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INTRODUCTION |
The collagens are a family of extracellular matrix proteins that
play a dominant role in maintaining the structural integrity of various
tissues. A well coordinated deposition of the components of the
extracellular matrix is essential to achieve and maintain their
physiological function (for reviews, see Refs. 1-3).
Type II collagen is the main structural component of hyaline cartilages
and forms their fibrous scaffold, which interacts with various types of
proteoglycan. It forms networks of thin fibrils that differ in
morphology from the much thicker fibrils of type I collagen, the main
fibril-forming collagen in most other tissues (1-3). Many factors such
as the presence of various proteoglycans (4-6) and interactions
between collagen types (7-9) influence collagen fibril formation and
the architecture of the resulting fibrils formed. Thus the differences
in fibril architecture between tissues are not necessarily dependent on
differences between the various collagen types themselves. However,
fibril formation experiments with purified collagens in
vitro have demonstrated that the differences in structure between
type II and type I collagen molecules appear to be sufficient to
explain many of the characteristic differences between these two types
of fibril present in tissues (10). The kinetics for the assembly of
type II collagen fibrils in such experiments differed markedly from
those for the assembly of type I collagen, and the critical
concentration for type II collagen at 37 °C was about 50 times
greater (10). In addition, the type II collagen fibrils formed in
vitro were much thinner than those of type I collagen and formed
three-dimensional networks (10).
The extents of hydroxylation of lysine residues and glycosylation of
hydroxylysine residues in type II collagen are much higher than those
in the other two major fibril-forming collagens, types I and
III (11). Thus the differences in fibril morphology between types II
and I might be related to these differences. The functions of the
hydroxylysine-linked carbohydrate units are nevertheless poorly
understood at present. As these are the most extrusive groups on the
surface of the collagen molecule, it has been suggested that they may
influence fibril assembly (12). Fibril formation experiments with type
I collagen in vitro have demonstrated that this collagen
with an experimentally produced increased degree of lysine
hydroxylation and hydroxylysine glycosylation formed thinner fibrils
than the same protein with a normal degree of these modifications, but
only a minor extent of overglycosylation could be achieved in these
experiments (13). In agreement with these data, type II collagen from
the annulus fibrosus, which has a slightly higher extent of lysine
hydroxylation and hydroxylysine glycosylation than that from articular
cartilage, formed fibrils with a smaller diameter (14). The possibility
has not been excluded, however, that the presence of minor
amounts of proteoglycan in the collagen preparations may have
contributed to these differences.
To allow study in detail of the effects of the extents of lysine
hydroxylation and hydroxylysine glycosylation on fibril formation, molecular collagen species are required that are genetically identical but differ markedly with respect to these modifications. It has recently been reported that recombinant human collagens with stable triple helices can be produced in insect cells by coinfection with
baculoviruses coding for the polypeptide chains of the collagen to be
produced and the two types of subunit of prolyl 4-hydroxylase, the key
enzyme of collagen synthesis (15, 16). The use of an additional
baculovirus coding for lysyl hydroxylase (17) markedly increased the
level of lysine hydroxylation and the amount of glycosylated
hydroxylysine residues, and it was possible to obtain pepsin-treated
recombinant human type II collagen preparations in which the
hydroxylysine residue content ranged from 1 to 21/1,000 and that of
glycosylated hydroxylysine residues from less than 1 to 9/1,000 (18).
We used such recombinant type II collagen preparations here to
study their in vitro fibrillogenesis.
