(Received for publication, March 4, 1997)
From the Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
Prolyl 4-hydroxylase (proline hydroxylase, EC
1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens.
The vertebrate enzyme is an 2
2
tetramer, the
subunit of which is identical to protein
disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the
recently discovered
(II) subunit from human sources. The mRNA
for the
(II) subunit was found to be expressed in a variety of human
tissues, and the presence of the corresponding polypeptide and the
(
(II))2
2 tetramer was demonstrated in
cultured human WI-38 and HT-1080 cells. The type II tetramer was found
to represent about 30% of the total prolyl 4-hydroxylase in these
cells and about 5-15% in various chick embryo tissues. The results of
coexpression in insect cells argued strongly against the formation of a
mixed
(I)
(II)
2 tetramer. PDI/
polypeptide containing a histidine tag in its N terminus was found to form prolyl
4-hydroxylase tetramers as readily as the wild-type PDI/
polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear
to offer an excellent source for a simple large scale purification of
the recombinant enzyme. The properties of the purified human type II
enzyme were very similar to those of the type I enzyme, but the
Ki of the former for poly(L-proline)
was about 200-1000 times that of the latter. In agreement with this, a
minor difference, about 3-6-fold, was found between the two enzymes in
the Km values for three peptide substrates. The
existence of two forms of prolyl 4-hydroxylase in human cells
raises the possibility that mutations in one enzyme form may not be
lethal despite the central role of this enzyme in the synthesis of all collagens.
Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes
the hydroxylation of proline in -Xaa-Pro-Gly- triplets in collagens and
other proteins with collagen-like sequences. The enzyme plays a central
role in the synthesis of all collagens, as the 4-hydroxyproline
residues formed in the reaction are essential for the folding of the
newly synthesized collagen polypeptide chains into triple helical
molecules. The vertebrate enzyme is an 2
2
tetramer in which the
subunits contribute to most parts of the two
catalytic sites (for reviews, see Refs. 1-3). The
subunit is
identical to the enzyme protein disulfide-isomerase (PDI, EC
5.3.4.1)1 and has PDI activity even when
present in the prolyl 4-hydroxylase tetramer (4-6). The PDI
polypeptide also has several other functions (1-3, 7, 8).
Prolyl 4-hydroxylase had long been assumed to be of one type only, with
no isoenzymes (1-3), but recently an isoform of the subunit,
termed the
(II) subunit, was cloned from mouse tissues (9).
Correspondingly, the previously known
subunit is now called the
(I) subunit. The
(II) subunit was found to form an (
(II))2
2 tetramer with the PDI/
subunit when the two polypeptides were coexpressed in insect cells. The
properties of the new type II enzyme were found to be very similar to
those of the type I tetramer, with the distinct difference that it was
inhibited by poly(L-proline) only at very high
concentrations (9).
The subunit of prolyl 4-hydroxylase cloned from the nematode
Caenorhabditis elegans (10) has been found to have features of both types of mouse
subunit, suggesting that C. elegans may have only one type of
subunit (9). This forms
active prolyl 4-hydroxylase in insect cell coexpression experiments
with either the C. elegans or the human PDI/
polypeptide,
but surprisingly, the enzymes containing the C. elegans
subunit are
dimers (10, 11).
We report here that the existence of (II) subunit mRNA is not
limited to the mouse, as a corresponding mRNA is expressed in a
variety of human tissues. All the data so far available on the
existence of the type II prolyl 4-hydroxylase tetramer are based on
insect cell coexpression experiments, but we now demonstrate that this
enzyme is indeed present in cultured human fibroblasts and represents
about 30% of their total prolyl 4-hydroxylase activity. We also
studied whether the
(I) and
(II) subunits can form a mixed
(I)
(II)
2 tetramer, and whether any differences are
found between the type I and II enzymes in their Km
values for various peptide substrates, as the two mouse enzymes differ so markedly from each other with respect to inhibition by
poly(L-proline). A new affinity purification procedure was
developed that is based on the use of a histidine tag in the N terminus
of the PDI/
polypeptide, and this makes it possible to obtain large
amounts of any form of the recombinant enzyme by very simple steps.
