(Received for publication, May 31, 1995; and in revised form, July 28, 1995)
From the
At its C terminus, the collagen IV molecule bears a globular NC1
domain, to which two functions have been assigned. In the
macromolecular network of collagen IV, two molecules are connected via
their NC1 domains, which form a hexameric complex, stabilized by
intermolecular disulfide bonds. In addition, the NC1 domains are
thought to be responsible for chain selection and assembly. In order to
understand the role of the NC1 domains during these steps, hexameric
complexes were isolated and further investigated. SDS-polyacrylamide
gel electrophoresis and Western blot revealed disulfide-linked
1(IV)NC1 and
2(IV)NC1 homodimers but no heterodimers. The
hexamers were dissociated at low pH, separated into monomers and
dimers, and submitted to reconstitution experiments. Only
1(IV)NC1
dimers were able to reconstitute a hexameric complex.
1(IV)NC1 and
2(IV)NC1 monomers as well as the
2(IV)NC1 dimers showed only
a low tendency to form complexes. It is assumed that during formation
of the collagen IV network, lateral aggregation of the molecules via
the triple helical domains brings the C termini of two molecules into
close vicinity and that subsequently the weak interactions observed
between the NC1 subdomains provide the correct alignment for a
disulfide exchange. It is, however, questionable whether the low
affinity between the NC1 subdomains alone is sufficient for chain
assembly and alignment of the
(IV) chains before molecule
formation.
The main collagenous constituent of basement membranes is type IV collagen(1, 2) . Its molecules bear a globular NC1 domain at their C termini, and their triple helical region is characterized by frequent interruptions with non-triple helical segments, which provide them with a high flexibility(3) . The macromolecular structure of collagen IV is a network, in which the molecules are connected via like ends(4) . At the N terminus, the triple helical end regions of four molecules overlap by 25 nm, and this arrangement is covalently connected by intermolecular disulfide bridges and lysine-derived aldimine bonds, characteristic for collagens(5) . At the C terminus, the NC1 domains of two molecules become aggregated to form a hexameric complex, which is also stabilized by intermolecular disulfide bonds(6, 7) . In addition, the triple helical domains of the molecules are aligned laterally, and, in the electron microscope, superhelices between two or three molecules have been observed(8) .
Presently, six
different (IV) chains,
1(IV) to
6(IV), are
known(9, 10) . Their genes are arranged head-to-head
in three pairs to form the transcription units COL4A1-A2,
-A3-A4, and -A5-A6, which apparently give rise to three
isoforms at the protein
level(11, 12, 13, 14) . The isoform
with the chain composition [
1(IV)]
2(IV)
is ubiquitous to all basement membranes, whereas the
[
3(IV)]
4(IV) isoform is mainly
restricted to the glomerular basement membrane and has also been found
in less abundance in some other basement membranes(10) .
Whether the
5(IV) and
6(IV) chains also form a common
molecule is unclear. Both chains are present in several tissues.
However,
5(IV) is mainly expressed in kidney, whereas the highest
expression of
6(IV) has been observed in esophagus and
lung(14) .
Comparison of the amino acid sequences of
(IV) chains of different species, from nematodes to mammals,
including the recently characterized isoforms in man, revealed a
particularly well conserved primary structure of the NC1 domain,
indicating an important general function for this domain(2) .
Two functions have been associated with the NC1 domains. Their crucial
role in linking two molecules via their C-terminal ends in the
macromolecular network is obvious. In addition, the NC1 domains are
thought, by analogy with the C-terminal propeptides of the
fiber-forming collagens, to be responsible for chain selection and
assembly before an intact triple helical molecule can be generated.
In order to understand the role of the NC1 domains during these two steps, hexameric NC1 complexes were isolated from human placenta after collagenase treatment and, after dissociation at low pH, were separated into NC1 monomers and cross-linked dimers(6, 7) . The monomeric and dimeric NC1 domains were submitted to reconstitution experiments at physiological pH and ionic strength(6) .
