(Received for publication, January 26, 1996)
From the
Glycosyl-phosphatidylinositol-anchored hydrophobic placental
folate receptors (PFRs), which have an important functional role in
maternal-to-fetal transplacental folate transport, can be converted to
soluble hydrophilic forms by a placental metalloprotease. Using a
Triton X-114 temperature-induced phase separation assay to monitor
enzyme-mediated conversion of radiolabeled hydrophobic PFR into
hydrophilic PFR, a metalloenzyme was isolated to apparent homogeneity
from Triton X-114-solubilized human placenta using concanavalin
A-Sepharose and reverse-phase high performance liquid chromatography
(HPLC) as major purification steps. The purified hydrophobic enzyme
eluted as a single protein peak on reverse-phase HPLC and
SDS-polyacrylamide gel electrophoresis revealed a single 63,000 M species, which was reduced to 58,000 M
following deglycosylation, findings comparable
with amino acid analysis (M
59,000). The
metalloenzyme was activated by Mg
,
Zn
, Mn
, and Ca
,
optimally at physiologic pH; it also exhibited EDTA-sensitive
endoproteolytic cleavage of [
H]leucine-labeled
full-length nascent PFR polypeptide generated in vitro in the
absence of microsomes. Rabbit polyclonal anti-metalloprotease antiserum
specifically immunoprecipitated
I-metalloprotease and
recognized cross-reacting moieties on plasma membranes of normal human
hematopoietic progenitor cells and human cervical carcinoma cells, both
of which also express FR.
Folate receptors (FR) ()are cell surface
glycoproteins that bind physiologic serum 5-methyltetrahydrofolate with
high affinity and transport the vitamin into cells (reviewed in (1) ). Human placental FR (PFR) have recently been shown to
have a major functional role as modulators of maternal-to-fetal
transplacental folate transport(2) . Native hydrophobic PFR are
glycosyl-phosphatidylinositol (GPI)-anchored proteins, have a M
of 35,000, and require detergent for
solubilization out of membranes(3) . Because of the GPI anchor,
PFR are susceptible to cleavage to soluble forms by GPI anchor-specific
phospholipases C and D(3) . However, through studies on
detergent-solubilized crude placenta (containing cytosolic, nuclear,
and membrane proteins), we also identified a specific
Mg
-dependent enzyme that converted hydrophobic PFR to
hydrophilic forms that retained ligand binding capacity(4) .
The functional nature of this metalloenzyme in chorionic villi cultured
under serum-free conditions has been demonstrated(5) . Because
hydrophilic and GPI-anchored FR from nasopharyngeal carcinoma (KB)
cells (6) have similar amino-terminal amino acid
sequences(7) , the locus of cleavage by the placental
metalloenzyme has been provisionally assigned to the carboxyl-terminal
domain of the native PFR species(1, 5) . Furthermore,
isolation of hydrophilic PFR released into the growth medium of
chorionic villi identified a species that was much smaller on
amino acid analysis (22.5 kDa) when compared with native PFR but was of
comparable M
with the soluble folate binding
protein isolated from human milk (8) and the growth medium of
KB cells(9) . This lent further support to the conclusion that
the placental metalloenzyme was a protease; however, such data were
indirectly generated, and a direct demonstration of endoproteolytic
cleavage of the native PFR by the metalloenzyme was lacking. In
addition, all studies that demonstrated metalloenzyme activity have
relied on gel filtration in Triton X-100, which reliably separates the
hydrophobic (apparent 160 kDa) FR substrate from its hydrophilic (40
kDa) product(4, 5, 10) . However, gel
filtration is a cumbersome assay that is not easily adaptable to
analysis of multiple variables required in isolation of proteins. This
may explain why additional information on physico-chemical
characteristics of this metalloprotease or its localization has not
been generated.
Although FR in KB cells are
GPI-anchored(6) , in the membranes of another KB cell line, the
biosynthetically labeled [H]leucine-hydrophobic
FR fortuitously contained a full-length FR polypeptide that is
leucine-rich in its COOH-terminal end(10) . Therefore, when
these investigators identified an activity in washed, Triton
X-100-solubilized KB and placental membranes that converted
[
H]leucine-labeled hydrophobic FR to hydrophilic
forms, the assignment of this enzyme as a metalloprotease (as opposed
to a GPI-specific phospholipase) was direct. This unique KB cell FR
differed considerably from PFR, which, like other GPI-anchored
proteins, have lost their COOH-terminal hydrophobic polypeptide during
post-translational addition of the preformed GPI
anchor(11, 12) . Thus, it is still unclear whether the
putative placental metalloprotease, which endoproteolytically cleaved
the FR polypeptide substrate from KB cells(10) , is the same
enzyme that converts mature GPI-linked PFR (3, 5) to soluble forms.
