating factor (G-CSF) was from Amgen (Thousand Oaks, CA). Percoll and protein A-sepharose CL-4B were from Pharmacia (Uppsala, Sweden). Brefeldin A (BFA) and 2-mercaptoethanol were from Sigma-Aldrich Co. (St. Louis, MO). Geneticin, endoglycosidase H (Endo-H), N-glycosidase F (N-glyc), and CompleteTM (protease-inhibitor cocktail tablets) were from Boehringer Mannheim (Mannheim, Germany). Novex pre-cast gels (10–20% Tris-glycine gel) were from Novex (San Diego, CA). Polyclonal antisera against MPO [13 , 14 ] and soluble tumor necrosis factor (TNF) receptor 1 (sTNFR1) [15 ] were obtained by immunization of rabbits. Rabbit polyclonal antiserum to {alpha}1-microglobulin ({alpha}1-m) was a gift from Dr. Bo Åkerström (Lund, Sweden). A rabbit antiserum directed against the amino-terminal propeptide of MPO was raised against two peptides derived from the MPO propeptide (42TPQPSEGAAPAVLG56E and 72LVDKAYKERRESIKQRLRS91G) coupled via a C-terminal cysteine residue to maleimide-activated keyhole limpet hemocyanin, according to the manufacturer’s instructions (Pierce Chemical Company, Rockford, IL).

cDNA, mutagenesis, and construction of expression vectors
A deletion mutant, -pro, was constructed from cDNA encoding full-length MPO and inserted into the pREP vector for stable expression in K562 cells [11 ].

cDNA encoding human {alpha}1-m was constructed by polymerase chain reactions (PCR), using the cDNA for human {alpha}1-m-bikunin (HAM-pCRscript), generously provided by Dr. Bo Åkerström, as a template in a 20-cycle reaction. By design of the primers, the Kozak consensus leader sequence for maximal translational efficiency [16 ] was introduced 5' to the ATG initiation codon, and the flanking restriction enzyme sites EcoRI and XbaI were added for subsequent cloning into the plasmid. The primers were upstream 5'-GACTCCGAATTCGCCACCATGAGGAGCCTCGGGGCCCTGC (no. 5684) and downstream 5'-CTTCAGTCTAGATCATCTCGGGATTAAGATGGGC (no. 5215; start and stop codons in boldface and restriction enzyme sites underlined).

A cDNA for the chimeric protein, MPOpro/{alpha}1-m, the propeptide of MPO joined to {alpha}1-m, was constructed in a two-step "spliced overhang extension" PCR. In the primary PCR reactions, two separate amplifications with 100 ng DNA template, cDNA of full-length MPO [17 ], and {alpha}1-m-bikunin (HAM-pCRscript), respectively, produced two fragments, MPOpro (10 PCR cycles) and {alpha}1-m without the signal peptide (20 cycles), respectively. The Kozak sequence, start and stop codon and flanking sites for HindIII and BamHI, were used in designing the primers as described above. The PCR primers in the two amplifications were upstream 5'-GACTTCAAGCTTGCCACCATGGGGGTTCCCTTCTTCTCT (no. 3720) plus downstream 5'-CCCCACGTCCTGGTAGGCGCA (no. 3588) and upstream 5'-TGCGCCTACCAGGACGTGGGGGGCCCTGTGCCAACGCCGCC (no. 5311) plus downstream 5'-CTTCAGGGATCCTCATCTCGGGATTAAGATGGGC (no. 5310), respectively. The PCR products were isolated on agarose gel, mixed, and subjected to a second 20-cycle splicing, PCR amplification with primer nos. 3720 and 5310 to create MPOpro/{alpha}1-m. The resulting PCR product was digested by HindIII and BamHI, followed by isolation on an agarose gel and cloning into the desired expression vector.

cDNA for sTNFR1 was created with PCR technique as described above for the cDNA of {alpha}1-m. Thus, pADTNF-R [18 ] was used as a template with the upstream primer 5'-TTCAGGTACCGCCGCCACCATGGGCCTCTCCACCGTGCCT (no. 2200) and the downstream primer 5'-CTTCAGGGATCCTCAATTCTCAATCTGGGGTAGGCA (no. 3586). The construct was digested by KpnI and BamHI and cloned into pCDNA3.

cDNA for MPOpro/sTNFR1 was constructed in a two-step spliced overhang extension PCR by analogy with the creation of cDNA of MPOpro/{alpha}1-m. cDNA of full-length MPO [17 ] and pADTNF-R [18 ], respectively, were used as templates in the two primary PCR reactions with the following PCR primers: upstream 5'-GACTTCGGTACCGCCACCATGGGGGTTCCCTTCTTCTCT (no. 3587) plus downstream 5'-CCCCACGTCCTGGTAGGCGCA (no. 3588) and upstream 5'-TGCGCCTACCAGGACGTGGGGGATAGTGTGTGTCCCCAAGGA (no. 3591) plus downstream 5'-CTTCAGGGATCCTCAATTCTCAATCTGGGGTAGGCA (no. 3586), respectively. In the second PCR amplification splicing with primer nos. 3587 and 3586, the cDNA of fusion protein MPOpro/sTNFR1 was created with the restriction enzyme sites for KpnI and BamHI. Subsequently, the cDNA was cloned into the desired expression vector. All PCRs were performed in a Perkin-Elmer 480 thermal cycler using Pfu polymerase (Stratagene, San Diego, CA), according to the manufacturer’s instructions.