Screening of a human lung
gt10 cDNA library (CLONTECH) with BT 14.1, a
cDNA clone for the mouse
(II) subunit (9), as a probe yielded
one positive clone, H9, among 600,000 recombinants. Rescreening of the
same library with H9 as a probe gave 8 positive clones out of 600,000 recombinants. Four of them, L121, L142, L21, and L22, were
characterized further.
The nucleotide sequences were obtained by the dideoxynucleotide chain termination method (12) with T7 DNA polymerase (Pharmacia). Vector-specific or sequence-specific 17-mer primers synthesized in an Applied Biosystems DNA Synthesizer (Dept. of Biochemistry, University of Oulu) were used, and the sequences were determined for both strands. DNASIS and PROSIS version 6.00 sequence analysis software (Pharmacia) was used to compile the sequence data.
A human multitissue Northern blot (CLONTECH)
containing 2 µg of poly(A)+ RNA per sample isolated from
various human tissues was hybridized under the stringent conditions
suggested in the manufacturer's instructions. The probes used were
32P-labeled cDNA clones encoding either the whole human
(II) subunit or PA-58 and PA-59 (13) encoding almost all of the
human
(I) subunit. The autoradiography time was 3 days.
To construct a baculovirus transfer
vector for the human (II) subunit, a 5
fragment was amplified from
the
-DNA of L142. The cDNA-specific primers used were
IIiN5
(5
-TCAGGCGGCCGCGACAGCCAGACACTTCCCTC-3
), containing an artificial
NotI site, and
IIiE3
(5
-GGTGAAGAATTCGGCCTGCAC-3
), containing a natural EcoRI site. Polymerase chain reaction
was performed under the conditions recommended by the supplier of the
Taq polymerase (Promega), and the reactions were cycled 27 times as follows: denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 3 min. The product was digested with NotI and EcoRI restriction
enzymes to give a fragment that extended from nucleotide 166 to 251. The next fragment was prepared by digesting L142 with EcoRI
and SacI. The resulting EcoRI-SacI
fragment, covering nucleotides 252-873, was ligated together with the
above NotI-EcoRI fragment to the
NotI-SacI site of the pBluescript vector
(Stratagene) and termed HuNS-BS. Clone H9 was digested with
SacI and PstI to give a fragment encompassing nucleotides 874-1426. Clone L21 was digested with PstI, and
the resulting fragment, covering nucleotides 1427-2021, was ligated together with the SacI-PstI fragment to the
SacI-PstI site of pBluescript, and the construct
was termed I3
SPP-BS. The NotI-SacI fragment from
HuNS-BS, and the SacI-PstI fragment from
I3
SPP-BS were then ligated to the NotI-PstI site
of pBluescript, and the construct was termed
(II)human-BS. Finally,
the
(II)human-BS was digested with NotI and
EcoRV, and the resulting fragment was ligated to the
NotI-SmaI site of the transfer vector pVL1392
(14).
A histidine affinity tag was generated in the N terminus of the human
PDI/ polypeptide. Six histidine codons were created by polymerase
chain reaction downstream of the codon coding for the last amino acid
of the signal peptide in a full-length cDNA for the human PDI/
polypeptide (4). The cDNA was digested with EcoRI and
BamHI and ligated to pVL1392.
Spodoptera frugiperda Sf9 insect cells (Invitrogen) were
cultured in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) at 27 °C, either as monolayers or in suspension in
spinner flasks (Techne Laboratories, Princeton, NJ). The pVL constructs
were cotransfected into Sf9 insect cells with a modified Autographa californica nuclear polyhedrosis virus DNA using
the BaculoGold transfection kit (Pharmingen). The resultant viral pools
were collected 4 days later, amplified, and plaque-purified (15). The
recombinant viruses were termed (II) and His-PDI. The viruses human
59(I), coding for the
(I) subunit, and human PDI/
, coding for
the PDI/
polypeptide, have been described previously (16).