The biological significance of the conversion of placental hydrophobic to hydrophilic FR is unclear. In the case of FR on malignant and normal cells, the conversion of hydrophobic FR to hydrophilic FR as mediated by a metalloprotease or GPI-specific phospholipase can potentially be an important mechanism for post-translational regulation of the expression of FR on the cell surface. Although a major role for the metalloprotease was not identified in the placenta at term(2) , its activity could easily determine the extent of acquisition of folates by FR on trophoblasts, thereby influencing placental growth and development. However, no probes (antibodies or cDNA) are as yet available to study such PFR-directed enzymes. Because the GPI-linked hydrophobic PFR sequesters in the micellar phase at the cloud point of Triton X-114(3) , whereas the hydrophilic PFR product sequesters in the aqueous phase, and the EDTA-sensitive enzyme in solubilized human placenta can completely cleave all native hydrophobic GPI-anchored FR into hydrophilic forms(3, 4, 13) , we developed a rapid assay for the metalloenzyme using Triton X-114 and focused on developing a purification strategy. We now report on the isolation and characterization of this enzyme and show that moieties that share epitopes with this metalloprotease are present on the surface of malignant and normal cells that co-express the FR substrate.
Figure 1:
Rate of conversion
of hydrophobic PFR to hydrophilic PFR by crude metalloenzyme as a
function of dose (A) and incubation time (B). A, to tubes containing 9.04 pmol of
[H]PteGlu-labeled hydrophobic PFR increasing
concentrations of crude solubilized metalloenzyme were added in the
absence (closed circles) or the presence (open
circles) of 60 mM EDTA. After incubation at 37 °C and
temperature-induced phase separation at the cloud point of Triton
X-114, the aqueous phase containing hydrophilic PFR (product) was
separated from the substrate retained in the micellar phase, and the
percentage of conversion was determined. B, two concentrations
of detergent-rich metalloenzyme, 0.4 (closed squares) and 2 mg (open squares), were incubated with 9.04 pmol of
[
H]PteGlu-labeled hydrophobic PFR for various
times indicated at 37 °C. The results are the means of triplicate
experiments, and each value did not deviate more than 5% from the
mean.
The optimal duration of incubation at 37 °C was determined for two levels of crude metalloenzyme (Fig. 1B). The basal conversion recorded for the higher level of metalloenzyme likely occurred during the incubation at 37 °C to effect temperature-induced phase separation. Following incubation for 2 h, there was 60 and 90% conversion of hydrophobic PFR to hydrophilic PFR at 400 and 2000 µg of metalloenzyme, respectively. Thus, 2 h was optimum, with no additional gain even after incubation for 16-24 h, consistent with earlier data(4) .
In preliminary studies (data not shown), an
activity that bound to and was eluted from ConA-Sepharose was noted to
be almost (>95%) entirely sensitive to EDTA; SDS-PAGE of this
preparation revealed a major protein that migrated at 63,000 M. When the ConA-Sepharose eluate was subjected to
HPLC gel filtration in Triton X-114 and each eluted fraction was
individually tested for metalloenzyme activity, there was a major
protein peak with activity that also migrated on SDS-PAGE at 63 kDa.
Thus, this species was the likely metalloenzyme.
Figure 2: Reverse-phase HPLC, SDS-PAGE, and functional analysis of the purified metalloprotease. A, the HPLC-purified metalloenzyme (eluted by 51% buffer B) was reanalyzed under similar conditions by reverse-phase HPLC, and each fraction was spectrophotometrically analyzed for protein. B, SDS-PAGE (7.5%) of reverse-phase HPLC-purified metalloenzyme before (lane 1) and after (lane 2) deglycosylation with recombinant glycopeptidase F. Each well was loaded with 15 µg of protein and stained with Coomassie Blue. C, dose-response curve of the purified sample using the temperature-induced phase separation assay in Triton X-114.