Green fluorescent protein (GFP) and MPOpro/GFP constructs
Constructs for expression in promyelocytic cell line cells used a pEF vector [12 ], kindly provided by Dr. Mary Dinauer (Indiana University, Indianapolis) and used previously for studies of gp91phox biosynthesis [19 , 20 ]. The GFP construct was derived from pADRSVhGFPpA, provided by Dr. Bev Davidson (Vector Core, University of Iowa, Iowa City).

Stably transfected cell lines
32D cl3 cells [21 , 22 ] were transfected with pcDNA3 constructs using the Bio-Rad Gene PulserTM (Bio-Rad, Hercules, CA) with electrical settings of 960 µF and 260–300 V, as described previously [23 , 24 ]. After electrophoresis, 1 mg/ml geneticin was added to select for recombinant clones containing the geneticin-resistance of pcDNA3. Individual antibiotic-resistant cell clones were expanded in suspension culture and screened for the expression of the transfected protein by biosynthetic radiolabeling. The clones with appropriate expression were chosen for the experiments described. Stably transfected K562 cells were generated using the expression vector pREP as previously described [11 , 25 ]. For stable transfection of PLB-985 cells, the vector pEF was used [12 ]. Cell cultures were incubated in 5% CO2 at 37°C in a fully humidified atmosphere. Exponentially growing cells were used in all experiments.

Biosynthetic radiolabeling
Biosynthetic radiolabeling of the newly synthesized proteins by stably transfected cell lines was carried out as described previously [11 , 24 25 26 27 ]. Cells were starved for 30 min followed by incubation with [35S]methionine/[35S]cysteine for radiolabeling of newly synthesized proteins (pulse-labeling). For chase of the radiolabeled protein variants, the cells were resuspended in complete nonradioactive medium after radiolabeling. At various time intervals, cells were withdrawn and subjected to analysis.

Peroxidase assay
The peroxidase activity of recombinant proteins was assessed in activity gels, as previously described [28 ]. Transfected cells were lysed in a nondenaturing sample buffer and separated in a native polyacrylamide gel. Samples were electrophoresed toward the cathode until the dye front was ~0.5 cm from the bottom of the gel. The gel was rinsed briefly in distilled water, and peroxidase activity was detected by staining with trimethylbenzidine HCl as described previously [28 ].

Subcellular fractionation
Subcellular fractionation was performed on continuous gradients of Percoll, as described previously [24 ], as well as in 10–60% sucrose [25 ]. Where indicated, the cell homogenate was centrifuged in a Percoll density gradient, after which nine fractions were collected with all the cytosol in fraction 9. The distribution of lysosomes and Golgi elements in the gradient was determined by assaying ß-hexosaminidase and galactosyl transferase [29 , 30 ]. The peak activities of ß-hexosaminidase and galactosyl transferase in Percoll fractions of 32D cells were localized in fractions 2 and 6, respectively. The 59-kDa heavy subunit of MPO expressed in 32D cells is localized predominantly to fractions 1–2, whereas the precursors of MPO are found in fractions 5–8 [17 ]. Subcellular fractionation in a sucrose gradient was performed as described [25 ]. Assays for cytochrome c reductase and ß-glucuronidase, markers for the ER and lysosome, respectively, were performed. The 59-kDa heavy subunit of normal MPO expressed in stably transfected K562 cells sediments predominantly in fractions 10–16 in this gradient, coincident with the lysosomal marker ß-glucuronidase, whereas the 90-kD precursors of MPO sediment in fractions 5–12, correlating well with the distribution of the ER markers CRT and cytochrome c reductase [25 ].

Immunoprecipitation
Immunoprecipitations from 32D cells were performed as described previously [5 ]. Immunoprecipitations from transfected K562 cells and PLB-985 cells used previously described buffers for solubilization [11 ].