Sf9 insect
cells were cultured as above. To produce an enzyme tetramer, the
59(I) or
(II) virus and the PDI/
or His-PDI virus were used in
a ratio of 1:1 or 2:1, and when attempting to produce an
(I)
(II)
2 tetramer, the
59(I),
(II), and
PDI/
viruses were used in the ratio 1:1:2 or 1:1:1. The cells were harvested 72 h after infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a solution of 0.1 M glycine, 0.1 M NaCl, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris, pH 7.8, and centrifuged at
10,000 × g for 20 min. The resulting supernatants were
analyzed by 8% SDS-PAGE or nondenaturing 7.5% PAGE and assayed for
enzyme activity. The cell pellets were further solubilized in 1% SDS
and analyzed by 8% SDS-PAGE. Western blot analysis was performed with
a monoclonal antibody L7B to the human
(I) subunit or a monoclonal
antibody K4 to the mouse
(II) subunit. These monoclonal antibodies
were generated by immunizing mice with denatured recombinant
(I) or
(II) polypeptides that had been purified by SDS-PAGE. The antibodies recognize the
(I) and
(II) subunit isoforms, respectively, both as native and denatured proteins from man, mouse, and chicken, but show
no cross-reactivity between isoforms.
Human lung fibroblasts (WI-38, ATCC CCL 75) and fibrosarcoma cells (HT-1080, ATCC CCL 121) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% newborn calf serum (Life Technologies, Inc.) at 37 °C as monolayers. The cells of confluent cultures were harvested, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a solution of 0.1 M glycine, 0.2 M NaCl, 50 µM dithiothreitol, 0.1% Triton X-100, 0.01% soybean trypsin inhibitor, and 20 mM Tris-HCl, pH adjusted to 7.5 at 4 °C, and centrifuged at 10,000 × g for 30 min. The resulting supernatants were analyzed by nondenaturing 8% PAGE followed by Western blotting using ECL. Total prolyl 4-hydroxylase activity was measured in aliquots of the supernatants, and the type II enzyme activity in aliquots of the supernatants that had been passed through small poly(L-proline) columns.
Assay of Type II Prolyl 4-Hydroxylase Activity in Tissues of 17-Day-old Chick EmbryosCalvaria, sternum, tendon, and liver tissues from 17-day-old chick embryos and whole chick embryos were homogenized in a solution of 0.1 M glycine, 0.2 M NaCl, 50 µM dithiothreitol, 0.1% Triton X-100, 0.01% soybean trypsin inhibitor, and 20 mM Tris-HCl buffer, pH adjusted to 7.5 at 4 °C, and centrifuged at 10,000 × g for 30 min. Aliquots of the supernatants were then used to assay the enzyme activities as above.
