(Received for publication, July 18, 1994; and in revised form, December 9, 1994)
From the
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.
Polymorphonuclear granulocytes (PMNs) ()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:HO
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.
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.
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. ()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 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 CRTMPO 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, (
)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
-[
C]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
C-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 C-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
C-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
C-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
C-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
C-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
C-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.
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. 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 and
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 CRTMPO 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.