Figure 3:
Analysis of various parameters of
metalloprotease activity. A, rate of conversion of I-hydrophobic PFR to hydrophilic PFR as a function of
time. 60 µg of metalloenzyme in 100 µl of 10 mM potassium phosphate, pH 7.5, containing 20 mM MgCl
was incubated with
I-hydrophobic PFR (350 fmol) for
various times indicated in the absence or the presence of 60 mM EDTA. Each data point represents the mean of experiments carried
out in duplicate; there was <5% variation from the mean in more than
three comparable experiments carried out with different preparations. B, dose-response curves using a fixed concentration of
I-hydrophobic PFR (350 fmol) and increasing
concentrations of purified metalloenzyme in the absence or the presence
of EDTA. C, determination of pH optimum for metalloenzyme
activity in the absence or the presence of 60 mM EDTA. D-F, characteristics of inhibition with 1,10-phenanthroline (D), and reactivation of metalloenzyme (50 µg) with
increasing concentrations of various cations (MgCl
,
MnCl
, CaCl
, and ZnCl
) after
inhibition with 60 mM EDTA (E) and 60 mM EGTA (F).
Following in vitro translation of
full-length 1-kilobase pair nascent PFR mRNA, 5-10% of total
[
H]leucine integrated into nascent PFR
polypeptide. The M
of this species was
30,000
on SDS-PAGE, and it eluted at apparent M
of
80,000 on Sephacryl S-200 gel filtration analysis in Triton X-100,
findings distinctly different from mature GPI-anchored PFR, which
migrated on SDS-PAGE at 44,000 M
and eluted at
apparent 160,000 M
, respectively(3) .
Furthermore, although
1 µl of anti-PFR antiserum
immunoprecipitated >90% of mature [
I]PteGlu (histamine derivative)-labeled
FR(3, 20, 21) , at least 20 µl of this
antiserum was required to specifically immunoprecipitate
[
H]leucine-labeled nascent PFR polypeptide (data
not shown). Thus, not unexpectedly,
[
H]leucine-labeled nascent FR polypeptide had
distinct differences on SDS-PAGE, gel filtration profile in Triton
X-100, and antigenic determinants when compared with mature PFR.
The
reverse-phase HPLC elution profile of in vitro translated and
[H]leucine-labeled nascent PFR polypeptide
generated in the absence of microsomes indicated a single sharp peak of
radioactivity that eluted at 5 min. When this species was incubated
with 50 µg of metalloenzyme, there was a clear-cut shift of the
peak to a retention time of 10 min (data not shown). This conversion
was EDTA-sensitive because the reaction was completely inhibited in its
presence. In addition, the total counts in the major peak eluting at 10
min (i.e. the product) constituted 73% of the total counts in
the EDTA-inhibited control (precursor), confirming that the alteration
in profile was a function of loss of net radioactivity from the nascent
PFR polypeptide. We could not locate the putative cleaved
[
H]leucine-labeled COOH-terminal fragment
released from the nascent polypeptide. Because the in vitro translation mixture itself contained several crude proteins, some
of which could be nonspecific proteases, it is possible that this
smaller hydrophobic species was degraded shortly after it was released
from the major body of the nascent PFR polypeptide by the
metalloprotease. Therefore, because it cleaved and shifted the elution
profile of [
H]leucine-labeled nascent PFR
polypeptide and reduced net cpm in the major fragment, these data
supported the conclusion that the metalloenzyme was a protease
(referred to hereafter as a metalloprotease).
Figure 4:
Fluorescence-activated cell sorting
analysis of cultured human cervical carcinoma cells. Cells were reacted
with nonimmune serum (solid lines), 1:10 diluted (dashed
line), and undiluted (dotted line) anti-metalloprotease
antiserum, respectively. 1 10
cells were
analyzed.
Normal human low density mononuclear cells enriched for hematopoietic progenitors are also known to express FR(22, 24) . When tested by fluorescence-activated cell sorting, nonimmune serum gave a mean channel fluorescence intensity value of 2. However, anti-PFR antiserum and anti-metalloprotease antiserum gave values of 81 and 30 units, respectively (data not shown). Thus, both cultured human cervical carcinoma cells and normal human hematopoietic progenitor cells co-expressed cross-reacting moieties to PFR and placental metalloprotease on cell membranes.