Protein Purification and N-terminal Amino Acid Sequence AnalysisThe type II prolyl 4-hydroxylase resulting from
coinfection with the viruses (II) and PDI/
was first purified by
a procedure consisting of anion exchange chromatography on a
DEAE-cellulose column (DE52 Whatman) and two gel filtrations. Sf9
insect cells harvested 72 h after infection were washed with a
solution of 0.15 M NaCl and 0.02 M phosphate,
pH 7.4, homogenized in a solution of 0.1 M glycine, 0.1 M NaCl, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris, pH 7.8, supplemented with 22 mM N-ethylmaleimide and 2.2 mM
phenylmethylsulfonyl fluoride, and centrifuged. The supernatant was
diluted 1:3 with a solution of 0.1 M glycine, 10 µM dithiothreitol, and 0.01 M Tris, pH 7.8, and applied to a DEAE-cellulose column (20 ml) equilibrated with 0.1 M glycine, 0.05 M NaCl, 10 µM
dithiothreitol, and 0.01 M Tris, pH 7.8 (buffer A). Unbound
material was washed off with the same buffer, and the bound proteins
were eluted with an increasing salt concentration generated by mixing
75 ml of buffer A and 75 ml of a solution of 0.1 M glycine,
0.4 M NaCl, 10 µM dithiothreitol, and 0.05 M Tris, pH 7.8 (buffer B). The 3-ml fractions containing
most of the prolyl 4-hydroxylase activity were pooled, concentrated by ultrafiltration (Millipore), and applied to a 1.5 × 90-cm
Ultrogel AcA34 (Serva) column, equilibrated, and eluted with a solution of 0.1 M glycine, 0.25 M NaCl, 10 µM dithiothreitol, and 0.01 M Tris, pH 7.8. Fractions of 3.5 ml were collected, and their absorbance at 280 nm was
measured. Several fractions were analyzed by 8% SDS-PAGE and assayed
for prolyl 4-hydroxylase activity, and a sample pool was combined and
concentrated as above for a second gel filtration on a 2.5 × 95-cm Bio-Gel A-0.5m fine (Bio-Rad) column equilibrated and eluted as
above. Fractions of 2.5 ml were collected, analyzed by 8% SDS-PAGE,
and assayed for prolyl 4-hydroxylase activity.
The type I and II prolyl 4-hydroxylase tetramers and the PDI/
polypeptide containing the histidine affinity tag were also purified by
a procedure consisting of a metal chelate affinity chromatography and a
gel filtration step. Insect cells expressing type I or type II prolyl
4-hydroxylase or the His-PDI polypeptide were harvested 72 h after
infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a 0.3 M
NaCl, 0.1 M glycine, 0.1% Triton X-100, and 0.01 M Tris buffer, pH 7.8, and centrifuged at 10,000 × g for 20 min. The supernatant was applied to a ProBond
column (Invitrogen) equilibrated with a 0.3 M NaCl, 0.1 M glycine, and 0.01 M Tris buffer, pH 7.8. Unbound material was washed off with the same buffer, and the bound
proteins were eluted with a 100 mM imidazole, 0.3 M NaCl, 0.1 M glycine, 10 µM
dithiothreitol, and 0.01 M Tris buffer, pH 7.8. Fractions containing the eluted proteins were pooled and applied to a 2.5 × 95-cm Bio-Gel A-0.5m fine (Bio-Rad) gel filtration column,
equilibrated, and eluted with a 0.3 M NaCl, 0.1 M glycine, 10 µM dithiothreitol, and 0.01 M Tris buffer, pH 7.8. Fractions of 3 ml were collected, their absorbance at 230 and 280 nm was measured, and they were analyzed
by 8% PAGE under nondenaturing conditions.
Fractions containing the prolyl 4-hydroxylase tetramer or the PDI/
dimer/monomer were pooled and analyzed by 8% SDS-PAGE and 7.5%
nondenaturing PAGE. Protein concentrations were estimated with the
Bio-Rad protein assay kit (Bio-Rad) according to the manufacturer's
instructions.
For the N-terminal sequencing the type II prolyl 4-hydroxylase was
run on 8% SDS-PAGE under reducing conditions and blotted onto a
polyvinylidene difluoride membrane. The membrane was stained with 0.1%
Coomassie R-250 in 50% methanol, and the band corresponding to the
(II) subunit was cut off. The N-terminal sequence was determined
using an Applied Biosystems model 477A on-line 120 liquid pulse protein
sequencer.
Prolyl 4-hydroxylase activity was
assayed by a method based either on the hydroxylation-coupled
decarboxylation of 2-oxo[1-14C]glutarate or on the
formation of hydroxy[14C]proline in protocollagen, a
biologically prepared, [14C]proline-labeled protein
substrate consisting of nonhydroxylated pro- chains of chick type I
procollagen (17). Km values were determined by
varying the concentration of one substrate in the presence of fixed
concentrations of the second while the concentrations of the other
substrates were held constant (18). The pure type II prolyl
4-hydroxylase tetramer was used as an enzyme source for the
Km determinations.
