From the
Parathyroid hormone (PTH), an 84-amino acid peptide, is the
major regulator of blood calcium homeostasis. Its mRNA, in addition to
encoding the mature peptide, also encodes a ``pre'' sequence
of 25 amino acids and a basic ``pro'' hexapeptide. To assess
which of the subtilisin-like prohormone convertases can process proPTH
to PTH we coinfected cells with a vaccinia virus construct expressing
human preproPTH and vaccinia virus constructs expressing furin, PC1 or
PC2. BSC-40 cells, having a constitutive secretory pathway, and GH4C1
cells, having a regulated secretory pathway, were used. PTH
biosynthetic products in cell extracts and media were purified by high
performance liquid chromatography, identified by radioimmunoassay, and
unambiguously defined as either proPTH or PTH by ion-spray mass
spectrometry. In both cell types, furin was the most effective in
processing proPTH to PTH. In all cases only PTH was released into the
medium. In addition, partially purified furin and PC1 were tested for
their ability to appropriately cleave a tridecapeptide spanning the
prohormone cleavage site found in proPTH. Here too furin was much more
effective at cleaving at the correct site. Northern blot analysis and
in situ hybridization showed that furin and preproPTH mRNA are
co-expressed in the parathyroid, whereas PC1, PC2, and PC5 are not and
PACE4 is expressed only at very low levels. Taken together these
studies strongly suggest that furin is the enzyme responsible for the
physiological processing of proPTH to PTH.
Parathyroid hormone (PTH),
In order to assess which of these enzymes can
process proPTH to PTH we coinfected cultured cell lines with a vaccinia
virus construct expressing human (h) preproPTH and vaccinia virus
constructs expressing either furin, PC1 or PC2. BSC-40 cells, having a
constitutive secretory pathway, and GH4C1 cells which, additionally,
have a regulated secretory pathway, were used. PTH biosynthetic
products were purified by HPLC and identified by mass spectroscopy.
In addition, partially purified preparations of furin and PC1 were
tested for their ability to appropriately cleave a 13-amino acid
peptide spanning the prohormone cleavage site in proPTH. To assess
which of the processing enzymes are expressed in the parathyroid,
Northern blot analysis and in situ hybridization were
conducted on parathyroid tissue.
The coinfection studies revealed
that in both cells of the constitutive and regulated pathway furin was
the most effective at processing proPTH to PTH. Colocalization studies
showed that furin is expressed in the parathyroid whereas PC1 and PC2
are not. Moreover, hfurin processes the -6 to +7 sequence of
hproPTH very efficiently in vitro. Therefore, furin is most
probably the enzyme responsible for the physiological processing of
proPTH to PTH.
Vaccinia Virus Constructs The purified recombinant vaccinia viruses (VV) used were as follows. The
vaccinia virus recombinant of human proPTH (VV:hPTH) was prepared using
a full-length human (h) preproPTH cDNA
(15) , VV:mPC1 used the
full-length cDNA insert of mouse PC1
(6) , VV:mPC2 was
constructed from the full-length cDNA insert of mouse PC2
(5) .
The recombinant VV:hfurin was prepared using the cDNA of human furin (a
gift from Dr. A. Rehemtulla, Genetics Institute) subcloned into the
pVV3 transfer vector as reported for VV:mPC1 and VV:mPC2
(16) .
The vaccinia virus recombinant of mouse proopiomelanocortin (VV:mPOMC)
was a gift from Dr. G. Thomas (Vollum Institute, Portland, OR). Vaccinia Virus Infections Cells, either BSC-40, an African green monkey epithelial-like cell line,
or GH4C1, a rat pituitary tumor cell line, were infected with a mixture
of VV:hPTH and either VV:mPOMC (control), VV:hfurin, VV:mPC1, or
VV:mPC2, as described previously
(17) . After the infection
period, the inoculum was replaced with Dulbecco's modified
Eagle's medium and cells were incubated for 17 h at 37 °C.
