©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calreticulin Functions as a Molecular Chaperone in the Biosynthesis of Myeloperoxidase (*)

(Received for publication, July 18, 1994; and in revised form, December 9, 1994)

William M. Nauseef (§) Sally J. McCormick Robert A. Clark

From the Department of Medicine, Veterans Administration Medical Center and the University of Iowa, College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible cross-linking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the posttranslational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells.


INTRODUCTION

Polymorphonuclear granulocytes (PMNs) (^1)are critical elements in human host defense against invading microorganisms(1, 2) . The most efficient microbicidal system employed by PMNs depends on two elements: reactive oxygen species generated by the NADPH-dependent oxidase (3) and myeloperoxidase (MPO), a heme-containing protein present in the azurophilic granules of PMNs(4, 5) . Within the phagolysosome of the activated PMN, these species interact to produce HOCl and exert microbicidal activity(5) .

MPO (donor:H(2)O(2) oxidoreductase, EC 1.11.1.7) has a molecular mass of 150 kDa and is composed of a pair of heavy-light protomers, each containing a glycosylated 59-kDa heavy subunit as well as a nonglycosylated 13.5-kDa light subunit(5, 6, 7) . Each molecule of MPO contains two heme groups, associated with the heavy subunit in each pair of heavy-light protomers.

Studies of the biosynthesis of MPO by cultured promyelocytic cell lines have demonstrated that MPO is the product of a single gene. The primary translation product undergoes cotranslational N-linked glycosylation to generate a 90-kDa heme-free precursor, apopro-MPO. The heme group is inserted into apopro-MPO to produce the 90-kDa enzymatically active precursor, pro-MPO. Subsequently, pro-MPO undergoes proteolytic maturation, whereby the 125-amino acid pro-sequence is removed, the remaining peptide is cleaved into heavy and light subunits, the heavy-light protomers are paired into the dimeric mature MPO, and the mature protein is targeted to the lysosome (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) .

Two peculiar features of MPO biosynthesis prompted these studies. First, the processing of MPO precursor to mature MPO is extremely slow, taking as long as 15 h to chase radiolabel from the precursor into mature protein(8) . Second, the processing and proteolytic maturation of MPO precursor to native MPO is blocked when cells are cultured in the presence of succinyl acetone, an inhibitor of heme synthesis(8, 20, 21) . Based on these observations, we reasoned that heme insertion into apopro-MPO may be a rate-limiting step in the relatively slow movement of MPO precursor in the ER to the final cellular destination of mature MPO in the lysosome. Accordingly, we examined whether a molecular chaperone participated in facilitating the critical folding of apopro-MPO to accommodate insertion of heme and subsequent conversion to pro-MPO.

We present data demonstrating that calreticulin (CRT), a calcium-binding protein present in the ER of a wide variety of cells (reviewed in (22) ), coprecipitated with apopro-MPO, the glycosylated heme-free precursor of MPO. In addition, CRT and apopro-MPO could be selectively and reversibly cross-linked. Pulse-chase experiments demonstrate that CRT interacted with the MPO precursor early in biosynthesis and that this interaction was transient. Taken together, these data indicate that CRT, like the structurally related ER protein calnexin, can function as a molecular chaperone. These observations expand the identified cellular activities in which CRT participates.


MATERIALS AND METHODS

Reagents

The human leukemia cell line PLB 985 was acquired from Dr. Timothy Ley (Washington University, St.Louis, MO) and maintained in RPMI 1640 medium supplemented with 2 mM glutamine, penicillin-streptomycin, and 5% heat-inactivated fetal calf serum with 5% Serum-plus (JRF Biosciences, Lenexa, KS). Cells were free of mycoplasma infection. Tissue culture medium was obtained from the University of Iowa Cancer Center. For biosynthetic labeling, RPMI Select Amine kit (Life Technologies, Inc.) was used to prepare methionine-free medium, which was supplemented with 1 mM pyruvate, 1 mM glutamine, antibiotics, and 10% dialyzed fetal calf serum. [S]Methionine (1, 320 Ci/mmol) and -[^14C]aminolevulinic acid (48.2 mCi/mmol) were obtained from Amersham Corp. and DuPont NEN, respectively. 3,3`-Dithiobis(sulfosuccinimidyl proprionate) (DTSSP) was obtained from Pierce. Rat monoclonal antibodies against hsp90 and GRP94 were obtained from StressGen Biotechnologies Corp. (Victoria, BC, Canada). Anti-hsp 90 antibody (clone 9D2) was raised against human hsp90 and identifies both free and complexed hsp90. Anti-GRP94 antibody (clone 9G10) recognizes GRP94 from chicken, rodents, and various mammalian cells. Protein A was purchased from Life Technologies, Inc. and radiolabeled with I at a core facility at the Department of Veterans Affairs Medical Center (Iowa City, IA). Additional chemicals and reagents were obtained from Sigma.