To isolate cDNA clones for
the human (II) subunit, a human lung cDNA library was screened
using BT14.1, a cDNA for the mouse
(II) subunit (9), as a probe.
One positive clone, H9, which codes for the central region of the
cDNA, was obtained. Rescreening of the same library with H9 as a
probe gave 8 positive clones, 4 of which were characterized
further.
The
cDNA clones cover 2194 nucleotides of the corresponding mRNA
(these cDNA sequences are not shown, but have been deposited in the
GenBankTM/EMBL Data Bank with accession number U90441) and
encode a 535-amino acid polypeptide, including a putative signal
peptide of 21 residues (Fig. 1). Sequencing of the N
terminus of the polypeptide indicated that its first amino acid is
glutamate (Fig. 1). The first amino acid of the mouse (II) subunit
was previously thought to be tryptophan (9), based on the computational
parameters of von Hejne (19), but it now seems more likely that this is likewise glutamate, as such a result would also be compatible with the
computational parameters.
The human (II), human
(I), and mouse
(II) polypeptides are
very similar in size,
(I) being three amino acids longer (Fig. 1).
All three polypeptides contain two potential attachment sites for
asparagine-linked oligosaccharides, but the position of the C-terminal
site in the
(II) and
(I) subunits differs by 4 residues (Fig. 1).
The five cysteine residues present in the human, mouse, and chick
(I) and the C. elegans
subunit are all conserved in
the human
(II) subunit, but the latter contains an additional cysteine, which is also present in the mouse
(II) subunit (Fig. 1).
The derived amino acid sequence of the human (II) subunit shows 64%
identity and 81% similarity to the sequence of the human
(I)
subunit and 93% identity and 96% similarity to the sequence of the
mouse
(II) subunit. The identity of the subunits is not distributed
equally, being highest within the C-terminal domains (Fig. 1).
Expression of the two types of subunit mRNA
in various human tissues was studied by Northern hybridization. The
sizes of the mRNAs for the human
(II) and
(I) subunits are
2.3 and 3.0 kilobases, respectively (Fig. 2). The
mRNA for the
(II) subunit was found to be expressed in a variety
of tissues, but distinct differences were found relative to the
expression pattern of that for the
(I) subunit, in that the relative
expression level of the latter was much higher in the skeletal muscle,
liver and kidney (Fig. 2).
The
To study whether the
(I) and
(II) subunits can form
(I)
(II)
2
mixed tetramers in addition to the
(
(I))2
2 type I and (
(II))2
2 type II tetramers (1-3, 9),
both types of
subunit were expressed in insect cells together with
the PDI/
polypeptide. The (
(I))2
2
tetramer is effectively inhibited by poly(L-proline) (1, 2)
and becomes bound to poly(L-proline) affinity columns (20).
The (
(II))2
2 tetramer differs distinctly
from the type I enzyme in that it is inhibited by
poly(L-proline) only at very high concentrations (9). It
thus can be expected that the type II enzyme will not become bound to
poly(L-proline) columns. If a mixed
(I)
(II)
2 enzyme existed, it could be expected
either to become bound to poly(L-proline), due to the
presence of the
(I) subunit, or to remain unbound.
Sf9 insect cells were coinfected with baculoviruses coding for either
the (I) subunit or the
(II) subunit together with viruses coding
for the PDI/
polypeptide, and a third set of cells was infected with
all three viruses. The cells were harvested 72 h after infection,
and Triton X-100-soluble proteins were analyzed by PAGE performed under
nondenaturing conditions. When the cells were coinfected with viruses
coding for either the
(I) subunit or the
(II) subunit together
with a virus coding for the PDI/
polypeptide, a type I or type II
enzyme tetramer was formed, the mobilities of these two types of
tetramer being essentially identical (Fig.
3A, lanes 1 and 4). An
enzyme tetramer was likewise formed when the cells were infected with
all three viruses (Fig. 3A, lane 7).