The cells were then incubated in Dulbecco's modified
Eagle's medium containing 0.01% bovine serum albumin for 3 h
after which cells and medium were harvested for further analysis. Reversed-phase High Performance Liquid Chromatography of Culture Media
and Cell Extracts Reversed-phase high performance liquid chromatography (RP-HPLC) of
culture media and cell extracts was performed using C
Both
culture media and cell extracts were separately subjected to an initial
RP-HPLC step using the µBondapak column which was eluted over 1 h
with a linear gradient of 28-48% aqueous acetonitrile containing
0.13% (v/v) heptafluorobutyric acid (C
Following incubation for 0, 30, 60, 90, 120, and 240
min, identical aliquots were taken, acidified with 50 µl of acetic
acid, and analyzed on an Exsil 300A/ODS column (25 cm
Determination of kinetic constants for the proPTH peptide was
accomplished by reversed-phase HPLC analysis followed by integration of
the peak area corresponding to the released COOH-terminal fragment
Ser-Val-Ser-Glu-Ile-Gln-Leu. Various concentrations of the proPTH
peptide ranging from 10 µ
M to 400 µ
M were
incubated in the presence of either hPC1 or hfurin in a total volume
ranging from 400 to 30 µl keeping the enzyme concentration
constant. Following incubation for 90 min (hPC1) or 15 min (hfurin),
the amount of the released COOH-terminal fragment,
Ser-Val-Ser-Glu-Ile-Gln-Leu, was determined after RP-HPLC, as described
above, and by amino acid analysis. The K
The hybridization conditions were as
follows. The cRNA probes were diluted in hybridization buffer (75%
formamide, 10% dextran sulfate, 3
Thus in cells having a constitutive
secretory pathway only ( i.e. BSC-40) and in cells having both
constitutive and regulated secretory pathways ( i.e. GH4C1),
exogenous expression of furin markedly stimulated processing of proPTH
to PTH (). In addition, exogenous expression of PC1
stimulated proPTH processing in GH4C1 cells.
In contrast, digestion of the
synthetic proPTH peptide by equal amounts (as assessed using the same
fluorogenic substrate) of hfurin or hPC1 enzymatic activity clearly
demonstrated as shown in Fig. 5 that the full proPTH recognition
sequence is very efficiently cleaved by hfurin as compared to hPC1.
Whereas the half-maximal value of appearance of the COOH-terminal
fragment was 154 min with hPC1, under the same conditions this was only
28 min for hfurin. In order to assess whether this was due to a better
recognition of the substrate, or an increase in catalytic efficiency or
both, the kinetic constants were determined with each enzyme. As shown
in Fig. 6, purified hPC1 exhibited an apparent
K
The isolation and characterization of several mammalian
subtilisin-like serine endoproteases which cleave carboxyl-terminal to
pairs of basic amino acids has added a new dimension to the analysis of
proprotein processing
(30, 31, 32, 33, 34) . These
enzymes include furin and PC1 (PC3) and PC2. Furin has a wide tissue
distribution, and is localized in the trans-Golgi network
(35) . It has a neutral pH optimum and functions within the
constitutive secretory pathway. In contrast, PC1 and PC2 function
within the regulated secretory pathway. These latter enzymes are
expressed only in endocrine and neural tissues and have been localized
to the secretory granule
(36) . These proteases display acidic
pH optima consistent with the secretory granule environment.
Although proPTH was one of the first prohormones to be described
(37, 38) and its processing to PTH has been extensively
analyzed, no study to date has addressed the issue of which of the
processing enzymes is responsible for its cleavage. The conversion of
endogenously labeled proPTH to PTH was previously shown to take place
within 15 min of synthesis in the trans-Golgi in cultured
bovine parathyroid gland slices in vitro (2) .
Complementary DNA encoding preproPTH was stably introduced into rat
fibroblast NIH 3T3 cells and the rat pituitary cell line GH4C1 with a
recombinant retrovirus
(39) . In both these cell-lines proPTH
was appropriately cleaved to PTH, and only PTH was released into the
medium. Therefore, it could be speculated on two counts, that the
enzyme responsible for cleaving proPTH to PTH was furin-like. First,
proPTH is processed in the trans-Golgi rather than in
secretory granules, and second, proPTH is appropriately processed in
cells of the constitutive as well as the regulated secretory pathway
type.
We demonstrate here that preproPTH expressed using a vaccinia
virus system in either BSC-40 cells or GH4C1 cells led to proPTH being
accurately cleaved to PTH and that the processed PTH molecule only was
released from the cell. In the BSC-40 cells coinfection of preproPTH
and furin constructs led to a clear increase in processing of proPTH to
PTH. In contrast, coinfection of preproPTH with either PC1 or PC2 did
not alter the rate from that of basal. In GH4C1 cells, whereas
coinfection with PC2 again was ineffective in stepping up the rate of
conversion, furin expression led to an even more marked processing than
that observed in the BSC-40 cells and PC1 coexpression caused a
conversion that was intermediate between that found for furin and PC2.
Thus, our studies confirm that proPTH cleavage takes place in cells
exhibiting either a constitutive or regulated pathway. Exogenous furin
expression causes an enhancement of this in both cell types, and
exogenous PC1 is somewhat effective in a cell type with a regulated
secretory pathway which therefore provides the appropriate environment
for it to be active
(17) . The finding that furin, an enzyme
normally associated with the constitutive pathway, processes proteins
destined for the regulated pathway does have precedents. For example,
the precursor to the neuroendocrine protein, 7B2, is processed by furin
within endocrine cells
(40) . Thus, although it is generally
accepted that furin cleaves proteins destined for the constitutive
pathway, our results emphasize that exceptions do exist.