To dissociate the hexameric NC1
complex into dimeric and monomeric NC1, the NC1-containing fraction
from the agarose A-1 eluate was dialyzed against 0.1 M ammonium formate, pH 3, and subsequently applied to a column (1
30 cm) of Superose 12 and separated by elution with the same
buffer. The fractions containing dimers and monomers were stored at
-20 °C.
SDS-PAGE ()and chromatographic
separation did not give evidence for the presence of
3(IV)-,
4(IV)-,
5(IV)-, and
6(IV)NC1 domains in the hexameric
complex isolated from placenta. Also, Western blot analysis with an
antiserum against the
5(IV)NC1 domain was negative.
To
reconstitute hexameric NC1 complexes, the fractions obtained from the
Superose 12 chromatography were dialyzed for 16 h at 4 °C against
0.1 M NaCl, 0.05 M sodium phosphate
buffer, pH 7.4, and filtered through a membrane with 0.45-nm pore
diameter (polyvinylidene difluoride, Millipore). 200 µl of each
fraction, with a protein concentration of 150-200 mg/ml, were
separated on a column (1
30 cm) of Superose 6, by elution with
the same phosphate buffer, pH 7.4, using a flow rate of 0.3 ml/min. The
individual fractions containing hexameric, dimeric, and monomeric NC1
domains were analyzed by SDS-PAGE.
Electron microscopy of the NC1 domains after rotary shadowing was performed as described previously(16) .
Figure 1:
SDS-PAGE of a hexameric NC1 complex
isolated from human placenta using the two-step collagenase procedure. 1, stained with Coomassie Blue. The positions of 1
dimers,
2 dimers, and
1(IV)NC1 and
2(IV)NC1 are
designated. 2, Western blot with an antiserum against
1(IV)NC1. 3, Western blot with an antiserum against
2(IV)NC1. M = lanes with molecular weight
markers.
The hexameric complex was dissociated at pH 3,
and the resulting mixture of monomeric and dimeric NC1 domains was
chromatographed on a Superose 12 column. The two peaks, containing
dimers and monomers, respectively, were divided into smaller fractions
and submitted to SDS-PAGE (Fig. 2). It is striking that for both
the dimers and the monomers, 1(IV)NC1 eluted earlier than the
2(IV)NC1 domains. There are fractions which contain only
1(IV)NC1 dimers and others with mainly
2(IV)NC1 dimers.
Densitometric analysis of gels from seven different preparations
revealed that 79% of the
1(IV)NC1 and 54% of the
2(IV)NC1
domains were cross-linked to form dimers, and that the two bands
observed for each type of dimer were of equal intensity. There is no
evidence for the presence of intramolecular disulfide bonds connecting
the
1(IV) chains within a collagen IV monomer via the
1(IV)NC1 domains. Their formation after end-to-end aggregation of
monomers to dimers is unlikely in view of the results of Siebold et
al.(7) but cannot be excluded completely.
Figure 2:
Chromatography on a Sepharose 12 column of
a hexameric NC1 complex after dissociation into dimers and monomers at
pH 3. SDS-PAGE demonstrates the composition of individual fractions.
The right lane shows the composition before separation.
Positions of 1(IV)NC1 and
2(IV)NC1 dimers and monomers are
indicated.
Figure 3:
Reconstitution of different NC1
preparations. The preparations were reconstituted at pH 7.4,
chromatographed on a Sepharose 6 column (left lane), and the
composition of the different fractions tested by SDS-PAGE (right
lane). A, native hexamers. B, 1(IV)NC1
dimers. C,
2(IV)NC1 dimers with minor content of
1(IV)NC1 dimers, arrows designate
1(IV) and
2(IV) dimers. Of the
1(IV) dimers, only the fraction with the
higher electrophoretic mobility is present. D,
1(IV)NC1
dimers as in B to which a mixture of
1(IV)NC1 and
2(IV)NC1 monomers has been added. E,
1(IV)NC1 and
2(IV)NC1 monomers. Lane 1, native hexamer for comparison; lane 2, mixture used for reconstitution; lane 3,
reconstituted hexamer; lanes 4 and 5, dimers and
monomers not incorporated in the hexameric
complex.