Biosynthetic Labeling

PLB 985 cells were grown at 37 °C in an atmosphere of 5% CO(2) in the medium described above. For biosynthetic labeling, cells were suspended at 5.0 times 10^5/ml in methionine-free RPMI with 10% dialyzed fetal calf serum for 60 min. After methionine depletion, 25 mCi/ml [S]methionine was added, and the cells were cultured for the specified time interval. At the end of the labeling period, cells were collected by centrifugation for subsequent analysis as previously described(9) . In general, 2.0 times 10^6 cells were solubilized in 450 µl of lysis buffer, and 150 µl of the solubilized cell lysate (approximately 6.7 times 10^5 cell equivalents) were used in a given immunoprecipitation. In the case of pulse-chase experiments, cells were resuspended in medium made 1 mM with cold methionine and chased for the specified time interval. In experiments using tunicamycin (TM), cells were suspended in medium containing a specific concentration of TM for 4 h prior to radiolabeling. Longer periods of exposure to TM result in differentiation of promyelocytic cells and concomitant cessation of MPO gene expression(7) . In experiments using succinyl acetone, cells were maintained in 250 mM succinyl acetone for 72 h prior to biosynthetic labeling. When cells were grown under these conditions, cell viability was normal, but peroxidase activity was reduced to 27.8 ± 1.8% of the control value (n = 11). The activity of lysosomal enzymes elastase and beta-glucuronidase were unchanged in succinyl acetone(8) .

Immunoprecipitation

Immunoprecipitations were done under either denaturing or nondenaturing conditions. For both conditions, 100 µl of lysed cells or 700 µl of culture supernatant were used for immunoprecipitations as previously described(9) . Samples were incubated with nonimmune serum for 30 min at 4 °C, followed by a 10% suspension of washed formalin-fixed, heat-killed, protein A-containing Staphylococcus aureus (Immunoprecipitin, Life Technologies, Inc.) to clear the sample of radiolabeled proteins, which might nonspecifically associate with the immunoprecipitates. The cleared sample was subsequently incubated with primary antiserum. CRT was immunoprecipitated using a monospecific rabbit antibody raised against purified recombinant human myeloid CRT expressed in baculovirus (22) . MPO-related peptides were recovered using a previously described monospecific rabbit polyclonal antibody to human MPO(23) . For immunoprecipitation of hsp90 and GRP94, the specific rat monoclonal antibody was used as the primary antibody, followed by rabbit anti-rat IgG antibody as the second antibody. The antigen-antibody complexes were recovered with protein A-bearing S. aureus, and the pellets were washed serially with 1 ml each of 0.5% Triton X-100 in Tris-buffered saline (TBS) (10 mM Tris buffer, pH 7.5, with 150 mM NaCl), 2 mM urea in 0.5% Triton X-100 in TBS, 1 mg/ml bovine serum albumin in 0.5% Triton X-100 in TBS, and TBS. After the final wash, the antigen-antibody complex was released from protein A by heating at 100 °C for 5 min in the presence of SDS sample buffer (62 mM Tris, 2 mM EDTA, 5% beta-mercaptoethanol, 2.3% SDS, pH 6.9).

When immunoprecipitations were done under denaturing conditions, the cleared lysate was denatured by making the sample 2% SDS and heating to 100 °C for 2 min. Prior to the addition of the primary antibody, the SDS concentration of the sample was reduced to 0.2% by the addition of dilution buffer (50 mM Tris-HCl, pH 7.4, 190 mM NaCl, 6 mM EDTA, and 2.5% Triton X-100) and cooled on ice. In contrast, immunoprecipitations done under nondenaturing conditions omitted the addition of SDS and heating and proceeded directly with addition of the primary antibody to the cleared sample, as described above.