The ability
of PC1 to process proPTH is consistent with our previous finding that
partially purified PC1 can correctly cleave a tridecapeptide spanning
the proPTH cleavage site
(24) . Here, we have extended those
studies to show that partially purified furin also appropriately
cleaves this peptide, but in a much more efficient manner. The
increased catalytic efficiency of furin appears to be due to both an
increase in affinity of the substrate and an increase in the rate of
processing of the substrate. Based upon the results obtained with
several different fluorogenic substrates (see ), one can
speculate that the apparent increase in affinity of the tridecapeptide
could be due to the presence of the basic residue occupying position P6
which essentially plays a role similar to the more frequently
encountered P4 Arg residue
(41) . On the other hand, we cannot
rule out the possibility that the COOH-terminal extension could also be
responsible for the increase in processing rate of the tridecapeptide
which in turn results in an increase in catalytic efficiency. This
conclusion, if warranted, could not have been obtained by studies
employing only the usual small peptidyl fluorogenic substrates. It
suggests that there are multiple subsites in the active site cleft of
convertases which are likely to interact with substrate residues
located on both sides of the bond to be processed. Along these lines,
it is of interest to note that in both the activation sequence of the
proenzyme forms of several of the convertases
(30, 42) ,
as well as in numerous sequences of various precursors cleaved by furin
(43) , one can observe the presence, as in the tridecapeptide
sequence of proPTH, of a hydrophobic aliphatic residue at position P2`.
Clearly, based upon these observations, further studies are warranted
to delineate the structural requirements of the recognition sites of
convertases.
Our present studies confirm those of others that
previously demonstrated that no proPTH is released from cells (or
indeed, found circulating in the blood)
(44) . This would
indicate that the prosequence exclusively serves an intracellular
function. It has been suggested that the prosequence can be considered
as part of a functional unit responsible for transport and cleavage of
the precursor (the preproPTH molecule) upon its entry into the
secretory pathway. Evidence for such a role came from studies
demonstrating that amino acids carboxyl-terminal to the signal peptide
play a role in the efficiency of cleavage of the signal peptide and the
sequence of the propeptide is compatible with this cleavage, whereas
the NH
Interestingly, no increase in the amount of PTH released into the
medium was observed in cells expressing exogenous furin versus control cells. This would indicate that in control cells
overexpressing PTH the available granules are already being maximally
utilized and when PTH production is increased by exogenous expression
of furin the secretory apparatus is unable to respond by increasing its
capacity further. It remains unknown whether this is characteristic of
normal cells or whether the use of the vaccinia expression system
influenced the outcome. Since, after a period of time, normal cellular
protein synthesis is shut down by the vaccinia infection, this may have
disrupted the formation of functional new secretory granules.
In the
parathyroid chief cell the amount of available intact PTH is thought to
be regulated in part by a blood calcium controlled intracellular
degradative pathway. Thus, under low extracellular levels of calcium
little degradation takes place
(46) . However, under high
extracellular calcium conditions, degradation of the hormone takes
place and fragments, predominantly comprising the middle and
carboxyl-terminal regions of the molecule are formed and released. The
enzyme(s) responsible for this metabolism of PTH is (are) unknown. It
has been estimated that the concentration of fragments within the
parathyroid gland is approximately 9-16% of the total content of
PTH moieties
(47) . We found no evidence for significant
fragmentation of the PTH molecule itself in any of the conditions
examined, either by overexpressing PTH alone or with any of the
convertases tested. The chromatography methods used and the midmolecule
radioimmunoassay employed would have been capable of detecting
fragments. Therefore, it is unlikely that this particular class of
enzyme plays a role in the intraglandular degradation of PTH.
By
both Northern blot and in situ hybridization analysis only
furin (and very low levels of PACE4), but not PC1, PC2, or PC5, was
found to be expressed in the parathyroid gland. This, taken together
with the results of the coinfection studies, points to furin as the
physiological mediator of proPTH processing. It remains to be
determined if the expression of furin is regulated in the parathyroid,
for example, by the major regulators of parathyroid gland activity,
calcium and 1,25-dihydroxyvitamin D. Studies have shown that the
conversion of proPTH to PTH is not regulated by extracellular calcium
(46) at least not in the short term, and therefore it would be
anticipated that parathyroid furin expression may not be regulated by
changes in this parameter.