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MATERIALS AND METHODS |
Production and Isolation of Recombinant Type II Collagens with
Low and High Hydroxylysine and Glycosylated Hydroxylysine
Content--
High Five insect cells (Invitrogen) were cultured in
TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum
(BioClear) in shaker flasks at 27 °C. For the production of
recombinant type II collagen with a low hydroxylysine content, the
cells were infected with viruses coding for rhproCIINIII and 4PH
as described previously (18), whereas for a high hydroxylysine content
they were coinfected with the above viruses and an additional virus
coding for human lysyl hydroxylase (17, 18). 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 0.2 M NaCl, 0.1% Triton X-100, and 0.05 M Tris
buffer, pH 7.4, incubated on ice for 30 min, and centrifuged at
16,000 × g for 30 min (18). The supernatant was
chromatographed on a DEAE cellulose column (DE-52, Whatman), equilibrated and eluted with a 0.2 M NaCl and 0.05 M Tris buffer, pH 7.4, the void volume being collected. The
pH of the sample was lowered to 2.0-2.5, and the sample was digested
with a final concentration of 150 µg/ml of pepsin for 1 h at
22 °C. The pepsin was irreversibly inactivated by neutralization of
the sample followed by overnight incubation on ice. The recombinant
collagen was precipitated by adding solid NaCl to a final concentration
of 4 M and centrifuging at 16,000 × g for
1 h. The pellet was dissolved in a 0.5 M NaCl, 0.5 M urea, and 0.05 M Tris buffer, pH 7.4, for 1 day. The sample was chromatographed on a Sephacryl HR-500 gel
filtration column (Amersham Pharmacia Biotech), eluted with a solution
of 0.2 M NaCl, and 0.05 M Tris, pH 7.4, dialyzed against 0.1 M acetic acid, and lyophilized.
In Vitro Fibril Formation--
To prepare stock solutions, the
collagen preparations were dissolved in 0.05% acetic acid at a
concentration of 1 mg/ml and centrifuged at 200,000 × g for 1 h at 4 °C. The upper half of the supernatant
was withdrawn and adjusted to a final concentration of 200 µg/ml,
checked with a Jasco J-500A spectropolarimeter. The collagen
self-assembly conditions followed a modification of the method
described by Williams et al. (19): briefly, 100 µg/ml of
type II collagen, 30 mM K2HPO4, and
135 mM NaCl, pH 7.4. The samples were then transferred to a
thermocontrolled quartz cuvette. Fibril formation was triggered by
increasing the incubation temperature to 34 °C. Optical density at
313 nm was monitored in steps of 2 min with a Perkin-Elmer Lambda2
photometer (connected to a personal computer). Each recording was
limited to 900 min. The sample was centrifuged at 5,000 × g for 30 min after the experiment, and the concentration of
the supernatant was measured by CD spectroscopy to determine the amount
of aggregated collagen. The experiments to study the effects of
collagen concentration were performed as above except that the collagen
concentration was varied, and the time was 600 min.
Electron Microscopy--
Aliquots of the assembly mixtures for
electron microscopy were incubated in parallel with the turbidity time
assay. After 1,000 min, samples of 4 × 3 µl were transferred to
formvar-coated copper grids using a micropipette, and the fibrils were
allowed to settle for 30 min. The buffer was then drained cautiously
with a filter paper, and three washing steps were performed. The
fibrils were stained with freshly prepared 1% uranyl acetate,
dissolved in distilled water for 2 min, washed 3 times more, and dried. The grids were examined using a Zeiss EM 109 electron microscope. Fibril width was measured with an image analysis system (Optoquant, Lübeck, Germany) using an internal distance of 10 periods in the
collagen banding pattern.
Low Angle Rotatory-shadowing Electron
Microscopy--
Recombinant type II collagen preparations were
dialyzed against a solution of 50% glycerol in 0.05% acetic acid for
16 h at 4 °C. The samples were sprayed onto freshly cleaved
mica using an air brush. The droplets on the mica were dried at room
temperature at 10
6 mm Hg for 12 h in a vacuum coater
(Edwards 306). The dried specimens were rotatory-shadowed with platinum
using an electron gun positioned at 6° to the mica surface and then
coated with a film of carbon generated by an electron gun positioned at
90° to the mica surface. The replica was floated on distilled water
and collected on a grid covered with a formvar film. The specimens were
studied with a Zeiss 109 transmission electron microscope.