Sequential immunoprecipitations were performed in two steps. First, cells were lysed after a specific period of biosynthetic radiolabeling, treated with DTSSP, and immunoprecipitated with antiserum to CRT, as described above. The CRT immunoprecipitates were heated at 100 °C in the presence of 2% SDS and 10 mM dithiothreitol (DTT) to disrupt the internal disulfide bond in the DTSSP and thereby dissociate the cross-linked proteins. The samples were cooled and diluted with TBS for a final concentration of 0.3% SDS and 1.6 mM DTT. The samples were then immunoprecipitated under denaturing conditions using antibodies against CRT, hsp90, GRP94, and MPO, respectively.

Cross-linking

Proteins were cross-linked with DTSSP according to the manufacturer's instructions. PLB 985 cells were washed in phosphate-buffered saline and resuspended in 100 µg/ml DTSSP with 1% Triton X-100 and 1 mM phenylmethanesulfonyl fluoride in phosphate-buffered saline. After standing for 30 min at room temperature, the cross-linking reaction was terminated by the addition of Tris-HCl (pH 7.4) (to make the final concentration 50 mM) and by incubation on ice for 5 min. Cell lysates were subjected to immunoprecipitation as described above. Except where noted, the cross-linked proteins were dissociated prior to separation in polyacrylamide gels.

Analysis of Immunoprecipitated Proteins

Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Except where noted, all samples were reduced prior to SDS-PAGE. When two-dimensional gel analysis was performed, the immunoprecipitate was soaked in lysis buffer and then electrophoresed into tube gels according to the method of O'Farrell(24) . Analysis in the first dimension was performed by electrophoresis toward the anode (isoelectric focusing, IEF) and toward the cathode (non-equilibrium pH gel electrophoresis) (NEPHGE). The IEF or NEPHGE tube gel was soaked in SDS sample buffer containing bromphenol blue and subsequently electrophoresed into a standard SDS slab gel, as previously done(23, 25) . The resultant slab gels were fixed, soaked in 1 mM sodium salicylate(26) , dried, and subjected to autoradiography. In some cases, the relative amounts of radioactivity in the gels of immunoprecipitated proteins were quantitated on a Phosphorimager SF (Molecular Dynamics).


RESULTS

To determine if CRT functioned as a molecular chaperone, we sought proteins that coprecipitated with CRT at various times during its biosynthesis. Experiments using PLB 985 cells or HL 60 cells gave identical results (data not shown), indicating the observed phenomena were not peculiar to PLB 985 cells but were representative of cultured human promyelocytic cells. PLB 985 cells synthesize CRT, which migrates as a single 60-kDa protein after SDS-PAGE(22) . Because the interactions between nascent proteins and their associated molecular chaperone are noncovalent and transient(27) , they are easily disrupted. For that reason immunoprecipitations performed under both highly stringent, denaturing conditions and less stringent, nondenaturing conditions were compared. PLB 985 cells were pulse labeled for 2 h, lysed, and immunoprecipitated with monospecific antiserum against CRT (Fig. 1A). Under denaturing conditions, the 60-kDa CRT was the predominant protein immunoprecipitated. A small amount of a 90-kDa protein, seen as a faint band after autoradiography, was coprecipitated even under these stringent conditions. However, when immunoprecipitation was performed under nondenaturing conditions, both CRT and a significant amount of the 90-kDa protein were immunoprecipitated.


Figure 1: Under nondenaturing conditions or after chemical cross-linking, CRT coprecipitated with a 90-kDa protein in PLB 985 cells. A, PLB 985 cells were pulse-labeled with [S]methionine for 2 h, lysed, and immunoprecipitated with antiserum for CRT under denaturing (D) or nondenaturing (ND) conditions, and the immunoprecipitates were separated by SDS-PAGE. Under both conditions, CRT migrated as a 60-kDa protein after SDS-PAGE. A 90-kDa protein coprecipitated with CRT, especially when immunoprecipitation was performed under nondenaturing conditions. B, radiolabeled PLB 985 cells were lysed, treated with DTSSP or sham treated, and immunoprecipitated with CRT antiserum under denaturing conditions. In the presence of DTSSP cross-linking, CRT antiserum precipitated CRT, a 90-kDa protein, and proteins in the 160-205-kDa range when the immunoprecipitate was electrophoresed under nonreducing conditions. When the cross-linked proteins immunoprecipitated by CRT antiserum were reduced prior to SDS-PAGE, only CRT and a 90-kDa protein were identified.