The finding of furin, but not of PC1,
PC2, or PC5 in the parathyroid, also bears upon the processing of the
other major secretory product (besides PTH) of this gland, namely,
chromogranin A (CgA). CgA, which contains several pairs of basic
residues, but no consensus furin site, is expressed widely in
neuroendocrine cells and is cleaved to generate active peptides such as
pancreastatin, which modulate hormone secretion in an autocrine or
paracrine manner. The extent of processing of CgA varies from one type
of neuroendocrine cell to the next. In the parathyroid the amount of
processing of CgA, although it does occur
(48) , is slight in
comparison to that in other neuroendocrine cells, e.g. the
In summary,
we have shown that furin is the most effective convertase in processing
proPTH to PTH, but is probably not involved in the further
intraglandular metabolism of the PTH molecule. Furin and preproPTH
mRNAs are coexpressed in the parathyroid, whereas PC1, PC2, and PC5 are
not. Therefore, the available data strongly suggest that furin is the
convertase responsible for the physiological processing of proPTH to
PTH. This is the only example characterized thus far of biosynthesis of
a peptide hormone which is dependent upon the action of furin.
A preliminary report
of this work was presented at the 76th Annual Meeting of the Endocrine
Society, Anaheim, CA. June 15-18, 1994.
We thank Weijia Dong, Dany Gauthier, Susan James,
Normand Rondeau, Diane Savaria, and Jasloveleen Sohi for their help
with various aspects of this study, and Judith Marshall and Karuna
Patel for preparation of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
an 84-amino
acid polypeptide, is the major regulator of blood calcium homeostasis.
Its messenger RNA, in addition to encoding the mature peptide, also
encodes a ``pre'' or signal sequence of 25 amino acids and a
basic ``pro'' peptide of 6 amino acids
(1) . The
signal sequence facilitates entry of the nascent peptide chain into the
endoplasmic reticulum and following entry into the intracisternal space
this peptide sequence is cleaved. ProPTH is then transported to the
trans-Golgi network where the propeptide is removed followed
by packaging of PTH into secretory granules
(2) . The enzyme or
enzymes responsible for this latter cleavage are unknown. However,
several mammalian subtilisin-like serine endoproteases have recently
been described which can function to process proproteins by cleaving at
pairs of basic residues. These include furin (PACE)
(3, 4) PC1 (PC3)
(5, 6, 7) , PC2
(5, 8, 9) , PACE4
(10) , PC4
(11, 12) , and PC5 (PC6)
(13, 14) . Furin
has a neutral pH optimum and contains a transmembrane region and is
thought to function in the trans-Golgi network, whereas PC1
and PC2 have more acidic pH optima and are thought to act within
secretory granules.
µ-Bondapak (Waters) and C
Vydac TP201 (Cole
Parmer, Chicago) columns, as described previously
(18) .
F
COOH) at
a flow rate of 1.5 ml/min. This gradient was chosen since PTH and all
known parathyroid cell derived fragments of PTH elute from the column
under these conditions
(19) . The presence of immunoreactive PTH
in column fractions was determined by radioimmunoassay with a goat
antiserum raised against
hPTH-
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84) and employing
I-Tyr
-hPTH-
(44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68) as tracer. ProPTH and PTH cross-react identically in this
radioimmunoassay. We previously determined that both proPTH and PTH are
recovered with 80-85% efficiency through successive RP-HPLC steps
(20, 21) . In the present studies, culture media derived
from BSC-40 and GH4C1 cells generated chromatograms in which
immunoreactive PTH coincided with single peaks of UV absorbance. For
these experiments fractions were dried and subjected to mass
spectrometric analysis without further purification. For all cell
extracts further RP-HPLC procedures were required to obtain material of
sufficient purity for mass spectrometry. For these experiments
fractions containing immunoreactive PTH were first subjected to RP-HPLC
using the Vydac column which in each case was eluted over 1 h with a
linear gradient of 8-48% aqueous acetonitrile containing 0.1%
(v/v) CF
COOH at a flow rate of 1.5 ml/min. At this stage
all samples derived from BSC-40 cell extracts yielded chromatograms
where PTH immunoreactivity was associated with single peaks of UV
absorbance. These materials were subjected to mass spectrometric
analysis. Further chromatography of material with PTH immunoreactivity
derived from extracts of GH4C1 cells was required prior to mass
spectrometry. In each case the PTH immunoreactivity could be resolved
into two fractions which were subjected separately to RP-HPLC on the
µBondapak column which was in each case eluted over 1 h with a
linear gradient of 16-56% aqueous acetonitrile containing 0.1%
CF
COOH at a flow rate of 1.5 ml/min. Ion Spray Mass Spectrometry Ion spray mass spectra of purified peptides were obtained using an API
III triple stage mass spectrometer (Sciex, Thornhill, Ontario, Canada).