GPI-anchored hydrophobic FR can be cleaved within its GPI
anchor by GPI-specific phospholipases C and/or
D(3, 6, 25, 26) , as well as
endoproteolytically by EDTA-sensitive enzymes (4, 5, 10) and an EDTA-insensitive enzyme(s)
(this report). Thus, cleavage of the GPI-anchored PFR substrate into
hydrophilic forms as monitored by our assay could represent activity of
any or all of these enzymes. Furthermore, in the initial purification
steps, there was some contamination of the crude solubilized
metalloprotease with endogenous hydrophobic PFR. These would reduce the
specific activity of I-hydrophobic PFR (the substrate)
and result in underestimation of the amount of metalloprotease. Thus,
except for the final purified preparation, the calculations to
determine yield during the earlier purification steps can only be
viewed as general estimates of relative improvement in the purity of
the metalloprotease.
The reverse-phase HPLC-isolated
metalloprotease, which exhibited biological activity in converting
hydrophobic PFR to hydrophilic forms, met several criteria for purity:
it exhibited a single protein peak on reverse-phase HPLC and a single
band of protein staining on SDS-PAGE at 63,000 M.
Furthermore, when this preparation was iodinated and similarly
analyzed, there was only a single iodinated species. Moreover, amino
acid analysis of the purified protein revealed a net M
of 59,000, which closely approximated the M
estimated following deglycosylation of the metalloprotease and
SDS-PAGE (58,000 M
). Whereas these data supported
the conclusion that the metalloprotease was apparently homogeneous,
immunofluorescence data indicated that moieties with shared epitopes
with placental metalloprotease were localized on plasma membranes of
normal and malignant cells in a similar distribution as the hydrophobic
FR substrate.
Major differences between the KB cell FR-directed
metalloprotease (10) and the PFR-directed metalloprotease (this
report) were characteristics related to cation dependence: thus, KB
cell metalloprotease was activated by Mn,
Ca
, and Zn
but not by
Mg
, whereas placental metalloenzyme was responsive to
Mg
in addition to the above cations. Although we had
earlier tested whether the placental metalloenzyme was activated by
Zn
and Mn
, our results failed to
assign these cations as activators(4) . Retrospective review of
those results within the context of the present data has identified the
most likely reason: earlier, because
I-hydrophobic PFR
was unavailable, we used the property of reversible [
H]PteGlu binding to hydrophobic PFR (as
opposed to covalent binding of the radioligand to hydrophobic
PFR in the present studies). These two cations (Zn
and Mn
) chemically precipitate free
[
H]PteGlu but do not have any such effect on
[
H]PteGlu that is covalently bound to hydrophobic
PFR. Thus, when these cations were added to activate the metalloenzyme,
most of the free radioligand would likely precipitate and thereby be
unavailable for noncovalent, albeit high affinity, interaction with
PFR. This would severely limit our capacity to detect conversion of
hydrophobic to hydrophilic forms of PFR. In fact, review of our
original ``raw'' data (4) revealed that the net
amount of soluble (nonprecipitated) radioligand applied to the gel
filtration column was reduced to less than 10% of the total
radioactivity added to the reaction mixture. However, the use of
[
H]PteGlu covalently labeled to hydrophobic PFR and
I-hydrophobic PFR circumvented these issues
and allowed reassignment of these cations as activators of the enzyme.
Nevertheless, the fact that Mg
activates the
placental (but not KB cell) metalloprotease suggests that these enzymes
may not be identical.
The strategy employed to prove that the
isolated metalloenzyme was a protease and not a GPI-specific
phospholipase was to determine if it cleaved in vitro synthesized [H]leucine-labeled nascent PFR
substrate, which was generated in the absence of microsomes. As pointed
out earlier(10) , based on the deduced amino acid sequence from
PFR cDNA(18) , there are 257 amino acid residues among which
there are a total of 22 leucine residues: five are within the signal
peptide (amino acids 1-25), and eight are in the hydrophobic
COOH-terminal domain (between amino acids 227 and 257), whereas the
other nine are distributed between amino acids 26 and 226. The nascent
PFR polypeptide translated in vitro in the absence of
microsomes would be full-length and not be truncated in its hydrophobic
COOH-terminal domain during addition of the
GPI-anchor(11, 12) . Therefore,
[
H]leucine would be biosynthetically incorporated
proportionately to its distribution in the nascent polypeptide, i.e. 23% in the signal peptide, 36% in the COOH-terminal
domain, and 41% in other regions of the polypeptide. Because the
COOH-terminal domain is hydrophobic, if EDTA-sensitive endoproteolytic
cleavage occurred either within or proximal to this region, the PFR
polypeptide would be expected to be converted to a relatively
hydrophilic form with loss of specific radioactivity in the major
fragment by up to 36% of the original value. In fact, the
metalloprotease did endoproteolytically alter the substrate in an
EDTA-sensitive manner, and the net recovered radioactivity was
30%
less than the original substrate.