Association of CRT and the 90-kDa protein was also directly demonstrated by the use of a reversible chemical cross-linker. Such cross-linking of associated proteins allows immunoprecipitations to be done under denaturing conditions, and the cross-linking can be easily reversed by incubating the immunoprecipitates with reducing agent prior to SDS-PAGE. Radiolabeled cells were lysed, treated with the water-soluble homobifunctional cross-linker DTSSP, and immunoprecipitated under denaturing conditions with antiserum to CRT. Cross-linked proteins migrated as high molecular weight complexes (160-205 kDa) when the CRT immunoprecipitate was electrophoresed under nonreducing conditions (Fig. 1B). In contrast, when the identical immunoprecipitate was reduced prior to SDS-PAGE, the high molecular weight species disappeared, leaving only CRT and the associated 90-kDa protein (Fig. 1B).

Undifferentiated PLB 985 cells are very active biosynthetically and make a number of proteins that migrate in the region of 90 kDa, including hsp90, GRP94, and the glycosylated precursors of MPO, apopro-MPO, and pro-MPO. To determine the identity of the 90-kDa protein, we performed sequential immunoprecipitations using antiserum to CRT in the first immunoprecipitation followed by antibodies to hsp90, GRP94, and MPO in the second immunoprecipitation. PLB 985 cells were labeled, lysed, treated with DTSSP, and immunoprecipitated under denaturing conditions with antiserum to CRT. When the immunoprecipitate was separated by SDS-PAGE under nonreducing conditions (-DTT), most of the cross-linked proteins remained associated with CRT, resulting in high molecular mass proteins migrating between 160 and 205 kDa (Fig. 2A). In addition to the high molecular weight complex, free CRT and an 85-kDa protein were separated by SDS-PAGE under nonreducing conditions. The internal disulfide bond in DTSSP was disrupted to disassociate the complex, and the samples immunoprecipitated with antibodies to CRT, hsp90, GRP94, or MPO (Fig. 2A). As an internal control, the antibody to CRT successfully precipitated CRT when used as the second antibody. Neither the antibody to hsp90 nor that to GRP94 precipitated a radiolabeled protein when used as the second antibody in the sequential immunoprecipitation. In contrast, the MPO antibody precipitated a 90-kDa protein from the sequential immunoprecipitation when used as the second antibody and separated in SDS-PAGE under reducing conditions. All three antibodies immunoprecipitated their target proteins from radiolabeled cells when used as the first antibody in the immunoprecipitation (Fig. 2B), demonstrating that the failure to precipitate hsp90 and GRP94 in the sequential immunoprecipitation reflected the absence of these proteins from the first immunoprecipitation, using CRT antiserum, rather than the inability of the cells to synthesize the proteins or of the antibodies to immunoprecipitate. MPO contains internal disulfide bonds(28) , and the precursor migrates as a 90-kDa protein under reducing conditions but as an 85-kDa protein under nonreducing conditions. (^2)The presence of a small amount of the 85-kDa protein in the nonreduced cross-linked sample (Fig. 2A) indicates that some of the coprecipitated MPO precursor was not cross-linked under these conditions. These data demonstrate that antiserum to CRT selectively immunoprecipitated the 90-kDa precursor of MPO.


Figure 2: CRT coprecipitated with the 90-kDa precursor of myeloperoxidase. A, cells were labeled as described in Fig. 1, lysed, cross-linked with DTSSP, and immunoprecipitated with antiserum to CRT. The cross-linked proteins were separated by SDS-PAGE under nonreducing conditions (-DTT). In the sequential immunoprecipitations, the cross-linking was disrupted, and the samples were immunoprecipitated with the second antibody directed against CRT, hsp90, GRP94, or MPO. The nonreduced samples contained the high molecular mass protein complexes (160-205 kDa), free CRT, and a protein of 85 kDa. The only antisera precipitating proteins in the second immunoprecipitation were those for CRT and for MPO. Internal disulfide bonds in the MPO precursor caused it to migrate differently under reducing (90 kDa) and nonreducing (85 kDa) conditions. B, under the labeling conditions used, PLB 985 cells synthesize numerous proteins (Total), including hsp90, GRP94, and MPO, each of which was immunoprecipitated from labeled cells by the corresponding specific antisera. The cross-linked proteins precipitated by CRT antiserum were dissociated and subjected to two-dimensional analysis. C, the immunoprecipitate was analyzed both by IEF and by NEPHGE in the first dimension, followed by SDS-PAGE. Whereas CRT migrated into the IEF gel in the first dimension, the 90-kDa protein migrated as a single protein into the NEPHGE gel, indicating it was highly cationic. The behavior of this protein under these conditions was identical to that of the MPO precursor (lowerpanel).