Mass estimations were done essentially as described previously
(22) . The lyophilized peptide samples were redissolved in 10%
acetic acid and infused through a stainless steel capillary (100-µm
internal diameter) at a flow rate of 1 µl/min. A stream of air was
introduced to assist in the formation of submicron droplets
(23) . These droplets were evaporated at the interface by
nitrogen gas, producing a series of multiply charged ions which are
mass analyzed. Simple algorithms correlate the charges produced by
peptides and proteins to their molecular weights. The mass to charge
ratio ( m/z) of each of these ions produces a molecular weight
estimate. These estimates are averaged to give an observed mass. In Vitro Cleavage of Human ProPTH Peptide
Recombinant hPC1 and hfurin
The two human endoproteases,
hPC1 and hfurin, were obtained from the medium of somatomammotroph
GH4C1 cells following infection with the respective recombinant
vaccinia virus as described elsewhere
(24) . The two proteases
were partially purified using anion exchange chromatography prior to
use and the enzymatic activity of each recombinant enzyme was assayed
using the fluorogenic substrate pGlu-Arg-Thr-Lys-Arg-MCA (Peptides
International, Louisville, KY).
Digestion of Synthetic Human ProPTH Peptide by hPC1 and
hfurin
The synthetic tridecapeptide (proPTH(-6-+7)),
corresponding to amino acids -6 to +7 of the human proPTH
molecule (with +1 designating the first amino acid of the mature
84-amino acid molecule), was kindly synthesized and purified by Drs. S.
Sakakibara and T. Kimura (Peptide Institute, Osaka, Japan). A
100-µg sample was incubated for various time intervals with 18
µl of purified hfurin (containing an activity of 50 nmol/h of
released AMC from pGlu-Arg-Thr-Lys-Arg-MCA (70 µ
M)) at 25
°C in 300 µl of 50 m
M sodium acetate, pH 7.0,
containing 2 m
M CaCland 1 µ
M guanidinoethylmercaptosuccinic acid (Calbiochem Corp., La Jolla,
CA). Similarly, incubations were also done using 50 µl of purified
hPC1 (corresponding to an activity of 50 nmol/h of released AMC from
pGlu-Arg-Thr-Lys-Arg-MCA (70 µ
M)) at 25 °C in 300
µl of 50 m
M sodium acetate, pH 6, containing 5 m
M CaCl
and 1 µ
M guanidinoethylmercaptosuccinic acid. All fluorescence measurements
were made with a Perkin-Elmer MPF-3L spectrofluorometer using an
excitation wavelength set at 370 nm and an emission wavelength set at
460 nm in order to measure the AMC released, as described previously
(25) .
0.46 cm).
The buffer system consisted of an aqueous 0.1% (v/v) CF
COOH
solution and an organic phase containing 0.1% (v/v) CF
COOH
in acetonitrile. Elution was carried out by using a linear gradient of
1% organic phase/min following a 5-min initial isocratic step; the flow
rate was 1 ml/min. The elution pattern was monitored by measuring the
UV absorbance at 225 nm. The peptides detected were automatically
collected and kept at -20 °C until characterized by amino
acid analysis as described previously
(26) .
Determination of Kinetic Constants
K
In
order to compare these constants between small fluorogenic peptidyl
substrates and those obtained with the tridecapeptide, a fluorogenic
substrate corresponding to benzylocarbonyl
( Z)-Val-Lys-Lys-Arg-MCA (Enzyme System Products, Livermore,
CA) was used. Incubations for determination of kinetic constants were
typically carried out in the presence of various concentrations of
fluorogenic peptide substrates from 20 µ
M to 2 m
M for each enzyme in the above mentioned buffers at 25 °C for 4
h before stopping the reaction by the addition of 50 µl of acetic
acid. The amount of AMC released was determined by spectrofluorometry.