Western blotting with monoclonal antibodies specific to the isolated
(I) and
(II) subunits was used to distinguish between the types
of tetramer. The antibody to the
(I) subunit stained the type I
tetramer (Fig. 3B, lane 1) but not the type II
tetramer (Fig. 3B, lane 4), whereas the antibody
to the
(II) subunit stained the type II tetramer (Fig.
3C, lane 4) but not the type I tetramer (Fig.
3C, lane 1).
The type I tetramer became efficiently bound to a
poly(L-proline) affinity column, as no enzyme could be
detected in the column effluent (Fig. 3, A and B,
lanes 2), and could be eluted with poly(L-proline) (Fig. 3, A and B,
lanes 3). The type II tetramer was found in the column
effluent (Fig. 3, A and C, lanes 5),
and no additional amounts could be eluted from the column with
poly(L-proline) (Fig. 3, A and C,
lanes 6). When the tetramer formed during infection with
viruses coding for both types of subunit was studied as above, a
Coomassie-stained enzyme band was seen in both the column effluent
(Fig. 3A, lane 8) and the eluate (Fig.
3A, lane 9). The band in the column effluent
could be stained by the antibody to the
(II) subunit (Fig.
3C, lane 8) but not that to
(I) (Fig. 3B, lane 8), thus ruling out the presence of
(I)
(II)
2 in the effluent. The band in the column
eluate could be stained by the antibody to the
(I) subunit (Fig.
3B, lane 9) but not to
(II) (Fig.
3C, lane 9) thus ruling out the presence of
(I)
(II)
2 in the eluate. The absence of the
(I)
(II)
2 tetramer cannot be due to the low level
of infection of Sf9 cells by three viruses simultaneously, as we have
recently shown that these cells can be efficiently infected by three
viruses (21).
Confluent cultures of
human lung fibroblasts (WI-38) and fibrosarcoma cells (HT-1080) were
homogenized, and Triton X-100-soluble proteins were analyzed by PAGE
performed under nondenaturing conditions followed by Western blotting
using ECL with monoclonal antibodies specific to the (II) and
(I)
subunits (as demonstrated above). Bands corresponding to both types of
enzyme tetramer were found in extracts from both cell types (Fig.
4). Quantification of the Western blots using ECL which
had been standardized against known amounts of both types of enzyme
tetramer indicated that the amount of type II enzyme is lower than that
of type I enzyme in both cell types, being about 30% of total prolyl
4-hydroxylase (details not shown).
Ratio of Type II Enzyme Activity to Total Prolyl 4-Hydroxylase Activity in Human WI-38 Fibroblasts and HT-1080 Cells and Certain Chick Embryo Tissues
Total prolyl 4-hydroxylase activity, i.e. the sum of the type I and type II enzyme activities, was measured in Triton X-100 extracts from cell or tissue homogenates using proline-labeled protocollagen as a substrate. The activity of the type II enzyme was measured by passing aliquots of Triton X-100 extracts through poly(L-proline) affinity columns and determining the enzyme activity in column effluents. The values were corrected for dilution, and the type I enzyme activity was estimated by subtracting the type II activity from the total activity. The type II enzyme activity was found to represent about 30% of the total enzyme activity in confluent cultures of human lung fibroblasts and HT-1080 cells (details not shown). The corresponding percentage in Triton extracts from homogenates of 17-day-old whole chick embryos was 10%, and the percentages in Triton extracts from homogenates of chick embryo tissues were 13% in calvaria, 8% in sternum, 10% in tendon, and 5% in liver (details not shown).
Purification of the Human Type II Prolyl 4-Hydroxylase TetramerAs the type II enzyme does not become bound to
poly(L-proline), this enzyme could not be purified by the
standard affinity column procedure (20, 22) developed for type I. It
was therefore initially purified using an ion-exchange chromatography
procedure consisting of DEAE-cellulose chromatography and two gel
filtrations. The enzyme purified by this procedure was pure as judged
by Coomassie staining of 8% SDS-PAGE performed under reducing
conditions (Fig. 5A) and 7.5% PAGE performed
under nondenaturing conditions (Fig. 5B).