To verify that the 90-kDa protein coprecipitated with CRT was related to MPO and that no additional 90-kDa proteins were coprecipitated, the cross-linked proteins precipitated by the CRT antiserum were reduced, to dissociate the linked proteins, and then subjected to analysis in two-dimensional gels (Fig. 2C). The precipitated proteins were separated in the first dimension by IEF or by NEPHGE, and the resultant gel was subjected to SDS-PAGE. In the first dimension analysis, all of the 90-kDa protein coprecipitated with CRT migrated under NEPHGE conditions, indicating its strongly cationic character. The behavior of this protein under these conditions was identical to that of the 90-kDa precursor of MPO (Fig. 2C, lowerpanel). In contrast, CRT migrated into the IEF gel, as expected given its isoelectric point. No 90-kDa protein was detected after IEF times SDS-PAGE.

When biosynthetically labeled PLB 985 cells were immunoprecipitated with MPO antiserum under nondenaturing conditions, no radiolabeled proteins unrelated to MPO were immunoprecipitated. CRT has a very long half-life, in excess of 50 h(22) , and one would anticipate that this pool of preexistent CRT would result in CRT of low specific activity after biosynthetic labeling. When unlabeled cells were immunoprecipitated with MPO antiserum and the electroblotted immunoprecipitates were probed with CRT antiserum, approximately 10% of the total cellular CRT was detectable in the immunoprecipitate (data not shown). These findings are essentially the same as those for calnexin and its associated proteins(29, 30, 31, 32) .

To determine if the glycosylation state of the MPO precursor influenced its interaction with CRT, we examined the effect of TM on the formation of the CRTbulletMPO precursor complex. In many instances, conditions that produce misfolded proteins increase the expression of molecular chaperones(33, 34, 35) . In the presence of TM, an antibiotic that inhibits N-linked glycosylation(36) , the resultant nonglycosylated proteins often fail to fold properly, and their presence in the ER induces the expression of molecular chaperones(33, 34, 35, 37) . In contrast to its effect on GRP94 synthesis, (^3)TM did not increase the rate of CRT biosynthesis (Fig. 3A). In addition, antiserum to CRT failed to coprecipitate any 90-kDa protein from cells grown in the presence of TM (Fig. 3A, lanes1 and 2). The failure of the 90-kDa protein to coprecipitate with CRT suggests that TM inhibited either the production of the 90-kDa protein or its association with CRT. As previously shown(7) , TM-treated PLB 985 cells synthesize the 80-kDa nonglycosylated form of the MPO precursor (Fig. 3B). These data indicate that CRT associated specifically with the glycosylated form of the MPO precursor and not with the nonglycosylated 80-kDa precursor.


Figure 3: CRT associated only with the glycosylated form of MPO. A, radiolabeled PLB 985 cells grown in the absence or presence of 5 µg/ml TM were cross-linked with DTSSP or sham treated prior to immunoprecipitation with CRT under denaturing conditions. In the presence of TM, no MPO precursor of MPO was associated with CRT even after chemical cross-linking. B, PLB 985 cells radiolabeled in the presence of 0, 2.0 µg/ml or 5 µg/ml TM and immunoprecipitated with antiserum for MPO under denaturing conditions were compared. In the absence of TM, the fully glycosylated 90-kDa MPO precursor was synthesized. In contrast, inhibition of N-linked glycosylation by TM resulted in synthesis of the nonglycosylated 80-kDa form.