Immunoelectron microscopy
32D cells stably transfected with {alpha}1-m or with MPOpro/{alpha}1-m were fixed for 24 h in 4% paraformaldehyde in 0.1 M PHEM buffer (pH 6.9) and then processed for ultrathin cryosectioning as described previously [31 ]. Cryosections (45 nm) were cut at -125°C using diamond knives (Drukker Cuijk, The Netherlands) in an ultracryomicrotome (Leica Aktiengesellschaft, Vienna, Austria) and were transferred with a mixture of sucrose and cellulose onto formvar-coated copper grids [32 ]. The grids were placed on 35-mm Petri dishes containing 2% gelatin. Double-immunolabeling was performed using a previously described procedure [33 ] with 10- and 15-nm protein A-conjugated colloidal gold probes (EM Lab, Utrecht University, The Netherlands). Staining used rat monoclonal ID4B against mouse lysosome-associated membrane protein 1 (LAMP-1; CD107a; Pharmingen Leiden, The Netherlands) and rabbit anti-{alpha}1-m. After immunolabeling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands). For the controls, the primary antibody was replaced by a nonrelevant rabbit antiserum.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{alpha}1-m is sorted for storage in myeloid cells
The fate of {alpha}1-m was investigated in stably transfected 32D and K562 cells. The {alpha}1-m of plasma is mainly manufactured normally by liver cells and secreted after cleavage from a precursor protein [34 , 35 ]. Two forms of newly synthesized {alpha}1-m with different molecular sizes were observed during radiolabeling of transfected 32D cells. A significant fraction of the synthesized high molecular-size species was secreted rapidly into the culture medium (Fig. 1 A ). The remaining intracellular forms of {alpha}1-m were degraded slowly, as judged by chase of the radiolabeled material (Fig. 1A) . To further investigate the fate of the intracellularly retained {alpha}1-m, subcellular fractionation experiments were performed. After 30 min of pulse-radiolabeling, most of the radiolabeled {alpha}1-m was present in light fractions (nos. 6–8), corresponding to ER and Golgi (Fig. 1B) . With time, however, labeled {alpha}1-m appeared in dense fractions (nos. 1–2), indicating transfer to storage granules. These data show unexpectedly that a nonmyeloid, normally secreted protein can be sorted for storage when expressed in myeloid cells.



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  		Figure 1. Targeting {alpha}1-m to dense cytoplasmic organelles. 32D cells expressing {alpha}1-m were radiolabeled biosynthetically for 30 min, followed by chase of the label for the indicated time periods. At depicted time points (A), 20 x 106 cells were removed and after lysis, subjected to immunoprecipitation with anti-{alpha}1-m. In addition, {alpha}1-m was precipitated from the incubation medium at each chase time point. At indicated time periods (B), 100 x 106 cells were removed and homogenized, after which subcellular fractionation of the postnuclear supernatant was performed by centrifugation in Percoll, with subsequent collection of nine subcellular fractions, fraction 9 containing all cytosol. The fractions were lysed and subjected to immunoprecipitation with anti-{alpha}1-m. The immunoprecipitates were analyzed by SDS-PAGE followed by fluorography. The fluorograms were exposed for 11 and 10 days, respectively. The positions of {alpha}1-m are indicated to the right with arrows. Peak activities of ß-hexosaminidase, fraction 2, and galactosyl transferase, fraction 6, indicate the position of lysosomes and Golgi elements, respectively.

 
To investigate the role of the propeptide of MPO in intracellular targeting, we examined the influence of the addition of the MPOpro sequence to the amino terminus of {alpha}1-m on its fate in stably transfected 32D and K562 cells. Two forms of MPOpro/{alpha}1-m were synthesized in 32D cells: The 43-kDa species was predominant intracellularly, and the 47-kDa protein was predominant extracellularly (Fig. 2 A ). Both forms diminished during chase of the radiolabel, and the coincident appearance of a 32-kDa protein was not recognized by the anti-MPOpro antibody (unpublished results) and therefore presumably represented free {alpha}1-m. Chloroquine treatment to block lysosomal hydrolysis did not inhibit the release of {alpha}1-m from the chimera (unpublished results), suggesting that the cleavage was not associated with the lysosome. However, processing and secretion of MPOpro/{alpha}1-m were inhibited by BFA (Fig. 2B) , which blocks egress of protein from ER, indicating that these two events occurred in a Golgi or post-Golgi compartment, access to which should be prevented by BFA [36 , 37 ]. Upon subcellular fractionation, most of the MPOpro/{alpha}1-m synthesized early was in the light fractions corresponding to the ER and the Golgi, but in contrast to {alpha}1-m (Fig. 1B) , only a small fraction of intact MPOpro/{alpha}1-m was ever visible in dense fractions (Fig. 2C) . Instead, a lower molecular-weight form, corresponding to cleaved {alpha}1-m (compare with Fig. 2A ), appeared in dense fractions (Fig. 2C) , indicating processing of MPOpro/{alpha}1-m to {alpha}1-m during transfer to dense organelles. Thus, the addition of the MPOpro peptide sequence at the amino terminus of {alpha}1-m delayed but did not inhibit its delivery to the dense organelles.