To make the purification simpler and more efficient, a histidine
affinity tag was constructed to the N terminus of the mature PDI/
polypeptide. Sf9 cells were then coinfected with viruses coding for
either the
(I) or the
(II) subunit and the His-PDI polypeptide,
and the Triton X-100-soluble proteins were studied by PAGE performed
under nondenaturing conditions. Both types of
subunit were found to
form an enzyme tetramer with the His-PDI polypeptide (Fig.
6, A and B, lanes 1) as
efficiently as with the wild-type PDI/
polypeptide (Table
I). The Triton X-100 extracts were then applied to a
ProBond column (Invitrogen), the unbound material was washed off with
the column equilibration buffer, and the bound proteins were eluted
with imidazole. No enzyme tetramer was found in the flow-through
fractions (Fig. 6, A and B, lanes 2),
whereas both types of enzyme tetramer and the His-PDI dimer and monomer
were present in the column eluates (Fig. 6, A and B, lanes 3). The enzyme tetramers (Fig. 6,
A and B, lanes 4) could then be
separated from the His-PDI polypeptide by gel filtration, during which
the latter was converted from dimers to monomers (Fig. 6A,
lane 5). The final preparations of both types of enzyme tetramer were also pure when analyzed by SDS-PAGE and Coomassie staining (Fig. 6C).
|
In agreement with data on mouse type II prolyl 4-hydroxylase (9), the Km values of the human type II enzyme for Fe2+, 2-oxoglutarate, and ascorbate were identical to those of the human type I enzyme (Table II). A marked difference was found between the two human enzymes in inhibition by poly(L-proline); however, the Ki values of the type II enzyme for poly(L-proline), Mr 7700, being about 200 times greater than those of the type I enzyme and those for Mr 44,000 about 1000 times greater (Table II). Small but significant differences were found between the human type II and type I enzymes in their Km values for three peptide substrates, in that all these values were 3-6 times greater in the case of the type II enzyme than for the type I enzyme (Table II). Additional experiments (details not shown) demonstrated that the type II enzyme, like the type I enzyme, did not catalyze any formation of 3-hydroxyproline when [14C]proline-labeled protocollagen was used as a substrate and the hydrolyzed reaction products were separated using an amino acid analyzer as described previously (17).
|
The data reported here indicate that the existence of an mRNA
for the (II) subunit of prolyl 4-hydroxylase is not limited to the
mouse (9), as an mRNA coding for a highly similar
(II) subunit
was also found in human tissues. Furthermore, the present data indicate
that the
(II) subunit is translated into the corresponding polypeptide in human cells. The
(II) subunit mRNA was found to be expressed in a variety of tissues, but distinct differences were
found in the expression patterns of the
(II) and
(I) subunit mRNAs between tissues.
Quantification of the proportions of the two types of prolyl
4-hydroxylase tetramer by Western blotting in cultured human WI-38 lung
fibroblasts and HT-1080 fibrosarcoma cells indicated that the type II
tetramer represents about 30% of the total enzyme protein in these two
cell types. Correspondingly, about 30% of the total prolyl
4-hydroxylase activity in extracts from these two cell types was found
in the flow-through fractions of poly(L-proline) affinity
columns, suggesting that the ((II))2
2
tetramer represents about 30% of the total prolyl 4-hydroxylase
activity. The type II prolyl 4-hydroxylase is also likely to be present
in chick embryo tissues, as a fraction of the total enzyme activity was found to pass through the poly(L-proline) affinity column
in the case of all the chick embryo tissues studied. Nevertheless, the proportion of type II enzyme activity may be lower in chick embryo tissues than in human fibroblasts, about 5-15% of total prolyl 4-hydroxylase activity. This percentage agrees with early reports indicating that up to at least 80% of the total prolyl 4-hydroxylase activity present in crude extracts from whole chick embryos is bound to
a poly(L-proline) affinity column (20), and that at least
80% of the total prolyl 4-hydroxylase activity present in extracts
from whole chick embryo homogenates is inhibited by
poly(L-proline) (23).
