From the Collagen Research Unit, Biocenter and
Department of Medical Biochemistry, and § Department of
Pathology, University of Oulu, FIN-90220 Oulu, Finland
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ABSTRACT |
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Procollagen-proline dioxygenase (EC 1.14.11.2),
an 2
2 tetramer in vertebrates,
plays a central role in the synthesis of all collagens. Recently an
isoform of the
subunit, the
(II) subunit, was characterized in
man and mouse and found to form a tetramer with the same
subunit as
the previously known
(I) subunit. We report here that the
(
(I))2
2 type I tetramer is the main
enzyme form in most cell types and tissues and that its contribution to
total prolyl 4-hydroxylase activity in cultured cells increases in
confluence. Surprisingly, however, the
(
(II))2
2 type II enzyme was found to
represent at least about 70% of the total prolyl 4-hydroxylase
activity in cultured mouse chondrocytes and about 80% in mouse
cartilage, the corresponding percentage in mouse bone being about 45%
and that in many other mouse tissues about 10% or less.
Immunofluorescence studies on samples from a fetal human foot confirmed
these data and additionally indicated that the type II enzyme
represents the main or only enzyme form in capillary endothelial cells.
Thus the type II prolyl 4-hydroxylase is likely to play a major role in
the development of cartilages and cartilaginous bones and also of
capillaries.
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INTRODUCTION |
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Procollagen-proline dioxygenase (EC 1.14.11.2) plays a central
role in the synthesis of all collagens, as the 4-hydroxyproline residues formed in its reaction are essential for the formation of the
collagen triple helix at body temperature. The enzyme requires Fe2+, 2-oxoglutarate, O2, and ascorbate and
acts on proline residues in -Xaa-Pro-Gly- sequences. The vertebrate
enzyme is an 2
2 tetramer in which
sequences contributing to the two catalytic sites are located in the
subunits, and the
subunit is identical to protein disulfide-isomerase (EC 5.3.4.1) (for reviews, see Refs. 1-3).
Prolyl 4-hydroxylase had long been assumed to be of one type only, but
an isoform of the subunit termed the
(II) subunit, has recently
been cloned and characterized from mouse (4) and human (5) tissues.
This
subunit was found to form a
(
(II))2
2 tetramer, the type II enzyme,
with the protein disulfide-isomerase polypeptide (4, 5). The
previously known
subunit (6) and enzyme form are now
correspondingly called the
(I) subunit and the type I enzyme (4).
Data on coexpression in insect cells strongly argue against the
existence of a mixed
(I)
(II)
2 tetramer (5). The
properties of the type II enzyme are very similar to those of the type
I enzyme, with the distinct difference that it is inhibited by
poly(L-proline) only at very high concentrations and does
not become bound to poly(L-proline) affinity columns (4,
5).
The type II enzyme was recently shown to represent about 30% of the total prolyl 4-hydroxylase activity in cultured human WI-38 lung fibroblasts and HT-1080 fibrosarcoma cells (5). No other data are currently available on the contribution of the two enzyme forms to the total prolyl 4-hydroxylase activity in various cells. We report here that the type I enzyme is the main form in most cell types and tissues and that its proportion of the total prolyl 4-hydroxylase activity in cultured cells increases in confluence. Surprisingly, however, the type II prolyl 4-hydroxylase was found to be the main enzyme form in cultured chondrocytes and in cartilage and also in capillary endothelial cells.
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EXPERIMENTAL PROCEDURES |
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Cell Cultures-- The cell lines used here were fetal human skin fibroblasts (ATCC CRL-1475), adult human skin fibroblasts (ATCC CRL-1987), human embryonic lung fibroblasts (WI-38, ATCC CCL-75), simian virus 40-transformed WI-38 cells (Va13/WI-38, ATCC CCL 75.1), human embryonal rhabdomyosarcoma cells (RD, ATCC CCL 136), mouse embryonal fibroblasts (3T3, ATCC CCL-92), and mouse chondrocytes, which were obtained from the heads of the ribs of 7-day-old mice. The rib cartilage was minced with a knife and digested with collagenase in Dulbecco's modified Eagle's medium without L-glutamine for 8 h at 37 °C with shaking. The chondrocytes were centrifuged and washed with a solution of 0.15 M NaCl and 0.02 M phosphate (PBS). All cell lines were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% newborn calf serum (Life Technologies, Inc.) and 50 µg of ascorbic acid/ml at 37 °C. The chondrocytes were used in the second passage.
Measurement of Type I and Type II Prolyl 4-Hydroxylase Activity in Cells and Mouse Tissues-- The cells of logarithmic phase or confluent cultures were harvested and washed with PBS, pH 7.4. Cells from 1-10 10-cm plates (diameter) were pooled, 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. Kidney, heart, liver, skeletal muscle, skin, bone (femur and tibiae), and rib cartilage tissues from 1-2-month-old mice were homogenized and centrifuged as above. Tissue from one to three mice (depending on the tissue) was pooled to form one sample. Total prolyl 4-hydroxylase activity was measured in aliquots of the supernatants, and other aliquots were passed through small poly(L-proline) columns as described (5). The type II enzyme activity was then measured in samples of the column effluents, and the type I enzyme activity was calculated as the difference between the total activity and the type II activity after correction for dilution (5).
Immunofluorescence Staining--
A foot specimen from an
apparently healthy 17-week-old gestational male human fetus (described
in Ref. 7) was available for indirect immunofluorescence studies. This
tissue had been immediately frozen in liquid nitrogen and stored at
70 °C. Samples were cut into 5-µm cryosections on SuperFrost
Plus glass slides (Menzel Gläzer, Braunschweig, Germany), and the
sections were fixed in precooled methanol for 10 min at
20 °C.