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  		Figure 2. Processing MPOpro/{alpha}1-m with cleavage of {alpha}1-m and targeting to dense cytoplasmic organelles. 32D cells expressing MPOpro/{alpha}1-m were radiolabled biosynthetically for 30 min followed by chase of the label for the indicated time periods. At depicted time points (A), 20 x 106 cells were removed and after lysis, subjected to immunoprecipitation with anti-{alpha}1-m. In addition, MPOpro/{alpha}1-m was precipitated from the incubation medium at each chase time point. The same experiment was carried out in the presence of 5 µg/mL BFA (B), present during starvation, radiolabeling, and chase of the label. At indicated time points (C), 100 x 106 cells were removed and homogenized, after which subcellular fractionation of the postnuclear supernatant was performed by centrifugation in Percoll, as described in the legend to Figure 1 . The immunoprecipitates were analyzed by SDS-PAGE and fluorography. The fluorograms were exposed for 11, 7, and 11 days, respectively. The positions of MPOpro/{alpha}1-m and {alpha}1-m are indicated with arrows to the right. Peak activities of ß-hexosaminidase, fraction 2, and galactosyl transferase, fraction 6, indicate the position of lysosomes and Golgi elements, respectively.

 
Reasoning that the transient nature of the MPOpro/{alpha}1-m in 32D cells reflected a limitation of the expression system rather than an intrinsic property of the construct, we compared the fate of wild-type and chimeric {alpha}1-m in K562 cells. Transfected K562 cells secreted the 28-kDa wild-type {alpha}1-m, whereas the majority of the 43-kDa chimeric species remained intracellular and was not secreted at the same rate as the parent protein (unpublished results). In analysis of subcellular fractions separated on 10–60% gradients of sucrose, the MPOpro/{alpha}1-m did not cosediment with dense organelles containing mature MPO when expressed but rather were retained in the ER as judged by cosedimentation of biochemical markers for ER (unpublished results). MPOpro/{alpha}1-m was not retained in the ER by association with CRT or CLN (unpublished results).

Results from immunoelectron microscopy using double immunogold-labeling revealed that {alpha}1-m colocalized with the lysosomal marker LAMP-1 [38 , 39 ] in 32D cells transfected with {alpha}1-m (Fig. 3 A ). Specific labeling was associated with multivesicular bodies that correspond to the abnormal granules of 32D cells. These results are consistent with those from subcellular fractionation experiments demonstrating targeting of {alpha}1-m to dense organelles (Fig. 1B) . As seen in cells expressing {alpha}1-m alone, the {alpha}1-m of cells transfected with chimeric MPOpro/{alpha}1-m colocalized with LAMP-1 in multivesicular bodies (Fig. 3B) . This product likely represents {alpha}1-m released from the chimeric protein and accumulated in granules, supported by the finding of almost exclusively free {alpha}1-m in the dense organelles, as judged by results from radiolabeling experiments (Fig. 2C) . Taken together, these data demonstrated the destination of the {alpha}1-m released from the chimera to be dense, multivesicular bodies, but the specific localization of the intact chimeric MPOpro/{alpha}1-m was not identified.



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  		Figure 3. Colocalization of {alpha}1-m and LAMP-1. Ultrathin cryosections from 32D cells transfected with {alpha}1-m (A) or with MPOpro/{alpha}1-m (B) were double-labeled with rat anti-LAMP-1, followed by rabbit anti-rat immunoglobulin G (IgG) and protein A gold (10 nm), and with rabbit anti-{alpha}1-m, followed by protein A gold (15 nm). The pattern of labeling in both transfectants is very similar; {alpha}1-m and LAMP-1 colocate in small and large multivesicular bodies. Original bar = 200 nm.

 
The intracellular, newly synthesized, low molecular-size {alpha}1-m was sensitive to digestion with Endo-H, indicating presence of high mannose glycoforms produced in the ER (Fig. 4 A ). Conversely, the larger species of {alpha}1-m, present intra- and extracellularly, was resistant to Endo-H, indicating the presence of complex glycoforms normally produced in the Golgi complex by trimming mannose and adding new terminal sugars. The results indicate that the smaller size {alpha}1-m, after ER export, is converted to the larger size {alpha}1-m as a result of complex oligosaccharide formation. Digestion with N-glycosidase F to remove all N-linked carbohydrates revealed two intracellular species of {alpha}1-m with distinct protein core sizes, of which only the larger form was detected extracellularly (Fig. 4A) , indicating that proteolytic trimming of {alpha}1-m occurred in storage organelles but not during constitutive secretion. MPOpro/{alpha}1-m showed a much slower acquisition of complex carbohydrates occurring in the Golgi complex as compared with {alpha}1-m (compare Fig. 4 A and B). Thus, a partially preserved susceptibility to Endo-H, indicating lack of complex glycoforms, was observed after 3 h of chase of radiolabeled MPOpro/{alpha}1-m, a time when {alpha}1-m was fully Endo-H-resistant. These data are consistent with a retarded rate of carbohydrate processing in MPOpro/{alpha}1-m as compared with {alpha}1-m, most likely because of a prolonged ER retention of the chimera.