What is unexplained is the
paradoxical conversion of the [H]leucine-labeled
nascent PFR polypeptide to a more hydrophobic species by the
metalloprotease, findings that would not have been predicted based on
data generated with the mature protein. However, three lines of
evidence supported the premise that the
[
H]leucine-labeled nascent PFR polypeptide had a
different folded structure when compared with the native, mature, fully
processed (glycosylated and GPI-anchored) PFR. These included its
relatively poorer immunoprecipitation by anti-PFR antiserum and its
aberrant elution as an apparent
80-kDa species on gel filtration
in Triton X-100. In addition, we previously observed that following
gentle methods of iodination, even mature (hydrophilic) PFR are
extremely susceptible to unfolding with resultant exposure of core
hydrophobic amino acids, which interact with Triton X-100; this led to
major alterations in gel filtration profiles and reduced recognition by
anti-PFR antiserum (20) similar to that noted with nascent PFR
polypeptide. Therefore, it is possible that subsequent to cleavage of
the [
H]leucine-labeled nascent PFR polypeptide by
the metalloprotease, the exposure to organic solvents during HPLC may
have led to its further unfolding, leading to exposure and interaction
of additional core hydrophobic regions with the hydrophobic HPLC
column, as also noted previously(27) . This may explain why
cleavage of the nascent PFR polypeptide versus mature PFR by
the same metalloprotease led to the observed differences in the
hydrophobic properties of the product.
Several results indicated
that the metalloprotease was hydrophobic. These include the fact that
the crude EDTA-sensitive metalloprotease was solubilized from placental
membranes and then recovered in the micellar phase; the fact that the
metalloprotease was eluted by hydrophobic elution buffers from
reverse-phase HPLC; both the HPLC-purified noniodinated and iodinated metalloprotease sequestered in the micellar phase; and
evidence for the membrane localization of cross-reacting moieties in
close proximity to its hydrophobic substrate on normal and malignant
cells. Interestingly, however, when the placental metalloprotease was
originally purified, it was recovered as a hydrophilic species. Subsequent analysis revealed that prolonged storage of
the ConA-Sepharose elution for 1 week at 4 °C (during multiple
batch elutions) led to conversion of the initially hydrophobic
metalloprotease to a hydrophilic species. Thus, the basis for the
hydrophobicity of the metalloprotease must exist within either a lipid
tail (possibly a GPI anchor) or a short hydrophobic polypeptide tail
and that the activity responsible for its conversion to a hydrophilic
species resides in the ConA-Sepharose eluate; this warrants further
investigation.
Recent studies ()have identified a
15-20-fold up-regulation of FR in HeLa-IU
cells
secondary to a reduction in the extracellular folate concentration,
primarily due to a 6-fold increase in the synthetic rate of FR at the
translational level. Up-regulation of FR was also accompanied by a net
increase of hydrophilic FR in the growth medium, similar to KB
cells(28) . Based on identification of FR-directed putative
metalloproteases in HeLa-IU
cells, we plan to determine if
these moieties are functionally active in these cells and are also
regulated during regulation of FR.
Several metalloproteases have
been reported in various tissues with different biological functions
(reviewed in (29) ). Among two distinct metalloproteases with M that are comparable with that estimated for
hydrophobic PFR-directed metalloprotease, one from human cartilage (M
of 62,000) has proteolytic activity against
elastin and requires Zn
for optimum
activity(30) . Another from Leishmania major promastigotes (M
of 63,000) is a
membrane-associated glycoprotein that cleaves azocasein in a pH range
7.0-9.0(31) . Human placenta is also rich in a number of
metalloproteases such as collagenase(32) . Therefore, it will
be of significant interest to determine if additional substrates exist
for this newly isolated placental metalloprotease.