We reasoned that if CRT were acting as a molecular chaperone, it likely associated with the MPO precursor transiently and at a specific stage during its biosynthesis. To determine the timing of the association between CRT and the MPO precursor, PLB 985 cells were pulse labeled for 1 h and chased for 20, 40, 60, or 120 min. At the end of each chase interval, cells were lysed, treated with DTSSP or sham treated, and immunoprecipitated with antibody to CRT (Fig. 4). Over the period of chase, the relative amounts of MPO precursor and CRT were stable. However, the amount of CRT-associated MPO precursor was relatively stable at 20 min (20.0 + 3.5% of total 90-kDa synthesized), 40 min (22.0 + 5.6%), and 60 min (17.1 + 4.3%) but decreased significantly at 120 min (11.8 + 0.5%, compared with amount at 0 min of chase, p < 0.0009 for n = 3). This decay in CRT-associated MPO precursor is especially remarkable given the very slow processing of MPO previously reported(8, 17, 38) . These data indicate that the association between CRT and the MPO precursor was restricted to a relatively short period early in MPO biosynthesis.


Figure 4: CRT associated transiently with an MPO precursor early in MPO biosynthesis. PLB 985 cells were biosynthetically pulse-labeled with [S]methionine for 60 min and then collected (0-hr chase) or chased for 20, 40, 60, and 120 min. Lysed cells were cross-linked with DTSSP or sham treated and immunoprecipitated with antiserum against CRT. Over the period of the pulse chase, levels of CRT and total 90-kDa MPO precursor remained relatively stable. In contrast, the amount of MPO precursor associated with CRT remained stable for 60 min and then decayed rapidly to 11.8 ± 0.5% of the total MPO precursor at 120 min of chase (p < 0.009, n = 3). Results are representative of three separate experiments.



Both the heme-free precursor, apopro-MPO, and the enzymatically active heme-containing precursor, pro-MPO, are 90-kDa proteins(8, 9, 17) . Arnljots and Olsson (17) demonstrated that -[^14C]aminolevulinic acid (-ALA), a precursor in heme synthesis, is incorporated into the 90-kDa precursor of MPO after 3 h of labeling but that long periods of chase (i.e. 7-20 h) are needed for conversion to the heme-containing 59-kDa heavy subunit of mature MPO. Based on the observed kinetics of the interaction between CRT and the MPO precursor (Fig. 4), we anticipated that CRT might not associate with heme-containing species of MPO. To determine directly if CRT associated with any of the heme-containing forms of MPO, PLB 985 cells were biosynthetically labeled continuously for 20 h with -ALA. Under these conditions, label is incorporated into the heme groups in pro-MPO and in the heavy subunit of mature MPO (17) . Such a prolonged labeling interval was used to maximize incorporation of ^14C-labeled -ALA into MPO-related species and to ensure that the heme in the heavy subunit of the mature protein was labeled.

Although it is possible to label cells with ^14C-labeled -ALA continuously for 20 h, parallel labeling with [S]methionine is not possible because the low absolute concentration of methionine severely limits overall protein synthesis by the cells. To overcome this limitation but still assess the CRT-MPO association at early and late time points in biosynthesis, we examined [S]methionine-labeled proteins, which coprecipitated with CRT at two times: immediately after pulse labeling (no chase) and after a 16-h chase. Lysed cells were treated with DTSSP and immunoprecipitated with antiserum to either CRT or MPO (Fig. 5). From the [S]methionine pulse-labeled cells that were not chased, the 90-kDa form of MPO precursor was immunoprecipitated by MPO antiserum (Fig. 5A). From [S]methionine-labeled cells chased for 16 h, the antiserum to MPO immunoprecipitated the 90-kDa precursors as well as the 59- and 13.5-kDa subunits of mature MPO (Fig. 5A). From cells labeled with ^14C-labeled -ALA, MPO antiserum immunoprecipitated the heme-containing species, pro-MPO and the 59-kDa heavy subunit of mature MPO (Fig. 5B). The pattern of radiolabeled proteins immunoprecipitated with CRT antiserum differed from that seen when MPO antiserum was used to immunoprecipitate. From [S]methionine pulse-labeled cells, CRT antiserum precipitated CRT and a 90-kDa MPO precursor (Fig. 5A). In contrast to the results obtained in the absence of a chase period, little or no MPO precursor coprecipitated with CRT after the 16 h of chase, consistent with earlier findings (Fig. 4). In addition, CRT antiserum failed to immunoprecipitate any proteins labeled with ^14C-labeled -ALA (Fig. 5B). Neither of the heme-containing species, pro-MPO and mature MPO, was immunoprecipitated with CRT antiserum, indicating the [S]methionine-labeled protein coprecipitated with CRT was free of heme, i.e. apopro-MPO.