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  		Figure 4. Digestion of {alpha}1-m and MPOpro/{alpha}1-m with glycosidases. 32D cells expressing {alpha}1-m (A) or MPOpro/{alpha}1-m (B) were radiolabeled biosynthetically for 30 min followed by chase of the label for 3 h. At these time points, 100 x 106 cells were removed and after lysis, subjected to immunoprecipitation with anti-{alpha}1-m. In addition, the incubation medium was analyzed at the end of the chase period. Aliquoted immunoprecipitates were incubated with 0.2 units/mL endoglycosidase H (+ Endo-H) or 7 units/mL N-glyc (+ N-glycosidase F) or were served as controls (-). The samples were analyzed as described in the legend to Figure 1 . The fluorograms were exposed for 21 days.

 
sTNFR1 is secreted partially but not MPOpro/sTNFR1
The fate of sTNFR1 was investigated in stably transfected 32D cells. sTNFR1 is the extracellular part of the 55-kDa cell-surface receptor for TNF, lacking transmembrane and cytosolic domains [18 ]. The 26-kDa sTNFR1 was immunoprecipitated from transfectants after 30 min of biosynthetic radiolabeling, and secreted forms were recovered from the culture supernatant within 2 h (Fig. 5 ). No association of sTNFR1with dense organelles was observed in subcellular fractionation experiments (unpublished results), indicating that this protein is not targeted for storage but rather secreted and that some but not other proteins are routed for storage in myeloid cells. To determine whether the presence of the MPO propeptide at the N-terminal end of sTNFR1 would influence its subcellular fate, we also expressed cDNAs of MPOpro/sTNFR1 in the 32D cells. In contrast to 26-kDa sTNFR1, the 40-kDa MPOpro/sTNFR1 fusion protein was not processed during the chase period and was not secreted into the culture medium (Fig. 5) . BFA, an agent that disrupts the Golgi and blocks egress of newly synthesized proteins from the ER [36 , 37 ], did not influence the fate of the radiolabeled MPOpro/sTNFR1 (unpublished results), suggesting that the chimeric protein was retained efficiently and degraded slowly in a pre-Golgi compartment. This conclusion is also consistent with the finding of complex glycans on sTNFR1 (indicating Golgi passage) but not on MPOpro/sTNFR1 (indicating ER retention; unpublished results). Furthermore, the addition of NH4Cl or chloroquine, agents known to interfere with hydrolysis in lysosomes, did not prolong the half-life of MPOpro/sTNFR1, thus arguing against lysosomal degradation as influencing the fate of the fusion protein (unpublished results). Because the MPOpro/sTNFR1 was retained and degraded in the ER of 32D cells, experiments in this system did not elucidate a targeting role for MPOpro.



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  		Figure 5. Biosynthesis of sTNFR1 and MPOpro/sTNFR1 in 32D cells. 32D cells expressing sTNFR (top panel) or MPOpro/sTNFR (bottom panel) were radiolabeled biosynthetically for 30 min followed by chase of the label for the indicated time periods. At depicted time points, 20 x 106 cells were removed and after lysis, subjected to immunoprecipitation with anti-sTNFR1. The incubation medium was also assayed. Immunoprecipitates were analyzed by SDS-PAGE and fluorography. The positions of sTNFR1 or MPOpro/sTNFR1, respectively, are indicated with arrows. The fluorograms were exposed for 5 weeks.

 
MPOpro/sTNFR1 and sTNFR1 behaved similarly in K562 cells; the "steady state" distribution of MPOpro/sTNFR1 in subcellular fractions separated on 10–60% gradients of sucrose was analyzed by immunoblots with antisera against the propeptide or the parent protein. The MPOpro/sTNFR1 was retained in the fractions of the gradient enriched for ER elements (peak in fraction 9, range 7–10), whereas the 59-kDa heavy subunit of mature MPO made by pREP-MPO cells sedimented predominantly in fraction 16 (range 14–18; Fig. 6 ). Thus, these data indicate that the addition of the propeptide to the N-terminus of sTNFR1 caused retention of the fusion protein in the ER, independent of the cell line used for expression.



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  		Figure 6. Subcellular localization of MPO and MPOpro/sTNFR1 in K562 cells. Stable transfectants expressing normal MPO or MPOpro/sTNFR were cavitated and separated on the same 10–60% sucrose gradient. Fractions from the gradient were separated by SDS-PAGE, and duplicate immunoblots were probed with antibody against MPO (mature MPO) or with antibody against sTNFR1.

 
Different fates for GFP and MPOpro/GFP expressed in PLB-985 cells
Our results with both chimeric constructs in each of the eukaryotic expression systems indicated that the propeptide of MPO failed to redirect proteins normally secreted to a population of dense organelles. However, we wished to exclude the possibility that a myeloid-specific targeting system, not present in 32D or K562 cells, might be required for the normal function of the propeptide. To that end, we compared the relative intracellular disposition of GFP and MPOpro/GFP stably expressed in PLB-985 cells, a human promyelocytic cell line that constitutively synthesizes functionally normal MPO [40 ].