Other Assays--
Protein samples hydrolyzed in 6 M
HCl for 24 h at 110 °C under N2 were used for amino
acid analyses performed in an Applied Biosystems 421 or Beckman system
6300 analyzer. The glycosylated hydroxylysine content was determined by
hydrolyzing collagen samples in 2 M KOH for 24 h at
110 °C in 2-ml polypropylene reaction vials. Hydroxylysine and its
glycosides were separated by cation exchange chromatography on Dovex
50W-X8 as described previously (18, 20). A
glucosylgalactosylhydroxylysine standard (20) was used to quantify the
glucosylgalactosylhydroxylysine and galactosylhydroxylysine. For the
analysis of pepsinized collagen chains, the samples were studied by 8%
SDS-polyacrylamide gel electrophoresis under reducing conditions
followed by Coomassie staining.
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RESULTS AND DISCUSSION |
Content of Hydroxylysine and Its Glycosides in the Type II Collagen
Preparations--
The recombinant human type II procollagen
preparations produced in insect cells were converted to collagens by
digestion with pepsin at 22 °C for 1 h, and the pepsin-treated
collagens were purified. Two kinds of recombinant human type II
collagen preparation were used: low hydroxylysine collagen, produced
without recombinant lysyl hydroxylase, and high hydroxylysine collagen,
produced with recombinant lysyl hydroxylase. Both types were pure when
studied by SDS-polyacrylamide gel electrophoresis under reducing
conditions followed by Coomassie staining (Fig.
1) and also by amino acid analysis
(details not shown).

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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of purified recombinant high and low hydroxylysine type II
collagens. The positions of the molecular weight markers are
indicated on the left. Lane 1, high hydroxylysine type II
collagen; lane 2, low hydroxylysine type II collagen. The
arrow shows the position of the 1(II) chains. 1(II),
1 chain of type II collagen.
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The recombinant low hydroxylysine type II collagen preparation had low
levels of total hydroxylysine and its glycosides (Table I), whereas the recombinant high
hydroxylysine preparation had levels that were even higher than those
in nonrecombinant human type II collagen from articular cartilage
(Table I). As discussed elsewhere (18), the marked increase in the
quantity of glycosylated hydroxylysine residues obtained by
coexpression with lysyl hydroxylase must be due to the presence of a
high level of endogenous collagen glycosyltransferase activities in the
High Five insect cells.
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Table I
Hydroxylysine and its glycosides in the recombinant type II collagen
preparations
The recombinant type II collagens were prepared and purified as
described under "Materials and Methods."
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Fibril Formation in Vitro--
Formation of fibrils of the two
kinds of human recombinant type II collagen was studied using an assay
based on the increase in absorbance at 313 nm in a 100 µg/ml collagen
solution at 34 °C as a function of time. A marked difference was
found between the low and high hydroxylysine collagens, in that the
maximum absorbance of the former was reached within 5 min, whereas the absorbance of the latter increased for about 600 min (Fig.
2). The amount of low hydroxylysine
collagen incorporated into the fibrils was slightly smaller than that
of high hydroxylysine collagen but was more than 90% in both cases
(details not shown). Thus the maximum absorbance/microgram collagen
incorporated into the fibrils of the low hydroxylysine collagen was
about 6 times that observed with the high hydroxylysine collagen (Fig.
2).

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Fig. 2.
In vitro formation of fibrils of the
recombinant high (solid line) and low (dotted
line) hydroxylysine type II collagens as measured by
turbidity at 313 nm as a function of time.
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The critical concentrations for fibril formation also differed markedly
between the two types of recombinant human type II collagen, the
apparent critical concentration for the low hydroxylysine collagen
being less than 10 µg/ml, whereas that for the high hydroxylysine collagen was about 70 µg/ml (Fig.
3).

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Fig. 3.
Effect of collagen concentration on formation
of fibrils of the recombinant high ( ) and low ( ) hydroxylysine
type II collagens. The maximal absorbance values were corrected
for the portion of incorporated collagen.
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Electron Microscopy--
Electron micrographs of the two kinds of
recombinant type II collagen when visualized by rotatory shadowing
showed predominantly monomers of uniform length (details not shown). No
differences were detected between the low and high hydroxylysine
collagens with this technique.
Electron microscopy of the fibrils formed by the high hydroxylysine
collagen showed that they were typically very thin with essentially no
interfibril interaction or fibril aggregation (Fig. 4A). A few short, slightly
thicker fibrils could also be seen with sharp tips on both ends (Fig.