Figure 5: CRT associated with apopro-MPO. PLB 985 cells were pulse labeled with [S]methionine (S-met) for 1 h and chased for 0 or 16 h (A) or labeled continuously for 20 h with ^14C-labeled -ALA (B), lysed, cross-linked with DTSSP, and immunoprecipitated with antiserum to CRT or to MPO. The MPO antiserum immunoprecipitated [S]methionine radiolabeled 90-kDa MPO precursors from pulsed cells and both precursor and the 59- and 13.5-kDa subunits of mature MPO from cells that were chased after labeling. As previously shown, heme is inserted into the MPO precursor to generate pro-MPO, resulting in ^14C-labeled -ALA-labeled pro-MPO and the 59-kDa heavy subunit. CRT antiserum coprecipitated only small amounts of the [S]methionine radiolabeled 90-kDa MPO precursor after the chase period and failed to immunoprecipitate any heme-containing species labeled with ^14C-labeled -ALA.



We reasoned that if CRT associated with apopro-MPO, conditions that increase the relative amount of heme-free precursor would result in an increase in the amount of MPO-related precursor associated with CRT. Previously, we have shown that inhibition of heme synthesis in cells cultured in succinyl acetone selectively blocks the processing of MPO (8) . Under these conditions, apopro-MPO is unable to mature in the biosynthetic pathway. Consequently, there is a block in proteolytic processing of MPO precursor to mature MPO, a profound inhibition of peroxidase activity, and a decrease in the amount of MPO precursor secreted(8) . To examine the effects of succinyl acetone on the CRT-apopro-MPO interaction, cells were labeled for 2 h and chased for 15 h in the absence or presence of 250 µM succinyl acetone. Lysed cells were treated with DTSSP and immunoprecipitated with antiserum to MPO or to CRT (Fig. 6). In the presence of succinyl acetone, the amount of CRT-associated MPO precursor increased to 242% of control levels (n = 3), whereas CRT or MPO alone each increased to 163% of control.


Figure 6: Inhibition of heme synthesis with succinyl acetone increased the amount of MPO precursor associated with CRT. PLB 985 cells cultured for 72 h in the absence or presence of 250 µM succinyl acetone were pulse labeled with [S]methionine and chased for 15 h; the cell lysates cross-linked with DTSSP prior to immunoprecipitation with antiserum against MPO or CRT. In the presence of succinyl acetone, the amount of MPO precursor coprecipitated with CRT was increased, suggesting that the MPO species that associated with CRT was the heme-free, enzymatically inactive form, apopro-MPO. Ippt. Ab, immunoprecipitating antibody.




DISCUSSION

Taken together, these data demonstrate that CRT associated with apopro-MPO during biosynthesis of MPO in the human myeloid cell line PLB 985. The interaction between CRT and apopro-MPO occurred early in MPO biosynthesis and terminated before introduction of heme into the protein backbone. Thus, CRT was not associated with pro-MPO or with the subunits of mature MPO. Furthermore, the association between CRT and the MPO precursor was restricted to an interaction with fully glycosylated apopro-MPO; CRT did not interact with nonglycosylated MPO precursors.

CRT is a high capacity, low affinity, calcium-binding protein that resides in the ER (reviewed in (39) ). Although most studies of CRT have focused on its importance in cellular calcium homeostasis, recent evidence indicates that it participates in a wide range of cellular functions(40) , from associating with perforin in the lytic granules of cytolytic T cells (41) to controlling gene expression(42, 43) .