After rinsing with PBS, nonspecific antibody binding was blocked by
incubating the sections with 1% bovine serum albumin in PBS, pH 7.2, for 1 h at 22 °C. The samples were then incubated at 4 °C
overnight or at 22 °C for 1 h with a 1:100 diluted monoclonal
antibody L7B to the
(I) subunit of human prolyl 4-hydroxylase or K4
to the
(II) subunit of mouse prolyl 4-hydroxylase. These monoclonal antibodies have been generated by immunizing mice with denatured recombinant
(I) or
(II) polypeptides that had been purified by
SDS-polyacrylamide gel electrophoresis (5). They 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 (5). After thorough washing with PBS,
a 1:100 diluted tetramethylrhodamine isothiocyanate-conjugated rabbit
anti-mouse antibody (DAKO) was applied, and the samples were incubated
in the dark at 22 °C for 1 h. After washing with PBS, the
slides were mounted with glycergel (Dako) and examined under an
epifluorescence microscope (Leitz Aristoplan) equipped with a filter
for tetramethylrhodamine isothiocyanate fluorescence. Control sections
were stained with the secondary antibody alone. For better histological
analysis, frozen sections were stained with hematoxylin and eosin by
routine methods.
Enzyme Activity Assay--
Prolyl 4-hydroxylase activity
was assayed by a method based 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
(8). Because of the nonlinearity of this assay (8), the optimal sample
concentration was first determined in preliminary experiments for each
cell line and tissue, and the final assays were performed at three to
four sample concentrations on both sides of the optimum. Only values
obtained from the linear region were used in the final calculations,
and those shown for each sample pool are the means of two to six
independent measurements and their S.D. values or ranges (in the cases
of two samples). Protein concentrations were estimated with a Bio-Rad
protein assay kit (Bio-Rad) according to the manufacturer's
instructions.
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RESULTS AND DISCUSSION |
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Contribution of the Two Isoenzymes to Total Prolyl 4-Hydroxylase
Activity in Cultured Cells--
It has recently been demonstrated that
the recombinant human type I prolyl 4-hydroxylase tetramer present in a
crude protein extract from insect cells becomes completely bound to a
poly(L-proline) affinity column, whereas all the type II
enzyme is found in the column effluent (5). This allowed the
contribution of the type II enzyme activity to total prolyl
4-hydroxylase activity in crude cell extracts to be determined in a
sample of column effluent, while the type I enzyme activity could be
calculated by subtracting the type II enzyme activity from the total
activity determined before the column (5). The values obtained by this
method for the ratios of the two isoenzymes in cultured WI-38 and
HT-1080 cells were in complete agreement with those obtained for the
proportions of the (I) and
(II) subunits in extracts from these
cells by Western blotting (5). In the present work the
poly(L-proline) column method was used to measure the
proportions of the type I and II enzyme activities in various
samples.
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Contributions of the Two Isoenzymes to Total Prolyl 4-Hydroxylase Activity in Mouse Tissues-- In agreement with data obtained in cultured cells, the type I prolyl 4-hydroxylase was found to be the main enzyme form in most mouse tissues studied (Fig. 2). This isoenzyme represented about 90% or more of the total prolyl 4-hydroxylase activity in the kidneys, heart, liver, skeletal muscle, and skin. Bone and cartilage differed distinctly from the other tissues studied, however, in that the type I enzyme contributed only slightly more than half of the total activity in bone and only about 20% of that in cartilage (Fig. 2).
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Immunofluorescence Staining for the Two Isoenzymes in a Fetal Human
Foot--
The development of the bones in the foot begins by
condensation of undifferentiated mesenchymal cells into tightly packed cell islands prior to enchondral bone formation. These undifferentiated mesenchymal cells became strongly stained by a monoclonal antibody to
the prolyl 4-hydroxylase (I) subunit (Fig.
3A) but showed only a very
weak staining with a monoclonal antibody to the
(II) subunit (Fig.
3B). The chondrocytic cells in the center of the cell
islands, corresponding to the advancement of enchondral ossification, showed an intense staining with both the
(I) and
(II) subunit antibodies (Fig. 3, C and D). The cells at the
periphery of such islands, representing cells of the developing
perichondrium and synovial membrane, gave a strong staining with the
(I) subunit antibody but a distinctly weaker staining with the
(II) subunit antibody (Fig. 3, C and D). The
osteoblasts of a small ossification center in the phalangeal shaft also
expressed a strong immunoreaction to the
(I) subunit (Fig.
3E) and a weaker signal for the
(II) subunit (Fig.
3F).
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Conclusions--
The type I prolyl 4-hydroxylase was found to be
the main enzyme form in most cell types and tissues studied. However,
although it represented at least about 90% of total enzyme activity in the kidneys, heart, liver, skeletal muscle, and skin, the data do not
exclude the possibility that these tissues may also contain cell types
in which the type II enzyme is the predominant prolyl 4-hydroxylase
form. One such example was found here in the case of capillary
endothelial cells, which gave strong immunofluorescence with the
antibody to the (II) subunit but were negative in the staining for
the
(I) subunit.
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ACKNOWLEDGEMENTS |
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We thank Riitta Polojärvi, Jaana Träskelin, and Annikki Huhtela for their expert technical assistance and Mirka Vuoristo and Janna Saarela for help with the preparation of the mouse specimens.
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FOOTNOTES |
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* This work was supported by grants from the Research Council for Health within the Academy of Finland, the Jenny and Antti Wihuri Foundation, the University of Oulu Foundation, the Finnish Medical Society Duodecim, and FibroGen Inc., South San Francisco, CA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland. Tel.: 358-8-537-5801; Fax: 358-8-537-5810.
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REFERENCES |
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