4A). In contrast, the low hydroxylysine collagen formed
thick fibrils on a background of thin ones (Fig. 4B).

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Fig. 4.
Electron micrographs of the fibrils formed
in vitro by the recombinant high (A)
and low (B) hydroxylysine type II collagens.
Bar = 1 µm.
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To study the distribution of the fibril diameters in more detail, a
histogram of 200 measured fibril diameters was plotted against their
relative frequency (Fig. 5). Fibril
aggregates were excluded from these measurements, and thus the
histogram shows only diameters of single fibrils. The high
hydroxylysine collagen showed the highest frequency of fibrils with a
diameter of about 10 nm, whereas the low hydroxylysine collagen had the
highest frequency of fibrils with a diameter of about 20 nm, and some fibrils had diameters exceeding 60 nm (Fig. 5).

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Fig. 5.
Distribution of diameters of fibrils formed
by the recombinant high (dark gray) and low
(light gray) hydroxylysine type II collagens.
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All experiments reported here were carried out with type II collagen
samples prepared from the corresponding procollagens by removing
the N and C propeptides by digestion with pepsin at 22 °C for 1 h. Although the collagen triple helix is resistant to proteolytic
enzymes, such treatment is likely to degrade part of the N and C
telopeptides, the short nontriple-helical sequences at the ends of the
collagen molecules (1-3). Many previous studies have shown that
pepsin-treated collagens tend to form fibrils with a smaller
diameter and a lesser degree of a highly resolved cross-striation
pattern than collagens produced from procollagens by the cleavage
with procollagen N and C proteinases (see Ref. 8). Thus the fibrils
studied here are not identical to those formed in vivo, but
the pepsin treatment cannot explain any of the differences in fibril
assembly and morphology between the recombinant low and high
hydroxylysine type II collagens.
The glucosylgalactose moiety is highly hydrophilic and has a length of
about 1 nm (14). This moiety is oriented parallel to the backbone of
the collagen molecule and shields three or four amino acid residues
(14). The glucosylgalactose moiety can thus be expected to inhibit
lateral growth of collagen fibrils because it reduces the surface
available for hydrophobic interactions. This hypothesis is supported by
our morphological findings and also by the data on fibril formation,
because the final turbidity of the high hydroxylysine collagen was only
about one-sixth of that of the low hydroxylysine variety.
Turbidity is related to molecular mass per unit length in the case of
very long molecules. The difference in turbidity between the low and
high hydroxylysine collagens accounts for a three-fold difference in
fibril diameter. This is not fully in accordance with the morphometric
analysis of single fibrils, however, because only a portion of the
fibrils deviated in diameter. It is likely that interfibrillar
interactions or banding of unit fibrils contributed to the increased
light scattering, even though the electron micrographs seem to suggest
that the thick fibrils appear to be formed from thin unit fibrils.
Conclusions--
The data indicate the presence of marked
differences in fibril formation between recombinant human type II
collagens containing low and high amounts of hydroxylysine and its
glycosylated forms. The maximal absorbance of the low hydroxylysine
collagen was reached within 5 min, whereas the absorbance of the high
hydroxylysine collagen increased for about 600 min and was only about
one-sixth of that obtained with the former. Furthermore, the critical
concentration for fibril formation with the high hydroxylysine collagen
was about 10 times that with the low hydroxylysine collagen. The
morphology of the fibrils formed was also different, in that the high
hydroxylysine collagen formed very thin fibrils with essentially no
interfibril interaction or fibril aggregation, whereas the low
hydroxylysine collagen formed thick fibrils on a background of thin ones.
The hydroxylation of lysine residues and the glycosylation of
hydroxylysine residues vary markedly in extent between collagen types
and even within the same collagen type, between tissues and in a given
tissue in various physiological and pathological states (11, 12). The
collagen molecules present in the thin fibrils found in embryonic
tissues, for example, have higher extents of these modifications than
the thicker fibrils found in adult tissues. The present data indicate
that regulation of the extents of these modifications may play a major
role in governing collagen fibril formation and the morphology of the
fibrils formed in vivo.