We first confirmed that the MPOpro/GFP construct was synthesized in the ER of the PLB-985 cells by assessing its glycosylation status. Whereas GFP lacks carbohydrate, MPOpro/GFP was glycosylated, because the addition of asparagine 139 in the propeptide resulted in tunicamycin-inhibitable N-linked glycosylation of the recombinant protein in the ER (unpublished results). Despite synthesis of MPOpro/GFP in the ER, it did not associate with the molecular chaperones CRT or CLN during their biosynthesis. Lysates of pulse-chased MPOpro/GFP transfectants were immunoprecipitated under nondenaturing conditions with antibodies to MPO (examining endogenous MPO production), CRT (recovering CRT-associated MPO precursors), and GFP (precipitating MPOpro/GFP) and were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography (Fig. 7 ). Under these conditions, multicomponent protein complexes remain intact [26 , 27 ], but in contrast to MPO, MPOpro/GFP was not precipitated in a complex with CRT (Fig. 7) or CLN (unpublished results). Whereas GFP was expressed stably and remained intracellular in PLB-985 cells (unpublished results), MPOpro/GFP was degraded relatively rapidly in the ER by a process dependent on the proteasome, as judged from inhibition of degradation by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl (ALLnL; unpublished results). Thus, in the PLB-985 cell line—cells competent to synthesize and target endogenous MPO—the addition of the propeptide to GFP was not sufficient for directing GFP to granule storage.



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  		Figure 7. Coprecipitation of CRT and MPO or MPOpro/GFP. Wild-type PLB-985 cells (PLB-985) or transfected PLB-985 cells expressing MPOpro/GFP (4G4) were radiolabled biosynthetically and chased for 0 or 4 h. Cell lysates were immunoprecipitated with antibodies to MPO (M), CRT (C), or GFP (G) to recover MPO, CRT-associated MPO or proMPO, or MPOpro/GFP.

 
Fate of stably transfected prodeletion MPO mutant
To determine if the presence of the propeptide on the MPO precursor was essential for normal intracellular targeting, we examined the biosynthesis by K562 cells of a stably expressed deletion mutant lacking the 125 amino acid MPO propeptide. The (-pro)MPO construct encoded the signal peptide directly followed by the light and heavy subunits of mature MPO. Although the (-pro) construct interacted transiently with CLN and CRT during expression in K562 transfectants (Fig. 8 A ), heme was not incorporated (unpublished results), and peroxidase activity was lacking (Fig. 8B) . After 8 h of chase following biosynthetic radiolabeling, (-pro)MPO colocalized with biochemical markers for the ER in 10–60% sucrose gradients (unpublished results). The decay of (-pro)MPO under these conditions was inhibited by lactacystin and thus presumably mediated by the proteasome (unpublished results).



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  		Figure 8. Biosynthesis of prodeletion mutant in K562 cells. (A) Transfectants stably expressing wild-type MPO (top panel) or the (-pro) mutant (bottom panel) were radiolabled biosynthetically for 1 h and chased for 1–4 h. Cell lysates were immunoprecipitated with anti-MPO to recover all MPO-related protein ({alpha}MPO), sequentially with anti-CLN and anti-MPO ({alpha}CLN) to recover CLN-associated (-pro), or with anti-CRT and anti-MPO ({alpha}CRT) to recover CRT-associated (-pro). CLN and the MPO precursor migrate nearly identically and could not be distinguished in one-dimensional gels (top panel). However, (-pro) and CLN were separated and identified after SDS-PAGE (bottom panel). (B) Lysates from the parental K562 cells (K562), K562 transfectants stably expressing wild-type MPO (MPO), K562 transfectants stably expressing (-pro) [-(pro)], and PLB-985 cells (PLB) were separated by PAGE in a native system and stained for peroxidase activity.

 
These data indicate that deletion of the propeptide compromised structural and functional maturation of the precursor and its following export from the ER. However, the failure of (-pro)MPO to exit the ER could reflect misfolding of the mutant protein for any of several other reasons, including the impact of such a relatively large deletion on the structural organization of apoMPO, the specific absence of the proregion, and/or its failure to accommodate heme, a prerequisite for proteolytic processing [41 ]. Coexpression of the propeptide and (-pro)MPO likewise failed to correct any misfolding and overcome the maturation defect (unpublished results), suggesting that the propeptide did not function in trans under these experimental conditions and was not likely serving as an intramolecular chaperone [42 43 44 ].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present investigation, a nonhematopoietic protein such as the normally secretory {alpha}1-m was expressed in hematopoietic cell lines and sorted for storage in dense cytoplasmic organelles. We demonstrated as well that fusion of the propeptide of MPO to create chimeras altered the distribution of the unmodified protein and resulted in ER retention.