and V
and V
values were determined after
curve-fitting using Sigmaplot wv 1.02 software (Jandel Scientific, San
Rafael, CA). Northern Analysis Total RNA was prepared from bovine parathyroid glands by the guanidinium
isothiocyanate/cesium chloride method. Ten-microgram aliquots of RNA
were electrophoresed on 1.1% agarose-formaldehyde gels, blotted onto
Nylon membranes, and hybridized with random primer
P-labeled probes as described elsewhere
(27) . The
probes used were a 2.2-kilobase SmaI- SalI restriction
fragment encoding a human furin cDNA (gift of Dr. A. Rehemtulla,
Genetics Institute), a 325-base pair rat PC1 cDNA corresponding to the
segment 715-1039 of mPC1
(5, 6) , a 753-base pair rat
PC2 cDNA corresponding to the segment 559-1326 of mPC2
(5) , and
a 534bp rPACE4 cDNA corresponding to the segment 1153-1687 of
hPACE4
(10) . In Situ Hybridization Rat thyroids including the parathyroid glands were frozen in isopentane
at -35 °C. Frozen 10-µm sections were cut on a cryostat,
thaw-mounted on polylysine-coated glass slides, and stored at -70
°C until processed for in situ hybridization as described
previously
(28) . Hybridization was carried out with a
digoxigenin-labeled UTP cRNA probe (PTH) or with
S-UTP-labeled cRNA probes (prohormone convertases).
Antisense cRNA probes consisted of a 430-nucleotide rat (r) PTH probe
(29) kindly provided by Dr. G. Heinrich, a 590-nucleotide rat
(r) PC1 probe equivalent to segment 1841-2430 in mouse (m) PC1
(6) ; a 425 nucleotide rPC2 probe equivalent to segment
1574-1998 in mPC2
(5) ; a 1232-nucleotide rfurin probe
equivalent to segment 823-2053 in human (h) furin
(4) ; a
534-nucleotide rPACE4 probe equivalent to the segment 1153-1687
of hPACE4
(10) ; and a 837 nucleotide rPC5 segment
1089-1925
(13) .
SSC, 50 m
M sodium
phosphate, pH 7.4, 1
Denhardt's solution, 0.1 mg/ml yeast
tRNA, and 0.1 mg/ml salmon sperm DNA) with the addition of 1 m
M dithiothreitol. The slides were incubated for 16 h at 55 °C.
After hybridization the coverslips were removed and the sections were
treated with RNase A (40 µg/ml) for 45 min at 37 °C and then
washed sequentially in 2
, 1
and 0.5
SSC for 10
min, followed by a 1-h wash in 0.1
SSC at 60 °C. After
dehydration the sections were air-dried and subsequently dipped in
emulsion, and exposure times ranged from 10 to 15 days. Slides to which
digoxigenin-labeled UTP cRNA probe was hybridized were treated as
follows. Each section was incubated 2 h with a solution of 2
SSC, 0.05% Triton X-100, 2% normal sheep serum. The sections were
washed in 100 m
M Tris, pH 7.5, 150 m
M NaCl followed
by a 5-h incubation with alkaline phosphatase-conjugated
anti-digoxigenin antibody (diluted 1:1000 in 100 m
M Tris, pH
7.5, 150 m
M NaCl, 0.3% Triton X-100, 1% normal sheep serum).
The sections were washed two times for 10 min each in 100 m
M Tris, pH 7.5, 150 m
M NaCl and once in 100 m
M Tris, pH 9.5, 100 m
M NaCl, 50 m
M MgCl
. This was followed by an overnight incubation in
chromogen solution, consisting of 45 ml of nitro blue tetrazolium (75
mg/ml in dimethylformamide), 35 ml of
5-bromo-4-chloro-3-indolyl-phosphate, and 2.4 mg of levamisole per 10
ml of 100 m
M Tris, pH 9.5, 100 m
M NaCl, 50 m
M MgCl
. Incubation was performed in humidified chambers
in the dark. The chromagen reaction was stopped in 10 m
M Tris,
pH 8.0, 1 m
M EDTA. Controls for specificity of hybridization
were carried out using sense strand cRNA probes of similar length and
specific activity or by pretreatment of the sections with RNase A.
Coexpression of ProPTH and Prohormone Convertases in
BSC-40 and GH4C1 Cells, Effect of Furin, PC1, and PC2 on ProPTH
Processing
Purification of immunoreactive (i) PTH moieties from
both cell and media extracts of BSC-40 and GH4C1 cells was readily
accomplished by reversed phase RP-HPLC. On initial chromatography using
an acetonitrile/CF
COOH solvent system for both
cell extracts and media of BSC-40 cells, iPTH eluted as a major peak at
38% acetonitrile (Fig. 1, A and B). This is the
expected elution position of both proPTH and PTH in this system. A
similar elution pattern was seen when GH4C1 cell extracts and media
were purified using the same solvent system (Fig. 1, C and D). Insignificant amounts of iPTH eluted either
before or after this major peak. This was observed in cells and medium
for both the control (coinfection with VV:mPOMC) and experimental
(coinfection with VV:hfurin, VV:mPC1 and VV:mPC2) situations,
indicating that none of the enzymes tested caused significant
fragmentation of the PTH molecule at potential internal cleavage sites.