The present insect cell expression data argue strongly against the
presence of a protein containing the (I) and
(II) subunits in a
single molecule. No information is currently available on sequences in
the
subunits that are involved in the
2
2 tetramer assembly, but the C-terminal
domains of the
subunits, which show the highest degrees of amino
acid sequence identity between the
(I) and
(II) subunits and the
C. elegans
subunit (9, 10), are known to contain
residues involved in the binding of all the cosubstrates to a catalytic
site (24, 25). It seems probable that the regions involved in tetramer
assembly contain some sequences that prevent incorporation of the
(I) and
(II) subunits into the same molecule.
Although no information is available on the sequences in the subunits that are critical for tetramer assembly, several observations suggest that in the case of the PDI/
subunit some such sequences are
located close to the C-terminal domain of the polypeptide (11, 26). The
present data demonstrate that the N terminus of the PDI/
polypeptide
is not critical for tetramer assembly, as the His-PDI polypeptide was
found to form an active prolyl 4-hydroxylase as readily as the
wild-type PDI/
polypeptide. Our additional experiments have
demonstrated that the His-PDI polypeptide also efficiently forms an
dimer with the C. elegans prolyl 4-hydroxylase
subunit.2 The histidine-tagged forms of
prolyl 4-hydroxylase appear to offer an excellent source of the enzyme
for simple large scale purification in experiments such as attempts at
crystallization.
Poly(L-proline) has been regarded as a highly effective
competitive inhibitor with respect to the polypeptide substrate of prolyl 4-hydroxylases from all the vertebrate sources studied, and an
efficient polypeptide substrate for all plant prolyl 4-hydroxylases (1-3). It is therefore highly surprising that the human and mouse (9)
type II prolyl 4-hydroxylases are inhibited by
poly(L-proline) only at very high concentrations. This
property of the type II enzyme agrees with that reported for crude
preparations of prolyl 4-hydroxylase from the nematode Ascaris
lumbricoides (27) and for the recombinant C. elegans
prolyl 4-hydroxylase dimer (10, 11). As these findings suggest
that distinct differences are likely to exist in the structures of the
peptide binding sites of various prolyl 4-hydroxylases, a comparison
was made here between the Km values of the human
type I and type II enzymes for three peptide substrates: the
polypeptide (Pro-Pro-Gly)10, which is the most commonly
used synthetic peptide substrate for prolyl 4-hydroxylase, the
pentapeptide Gly-Val-Pro-Gly-Val, which has been introduced as a model
peptide for elastin (28), a protein that also contains 4-hydroxyproline
(1-3), and protocollagen, a biologically prepared collagenous
substrate for the enzyme. Small but significant differences were found
between the type I and type II enzymes in these experiments, in that
the Km values for all three peptide substrates with
the type II enzyme were about 3-6 times those with the type I enzyme.
Nevertheless, these differences are very small when compared with the
at least 200-1000-fold differences between their Ki
values for poly(L-proline).
Mutations have been characterized in the genes for many types of
collagen and for lysyl hydroxylase, a collagen hydroxylase closely
related to prolyl 4-hydroxylase in its catalytic properties (3,
29-32). No mutations have been identified in the gene coding for the
(I) subunit of prolyl 4-hydroxylase, and due to the central role of
this enzyme in the synthesis of all collagens, such mutations have
generally been assumed to be lethal. The present data indicating the
presence of two isoforms of prolyl 4-hydroxylase
subunit in human
tissues raises the possibility, however, that mutations in the gene
coding for one type may not be lethal, especially if cells are capable
of up-regulating the expression of the other type in cases when one
type is inactive.
We thank Riitta Polojärvi, Hanna-Mari Jauho, and Anne Kokko for their expert technical assistance.