Hematopoietic cells contain an array of granule compartments, whose composition reflects the timing and coordination of gene expression [2 ]. Therefore, specific sorting mechanisms for targeting to different granule subtypes may not be required. However, a mechanism is needed for retrieval of proteins from the secretory pathway to allow sorting for storage, because constitutive secretion also occurs in myeloid cells, as demonstrated for proforms of newly synthesized granule proteins [3 ]. We have demonstrated previously that LBP, a nonmyeloid protein that is normally secreted after biosynthesis in liver cells, can be retained and targeted for dense organelles when expressed in myeloid cells [5 ]. We now demonstrate that this is also the case also for {alpha}1-m, which was chosen for this study, because it is secreted constitutively by normal liver cells. Therefore, its sorting for storage in myeloid cells was unexpected, but this and our previous results suggest that retention for storage in myeloid cells may reflect cell-specific rather than protein-specific determinants. Thus, even nonmyeloid proteins can be targeted for storage when the conformation is acceptable to the sorting machinery. A precursor protein, {alpha}1-m-bikunin is synthesized in hepatocytes and cleaved in the Golgi followed by secretion of {alpha}1-m and bikunin [35 ]. Glycosylation may be important for secretion inasmuch as the secretion of carbohydrate-free {alpha}1-m from recombinant insect cells was reduced significantly [45 ]. Our results demonstrate that the {alpha}1-m synthesized in myeloid cells accomplished complex glycoforms in the Golgi, followed by retention for storage or partial secretion. We therefore have no reason to anticipate that abnormal glycosylation would be responsible for the intracellular retention for storage of {alpha}1-m in myeloid cells. Furthermore, it is noteworthy that {alpha}1-m belongs to the lipocalin protein family, and human neutrophils normally synthesize and store NGAL, another lipocalin, in specific granules [1 ]. Therefore, the targeting of {alpha}1-m in myeloid cells may reflect similarities in targeting sequences between these two structurally related proteins.

One essential observation is that myeloid cells can discriminate and selectively target some proteins for storage. The routing of myeloid and nonmyeloid proteins likely depends on their structural determinants inasmuch as not all soluble proteins delivered to the trans-Golgi network are sorted for storage in myeloid cells. For instance, the behavior of the soluble TNFR1 that was exported from ER and secreted contrasts with that of LBP [5 ] and {alpha}1-m, which were intracellularly retained. Similarly, we have observed that propeptide-deleted pro-MPO is secreted constitutively in myeloid cell lines without accumulation of the final product in granules [17 ]. This behavior is not a property peculiar to transfected, cultured cell lines; results from normal bone marrow cells have indicated that differences exist in sorting granule proteins between constitutive secretion and retrieval for storage in granules [46 ], e.g., a significant fraction of lysozyme was routed for constitutive secretion. The specific physicochemical properties that dictate targeting to specific storage compartments in myeloid cells remain to be elucidated.

Unlike endocrine, neuroendocrine, and exocrine cells, which manufacture granules for regulated secretion of their stored products, myeloid cells often use the endosomal pathway for manufacturing lysosome-related organelles. This involves transfer of coated vesicles from the TGN to late endosomes, often with the appearance of multivesicular bodies that form mature, lysosome-related storage organelles [47 ]. The endosomal pathway was involved in targeting {alpha}1-m for dense organelles as judged by the results from immunogold-labeling in 32D cells. Thus, {alpha}1-m colocalized with the lysosomal marker LAMP-1 [38 , 39 ], and much of the labeling was associated with organelles having the features of multivesicular bodies corresponding to the vacuole-like abnormal granules of 32D cells [48 ]. Azurophil granules of neutrophils are lysosome-like and may belong to the lysosome-related organelles [47 ], although they lack LAMP-1 and LAMP-2 [49 ]. Thus, all lysosome-related organelles, including azurophil granules, are defective in the Chediak-Higashi syndrome [47 ]. Endogenous granule proteins of 32D cells are more stable than exogenously introduced {alpha}1-m, which is eventually degraded in the dense organelles. This may result from abnormal packing of {alpha}1-m, which is normally not present in this environment, thus resulting in a lack of protection against proteases.

These studies were also designed to assess the potential role of the propeptide of MPO for the intracellular targeting of the enzyme. A deletion mutant in which the propeptide was eliminated from the MPO precursor aborted proper delivery of mature MPO to the dense organelles of the 32D cell line, despite the fact that constitutive secretion was not globally affected [17 ]. Moreover, the propeptide of MPO targets a fusion protein of MPOpro and lysozyme to the lysosomes of Chinese hamster ovary cells, followed by cleavage of the MPO propeptide from the lysozyme moiety [50 ]. Similarly, we tracked the subcellular fate of transfected chimeric constructs to determine if the addition of the propeptide of MPO would redirect proteins from the constitutive secretory pathway to sorting for storage in granules and thereby support a functional role for that domain in proper cellular targeting. Thus, we created chimeric proteins that would exhibit a "gain of function" if the propeptide in fact represented a targeting domain. A chimera of MPOpro and {alpha}1-m was targeted to dense organelles of 32D cells after cleavage of the {alpha}1-m moiety, but the potential information gained by adding the MPOpro to {alpha}1-m was undermined by our unexpected finding of targeting for storage of the normally secreted {alpha}1-m alone. It should be noted that a substantial portion of newly synthesized {alpha}1-m was released extracellularly (Fig. 1A) . Therefore, a decreased secretion to the medium is consistent with increased intracellular retention and sorting. However, the relatively slow appearance of complex carbohydrates on MPOpro/{alpha}1-m, as compared with {alpha}1-m alone, suggested that the half-life of MPOpro/{alpha}1-m in the ER was prolonged. Although this observation supported a role for the propeptide in a transient ER retention, this ER retention may not specifically reflect a targeting function for the propeptide. Clearly, the relative fates of {alpha}1-m and MPOpro/{alpha}1-m differed when expressed in K562 cells. In this human hematopoietic cell line, wild-type {alpha}1-m was secreted into the culture medium, whereas the fusion protein was trapped intracellularly, primarily within the ER, without detectable targeting for storage in dense organelles. Therefore, a role for the propeptide in sorting for storage in granules could be neither confirmed nor excluded from these experiments.