The hexamers
reconstituted from the unseparated mixture of monomers and dimers
showed the same composition as in the native complex (Fig. 3A). From the isolated dimers and monomers, only
the 1(IV)NC1 dimer was able to reform a hexameric complex (Fig. 3B). The hexameric fraction observed after
reconstitution of
2(IV)NC1 dimers is due to a small amount of
1(IV)NC1 dimers still present in the preparation (Fig. 3C). The
1(IV)NC1 dimers are, however, also
able to incorporate other NC1 constituents that are present during the
reconstitution procedure. This is shown in Fig. 3D,
where hexamers reconstituted from
1(IV)NC1 dimers in the presence
of
1(IV)NC1 and
2(IV)NC1 contained monomers, and it is
notable that
2(IV)NC1 was preferentially incorporated. A mixture
of
1(IV)NC1 and
2(IV)NC1 monomers showed no tendency to
aggregate (Fig. 3E).
Under the chromatographic conditions used, separation of tetrameric, trimeric, and dimeric NC1 complexes was only partially achieved, so that any such complexes which may have formed would have been overlooked. The preparations were therefore further investigated in the analytical ultracentrifuge. The data obtained confirmed on the whole the results described in Fig. 3. In Table 1, the values of the dominating materials are underlined but for dimers and mixtures of monomers, smaller and/or larger species were also visible. Their fractions were estimated from the sedimentation profiles. The sedimentation coefficients and molar masses are in good agreement with previously determined values(6) .
Monomeric NC1 did not form hexamers, although a
slight tendency to form aggregates could be seen clearly. For the
1 dimers, 80% recovery of intact hexamers was observed. The
2(IV) dimer fraction showed 30% hexamers. This is apparently due
to contamination with
1(IV) dimers (see Fig. 3C).
The hexameric complexes reconstituted from a native NC1 mixture or
from isolated 1(IV)NC1 dimers were investigated after rotary
shadowing in the electron microscope (Fig. 4). Comparison of the
reconstituted material with the native hexameric complex did not reveal
obvious differences. Hexamers, however, can easily be distinguished
from monomers and dimers.
Figure 4:
Electron micrographs of NC1 domains after
rotary shadowing. A, reconstituted native hexamers; B, mixture of monomeric and dimeric NC1 domains dissociated at
acidic pH; C, hexamers reconstituted from isolated
1(IV)NC1 dimers.
The hexameric NC1 complex represents the connecting element
between the C termini of two collagen molecules incorporated in the
macromolecular network of collagen IV(4) . The complex is
stabilized by intermolecular disulfide bonds between two like NC1
domains(7) . Only 1(IV)NC1 and
2(IV)NC1 homodimers
but no
1-
2 heterodimers have been found. Striking is the
occurrence of two species for the
1(IV)NC1 as well as for the
2(IV)NC1 dimers, which may be explained by two sets of
intermolecular disulfide bonds. These different sets may possibly
originate from the fact that each NC1 domain consists of a tandem
repeat of two homologous domains, and disulfide exchange could be
between either the first or second of these repeats(7) .