With medium from BSC-40 cells (Fig. 1 B and data not
shown), a single absorbance peak coeluted with the major iPTH peak.
These fractions were pooled and subjected to mass spectrometric
analysis (Fig. 2). In all cases only PTH itself was found to be
released from the cells; i.e. no proPTH was detectable in the
medium.
Figure 1:
Reversed-phase HPLC
of cell extracts and media derived from cultures of BSC-40 cells
( Panels A and B) and GH4C1 cells ( Panels C and D) infected with VV:hfurin and VV:hPTH. In each
instance the column was eluted with a linear gradient of aqueous
acetonitrile containing 0.13% (v/v) CF
COOH
throughout. Column eluates were monitored for UV absorbance at 210 nm
( continuous line) and column fractions were assayed for the
presence of immunoreactive hPTH ( solid bars). Note that for
the elution profiles of media samples ( Panels B and
D), a peak of PTH immunoreactivity coincided with a single
peak of UV absorbing material ( arrow) which was positively
identified as hPTH using mass spectrometric analysis (see Fig.
2).
Figure 2:
Selected
examples showing ion-spray mass spectra of immunoreactive PTH purified
from extracts of the medium ( A) and cells ( B and
C) derived from cultured GH4C1 cells infected with vaccinia
virus constructs bearing PTH and POMC (control) cDNAs. All major
immunoreactive species isolated from both BSC-40 and GH4C1 cells and
media were identified as either proPTH or PTH by mass spectrometric
analysis as described under ``Experimental Procedures.'' This
allowed unequivocal assignment of the molecular forms to be made. All
values agreed within two mass units of the theoretical values for
hproPTH (10,151 Da) and hPTH (9,424 Da) (15). Panel A shows
the mass spectrum of PTH from the culture medium ( M= 9,424.8). Panels B and C show the mass
spectra of proPTH ( M
= 10,152.4) and PTH
( M
= 9,425.9), respectively, purified from
cell extracts. In each case the molecular weight
( M
) was calculated from the observed mass values
of multiply charged ion signals using the equation M
= nm - n, where n is the
number of positive charges and m is the mass signal bearing
n positive charges.
In the case of BSC-40 cell extracts, the fractions obtained
from the initial RP-HPLC using CF
COOH as
counterion, and which correspond to the major peak of iPTH, were pooled
and rechromatographed using a solvent system containing
C
F
COOH. Absorbance peaks which were positive by
radioimmunoassay were individually collected and analyzed as described
above. This defined the absorbance peaks as representing either proPTH
or PTH (Fig. 2). In BSC-40 cell extracts, both proPTH and PTH
were present (Fig. 3). In the BSC-40 cells coinfected with either
VV:POMC (control) or VV:PC2, the least amount of processing was
observed (). Processing of proPTH in cells coinfected with
VV:PC1 was not different from that in the control, however, in cells
coinfected with furin, processing was markedly stimulated being almost
three times that in control cells ().
Figure 3:
Final reversed-phase HPLC of extracts of
BSC-40 cells infected with VV:hPTH and ( a) VV:PC-1,
( b) VV:PC-2, ( c) VV:furin, or ( d) VV:POMC
(control). Cell extracts were subjected to initial reversed-phase
chromatography using solvents containing 0.13% (v/v)
CF
COOH. Fractions containing PTH
immunoreactivity were subjected to further chromatography (shown here).
In each instance the column was eluted with a linear gradient of
aqueous acetonitrile containing 0.1% (v/v) CF
COOH
throughout and column eluates were monitored for UV absorbance at 210
nm ( continuous line). Column fractions were assayed for the
presence of immunoreactive hPTH and in each case two peaks of
immunoreactive hPTH were found to correspond to two peaks of UV
absorbing material. These corresponded in elution position to hproPTH
( open arrowhead) and hPTH ( arrow), and were
identified as such by mass spectrometric analysis (see Fig.
2).
In profiles
derived from the GH4C1 medium (Fig. 4), as for BSC-40 medium, a
single absorbance peak coeluted with the major peak of iPTH. These
fractions were pooled and subjected to mass spectrometric analysis
(Fig. 2). Again, as in the case of medium derived from BSC-40
cells, only the PTH molecular species was found in the medium and no
proPTH. It was of interest to note in the medium of both BSC-40 and
GH4C1 cells infected with the VV:POMC construct an absorbance peak
eluting at an acetonitrile concentration of 45% and having endorphin
immunoreactivity (see Fig. 4). This corresponds to POMC itself.