32D cells and K562 cells transfected with wild-type sTNFR1 or the MPOpro/sTNFR1 demonstrated the same differential phenotypes, namely secretion of the wild-type proteins, but intracellular retention of the fusion proteins without secretion to the same extent as that seen for the parental protein. Results from subcellular fractionation studies in K562 cells demonstrated that the chimeric species containing the MPO propeptide was retained in fractions that cosedimented with markers for the ER. The chimeric protein was not retained in the ER by association with molecular chaperones CRT and CLN (unpublished results), indicating that the presence of the propeptide was not sufficient to mediate interactions with those ER lectins. Last, data from studies comparing the subcellular localization of GFP and MPOpro/GFP in PLB-985 cells, human promyelocytic cells constitutively synthesizing, and packaging normal MPO [26 , 40 ] demonstrated that the addition of the propeptide to GFP caused ER retention followed by proteasome-dependent degradation, thus making it difficult to judge whether the propeptide could affect sorting at more distal locations of the secretory pathway.

We also examined the fate of the propeptide deletion mutant expressed in K562 cells. The (-pro)MPO construct failed to egress from the ER and decayed rapidly in a proteasome-dependent fashion. Coexpression of the propeptide with the (-pro)MPO construct failed to reconstitute proper targeting or function of the deletion mutant (unpublished results). These data suggest that deletion of the propeptide irreversibly compromised proper functioning of the MPO precursor, resulting in a species rapidly eliminated by the ER-associated degradation system and the proteasome and that the propeptide did not function as an intramolecular chaperone. Conversely, previous results [17 ] showed that (-pro)MPO expressed in 32D cells was secreted, albeit less than normal, without being targeted for storage, indicating a role of the propeptide for targeting for storage in granules in 32D cells [17 ]. The relative fates of (-pro)MPO in these two cell lines may parallel the extent of heme incorporation into the precursor; whereas there is limited incorporation of heme into (-pro)MPO expressed in 32D cells [17 ], the same construct expressed in K-562 cells lacked heme. If heme is necessary to allow ER export of (-pro)MPO, as is the case in the biosynthesis of wild-type MPO [41 ], one would anticipate that any compromise in heme insertion per se would create a phenotype similar to that of maturation arrest of the precursor.

Collectively, these data demonstrate that fusion of the propeptide of MPO altered subcellular targeting relative to that of the unmodified protein, but that the propeptide alone was not sufficient to target the reporter protein to an intracellular site where further proteolytic processing and maturation of MPO precursors occur. However, as the chimeric proteins were retained in the ER, we cannot exclude a role for the propeptide in directing MPO to dense organelles, as indicated by previous studies demonstrating that processing of MPO precursors and efficient sorting for storage require an intact propeptide [17 ]. A BLAST search of the Swissprot protein databank for sequences related to the MPO propeptide recovered only precursor forms of other members of the family of animal peroxidases, including eosinophil peroxidase, thyroid peroxidase, and lactoperoxidase, perhaps suggesting a function shared by these structurally related proteins. In that light, the propeptide may, in addition to being necessary for granule targeting, provide a mechanism for retention of the apopro-form of animal peroxidases in the ER, independent of association with the chaperones CRT and CLN, to facilitate heme insertion and proper folding and thus generate a precursor competent to exit the ER. Alternatively, ER retention may reflect abnormal folding followed by degradation, as seen for some of the chimeras investigated.

We conclude that nonmyeloid proteins that are normally secreted can be targeted for storage when expressed in myeloid cells, indicating the existence of tissue-specific retention mechanisms for intracellular storage, and that myeloid cells selectively route some proteins but not others for storage. In addition, the propeptide of MPO may play a role in retention mechanisms necessary for proper maturation and storage of MPO.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swedish Cancer Foundation, the Swedish Medical Research Fund (Project #11546), the Alfred Österlund Foundation, funds of the Lund University Hospital, and the Greta and Johan Kock Foundation. Work in the Nauseef lab was supported by a Merit Review Grant from the Veterans Administration and by the Public Health Service of the National Institutes of Health, HL 53592.

Received September 11, 2001; revised October 18, 2001; accepted October 22, 2001.


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