From earlier investigations(6) , it is known that the
compact and stable hexameric complex dissociates at low pH into its
monomeric and dimeric constituents and that readjustment to neutral pH
leads to reformation of the complex, whereby all constituents, dimers
and monomers, are again incorporated. It was therefore surprising that
the aggregation behavior of the separated monomers and dimers was so
different. At neutral pH, only the 1(IV)NC1 dimers were able to
reform a hexameric complex, which could not be differentiated from the
native complex in the electron microscope. The
1(IV)NC1 and
2(IV)NC1 monomers, as well as the
2(IV)NC1 dimers, showed
only a slight tendency to aggregate. Weak noncovalent interactions, as
revealed by ultracentrifugation analysis, may however be of decisive
importance for the disulfide exchange reaction which leads to stable
hexameric complexes connecting collagen IV molecules at their C termini (4, 5, 6, 7) . A precomplex with
correct arrangement for disulfide formation will greatly accelerate
formation of the final covalently linked complex even when the fraction
of the precomplex is small. This effect was demonstrated in many
studies in which disulfide formation was used as a probe for structure
and flexibility of proteins (for a recent work, see (17) ). The
precomplex only needs to be short-lived because dissociation of the
final complex is rendered impossible by the disulfide bridges. It is
obvious that the disulfide exchange between two
1(IV)NC1 domains
is accompanied by a drastic alteration of their aggregation behavior.
In the presence of a mixture of
1(IV)NC1 and
2(IV)NC1
monomers, mainly the latter were incorporated into the hexamers formed
by
1(IV)NC1 dimers, indicating a tendency to restore the native
ratio of
1(IV) to
2(IV) chains.
The observation that monomeric NC1 domains have no strong affinity for one another correlates with the behavior of monomeric collagen IV molecules secreted into the medium of collagen IV synthesizing cells (18) . The molecules in solution tend to form dimers and tetramers via their N termini. Dimers connected by the C termini have not been observed. Such dimers, however, can be extracted from the cell layer, where a minority of collagen IV molecules have already been laid down to form a network. We assume that lateral aggregation of the molecules with their triple helical domains may help to align the NC1 domains, so that disulfide exchange and subsequent formation of stable hexamers can occur. A putative model is given in Fig. 5. In the first step, two molecules aggregate at their C-terminal regions in an antiparallel manner. The NC1 domains have not yet found a partner. Subsequently, a third molecule attaches, bringing two C-terminal ends into close vicinity, so that the weak interaction between the NC1 domains is sufficient to form complexes in the correct arrangement for disulfide formation(2) . Another possibility could be an end-to-end alignment as an intracellular event, which may be facilitated by chaperones. However, this appears to be very unlikely. In collagen IV synthesizing cells in culture, all intracellular modifications including chain assembly, occur in an ordered manner. There is no reason why just the end-to-end aggregation should be impaired, so that in culture monomeric molecules are secreted instead of dimers.
Figure 5: Schematic representation of lateral aggregating collagen IV molecules leading to a highly ordered arrangement of two molecules with their C termini, so that an intermolecular disulfide exchange between NC1 domains can take place (2) . 1, two molecules aggregated at their C-terminal regions, NC1 domains have not yet found a partner; 2, a third molecule has attached, which brings two C-terminal ends into close vicinity; 3, weak interactions have led to a highly ordered hexameric complex which has been stabilized by disulfide exchange.
It was demonstrated previously for dimers of collagen IV
connected by their NC1 domains (19) and for procollagen IV (20) that refolding of the collagen triple helix after thermal
denaturation started at the C terminus. For collagen I it was
established that the C-terminal propeptides of two 1(IV) and one
2(IV) chains form a heterotrimeric complex, which has to be
stabilized by interchain disulfide bonds before folding of a triple
helix can occur(21, 22, 23, 24) . It
is questionable whether the NC1 domains in type IV collagen have a
similar recognition function because of their low affinity to each
other. Also, no interchain disulfide bonds have been observed by which
three NC1 domains, which had formed a complex, would be stabilized. One
could speculate that intramolecular disulfide bridges between NC1
domains are not formed since they would interfere with the formation of
the hexameric NC1 complex necessary for the organization of the
macromolecular network of collagen IV. The mechanism responsible for
selection of the
1(IV) and
2(IV) chains in a molecule is also
unknown, and further work is needed to elucidate the specificity of
chain recognition.