Figure 4:
Reversed-phase HPLC of media derived from
cultured GH4C1 cells infected with VV:hPTH together with ( a)
VV:PC-1, ( b) VV:PC-2, ( c) VV:furin, or ( c)
VV:POMC (control). The column was eluted in each instance with a linear
gradient of aqueous acetonitrile containing 0.13% (v/v)
CF
COOH and column eluates were monitored for UV
absorbance at 210 nm ( continuous line). Column fractions were
assayed for the presence of immunoreactive hPTH and in each case a peak
of hPTH immunoreactivity was found to coincide with a single peak of UV
absorbing material ( arrow) which was positively identified as
hPTH using mass spectrometric analysis (see Fig. 2). In the control
panel the peak of endorphin immunoreactivity corresponding to POMC is
indicated by the open arrowhead.
For the GH4C1 cell extracts, after the first chromatography step
using a CF
COOH solvent system (see Fig.
1 C), and a second step using solvents containing
CF
COOH (not shown), two pools corresponding in elution
positions to proPTH and PTH were separately rechromatographed using the
CF
COOH solvent system (data not shown). Absorbance peaks
were individually collected and those positive in the PTH
radioimmunoassay were analyzed as described and defined as either
proPTH or PTH (Fig. 2). In GH4C1 cells little processing was
evident in VV:PC2 coinfected cells, the ratio of PTH to proPTH being
similar to that of control VV:POMC coinfected cells. However, this
ratio was approximately twice that of the control in cells coinfected
with VV:PC1, and more than 400% of control in cells coinfected with
VV:furin ().
In Vitro Cleavage of ProPTH Peptide with hPC1 and
hfurin
In order to study the cleavage of proPTH by hfurin and
hPC1 further, the tridecapeptide proPTH(-6+7) as well
as a small related synthetic fluorogenic substrate
carbobenzoxy-Val-Lys-Lys-Arg-MCA were used as model peptides. As
shown in , this small synthetic substrate, lacking the
basic residue at P6 present in the tridecapeptide, failed to
discriminate between hfurin and hPC1 as, in both cases, similar
V
/ K
values were
obtained. Thus, despite representing the sequence from P4 to P1 of
proPTH, it was less efficiently cleaved by either enzyme than the
pGlu-Arg-Thr-Lys-Arg-MCA substrate as shown previously
(24) .
This is not unexpected as it does not contain the P4 Arg residue
present in the latter sequence.
value of 79 ± 14 µ
M and V
value of 34.8 ± 2.5 nmol of
the COOH-terminal fragment/h, whereas, purified hfurin exhibited
apparent values of 23 ± 8 µ
M and 129 ± 10
nmol of the COOH-terminal fragment/h, respectively. As shown in
, the major difference observed lies in the significant
increase of the V
observed upon incubation of
the tridecapeptide with hfurin; indeed, this value is 3-100-fold
higher than that for all other substrates analyzed with either hfurin
or hPC1. Thus, compared to hPC1 the increased catalytic efficiency of
hfurin with the proPTH tridecapeptide, appears to be due to both an
increase in the apparent affinity for the substrate (lower
K
), and an increase in the rate of
processing of the substrate (higher V
).
Expression of Prohormone Convertase mRNAs in Parathyroid
Tissue Assessed by Northern Blot Analysis
As shown in Fig. 7
furin transcripts were identified in both parathyroid (bovine
parathyroid gland) and thyroid (medullary thyroid cell line (TT)),
whereas, only thyroid contained PC1 and PC2 transcripts, with them
being absent in parathyroid. PACE4 mRNA was expressed at low levels in
both parathyroid and thyroid.
Expression of Prohormone Convertase mRNAs in Parathyroid
Tissue Assessed by in Situ Hybridization
As shown in Fig. 8
furin mRNA is colocalized in PTH-expressing cells in the rat
parathyroid gland. These cells do not express PC1, PC2, or PC5 mRNA,
but do express, at a very low level, PACE4 mRNA.
-terminal sequence of PTH which is constrained by its
critical role in conferring bioactivity, is incompatible with efficient
cleavage of the prepeptide
(45) . Hence the need for the
prosequence as a spacer between the pre- and PTH sequences.
-cell of the pancreas
(49) . Therefore, the modest cleavage
of CgA in the parathyroid is probably related to the lack of expression
of PC1 and PC2. The precise roles played by furin and PACE4 in CgA
processing in the parathyroid remain to be determined.
Table:
Conversion of proPTH to PTH
Table:
Kinetic constants for cleavage of various
flurogenic peptide substrates and synthetic hproPTH (-6-+7)
with hPCl and hfurin
F
COOH, heptafluorobutyric acid;
CF
COOH, trifluoroacetic acid; MCA,
4-methylcoumaryl-1-amide; AMC, 7-amino-4-methylcoumarin; CgA,
chromogranin A.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.