In most cells, CRT resides in the ER, retained there by the carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) sequence, a recognized ER retention signal(44, 45) . The lumen of the ER is the site of nascent polypeptide folding, a process often facilitated by various molecular chaperones(46, 47, 48, 49, 50) . CRT may modulate interactions between KDEL-containing proteins and the KDEL receptor by regulating calcium within the ER. In addition, CRT may directly participate in biosynthesis of proteins as they are processed in the ER(39, 51) . The genes for CRT and the molecular chaperones GRP78 and GRP94 share significant sequence homology in the 5`-flanking regions(52) , suggesting they may be coordinately regulated and/or their gene products might subserve similar functions. Like CRT, both GRP78 and GRP94 possess the ER retention sequence KDEL at the carboxyl terminus and bind calcium with high capacity and low affinity(44, 53) . Furthermore, Nigam et al.(54) have recently found GRP78, GRP94, and CRT among the seven ER proteins in homogenates of rat liver specifically eluted by ATP from a denatured protein affinity column. In support of a role for CRT in associating with specific proteins in the ER, CRT and flavin-containing monooxygenase were copurified from rabbit lung, prompting speculation that CRT may act as a chaperone for flavin-containing monooxygenase(51) .

Our data suggest that the role of CRT as a molecular chaperone is similar to that recently described for calnexin(29, 30, 31, 55, 56, 57, 58, 59, 60, 61) and different from that of GRP78 and related molecular chaperones. Calnexin associates only with glycosylated forms of specific proteins; nonglycosylated forms produced in the presence of TM do not interact with calnexin(29, 61) . Nonglycosylated proteins synthesized under these conditions are often misfolded and generally interact with GRP78, GRP94, or other molecular chaperones(33, 34, 35, 37) . In a fashion similar to that of calnexin, CRT interacted with glycosylated apopro-MPO but not with the 80-kDa nonglycosylated MPO precursors synthesized in the presence of TM. In fact, GRP94 coprecipitated with the nonglycosylated 80-kDa form of MPO precursor synthesized in the presence of TM.^3 CRT is a soluble protein within the lumen of the ER (39) , whereas calnexin is tethered there by a transmembrane domain. However, the four domains of calnexin that are homologous to CRT all reside within the ER lumen(59, 62) , suggesting that these regions in both proteins mediate their function as chaperones.

As a molecular chaperone in the ER, CRT occupies a niche between the soluble, stress-modulated species of the GRP78 family and the ER membrane-bound chaperone calnexin. In addition, the selective interaction of CRT with apopro-MPO suggests that in myeloid cells CRT may facilitate events peculiar to apopro-MPO. Just as calnexin may function to keep integrin subunits alpha(6) and beta(1) in a state competent for assembly into the functional heterodimer(31) , CRT may keep apopro-MPO in a conformation that allows insertion of heme into its polypeptide backbone and conversion of inactive precursor to the enzymatically active MPO precursor. This would be the first report of an interaction between CRT and a maturing protein. Furthermore, facilitation of heme insertion into MPO would constitute a novel function for a molecular chaperone. It is conceivable that CRT and calnexin may be representatives of a class of molecular chaperones that interact with selective proteins made in specially differentiated cells.

It is possible that the insertion of heme into apopro-MPO is the signal for dissociation of the CRTbulletMPO precursor complex. However, our data do not address directly what factors regulate CRT-apopro-MPO dissociation. Coordination and regulation of the interactions necessary for productive protein folding, insertion of a functional heme group, and assembly of newly synthesized polypeptide chains remain to be elucidated.


FOOTNOTES

*
This work was supported in part by merit review grants from the Dept. of Veterans Affairs (to W. M. N. and R. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Clinical Investigator in the Dept. of Veterans Affairs. To whom correspondence should be addressed: Dept. of Medicine, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242; Tel.: 319-356-1739; Fax: 319-356-4600.

(^1)
The abbreviations used are: PMNs, polymorphonuclear neutrophils; -ALA, -aminolevulinic acid; CRT, calreticulin; DTSSP, 3,3`-dithiobis(sulfosuccinimidyl proprionate); DTT, dithiothreitol; GRP94, glucose-regulated protein, 94 kDa; hsp90, heat shock protein, 90 kDa; IEF, isoelectric focusing; MPO, myeloperoxidase; NEPHGE, non-equilibrium pH gel electrophoresis; PAGE, polyacrylamide gel electrophoresis; TBS, tris-buffered saline; TM, tunicamycin; ER, endoplasmic reticulum.

(^2)
W. M. Nauseef, unpublished data.

(^3)
W. M. Nauseef and S. J. McCormick, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Elizabeth J. Luna for critical reading of the manuscript and helpful comments. We gratefully acknowledge the secretarial support of Deb Nollen in the preparation of this manuscript.


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